1
Expression and Secretion of Recombinant
Ovine Somatotropin in
Escherichia coli
A THESIS SUBMITTED TO
THE UNIVERSITY OF THE PUNJAB
IN FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN
BIOLOGICAL SCIENCES
By
Faiza Gul
School of Biological Sciences
University of the Punjab
Lahore, Pakistan
2012
i
In the Name of Allah, the Merciful, the Compassionate.
Read! In the Name of Thy Lord. [Quran 96:1]
ii
Dedicated to My Dear loving Parents
Mr & Mrs M.Ajmal Khan
iii
CERTIFICATE
This is to certify that the research work described in this thesis is the original work of Faiza
Gul and has been carried out under my supervision. I have personally gone through all the
data/results/materials reported in the manuscript and certify their correctness/authenticity. I further
certify that the material included in this thesis have not been used in part or full in a manuscript
already submitted or in the process of submission in partial/complete fulfillment of the award of any
other degree from any other institution. I also certify that the thesis has been prepared under my
supervision according to prescribed format and I endorse its evaluation for the award of Ph.D. degree
through the official procedures of the University.
(Prof. Dr. M. Waheed Akhtar) Research Supervisor
iv
ACKNOWLEDGEMENT
All Praise Allah Subhanahu wa Tala, Master of this Universe, and Master of
all mankind, truly without Him, man is at loss. Without His guidance there is
no light, without His protection there is no sanctuary and without His
Knowledge there is no real knowledge. Without doubt, one can not praise Al
Khaliq enough. He says “And I did not create the jinn and mankind except to
worship Me.” [Quran 51:56] Without doubt, all praises and thanks are to
Allah, the Ever-Lasting.
The writing of this dissertation has been the most significant academic
challenge I have had to face. Without the support, patience and guidance of
the following people, this study would not have been completed. It is to them
that I owe my deepest gratitude.
Above all, my sincere gratitude to my honourable supervisor Prof. Dr. M.
Waheed Akhtar, whose unsurpassed knowledge and untiring guidance have
seen this thesis through. I have heartfelt gratefulness to him for giving me the
opportunity to work in his laboratory. His profound insight, patience,
dynamic supervision and encouraging approach have granted me the
confidence to face the challenges of the Ph.D. I’m also thankful to our
Director General Dr. M. Akhtar and all the directors of School of Biological
Sciences, University of the Punjab, Lahore for their invaluable help and
guidance .
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I am deeply indebted to Dr. Saima Sadaf for her assistance and support. I also
appreciate the role Dr.Mahjabeen Saleem and Dr. Farhat Zaheer has played
in my PhD, for their moral support and help whenever I faced a problem. I
am also thankful to Dr.Naeem Rasheed for his valuable guidence specially for
primer designing.
I am thankful to (late) Dr. Mustaq Kaderbhai whose critical guidance,
suggestions and encouragement have been invaluable. It has been a great
pleasure to have guidence from such wonderful personality. His sudden
passing left me in great sorrow but his wife Dr.Naheed Kaderbhai supported
me and its all her continuous effort that I could finish the last part of my
work.as her numerous educational discussions, feedback and suggestions have
played a fundamental part in shaping my views, knowledge and
understanding. It has also been a pleasure to have worked with such
wonderful people. Sharing of ideas, thoughts and suggestions have vastly
contributed to the stance I have taken in this thesis. I thank both of them for
being a reflective and critical contributor.
My dear parents, who made me the very person I am, instilling core values of
hard-work, perseverance, resilience and patience - this PhD is really about
them and the fruits of their effort – for that I am forever indebted. My deepest
gratitude is also to my Ami Jan (Mrs.Neelam Durrani) for the support,
encouragement and comfort for when I needed it most. My loving husband
vi
Ali, without whose encouragement support and love ,this thesis could never
have continued – for the kind words, care and confidence in me, for this, my
mere expression of thanks does not suffice.
My brothers and sisters and their families have given me their unequivocal
support throughout. Rizwan, Arjumand, Rehan and Mehran, their enduring
comfort during all times is forever appreciated. The joyous times spent
together are the best memories. Special thanks to all my cousins, elders and
my in-laws in particular my new sister Sadia Durrani. Finally, to my late
dear grandparents especially my Daadi Jan, Masooma Khanum and late
father-in-law Mahmood Ahmed Khan Durrani, I know they would have been
proud of my accomplishments – thank you for the dreams and aspirations that
have enabled this work to have materialised.
I would also like to thank my dear friends, Roquyya “My twin” Gul, Sadaf
Zaidi, Nadia Azhar, Dr. Mahjabeen Saleem, Dr. Saadia Shazad Alam, Gul
Sher Muhammad Tahir , who have shown me tremendous support, above all,
they have been true friends, standing by me in all phases of my education and
career. I can never forget the tea time and walks on the jogging track.
Penultimately, I’d like to thank my little baby, Muhammad Ahmed who
taught me the skill of multi-tasking between nappy changes, feeds and my
write-up. I hope he too has learned something here.I am also appreciative of
all my other laboratory colleagues specially Annie, Hooria, Hina Farheen,
vii
Deeba, Altaf and Sajjad for the nice educational and fun time discussions. I
would also like to thank to all the technicians for their aid in anything I
needed. I am especially thankful to Muhammad “M.D. Sahib” Deen, Irfan
Sahab and Afzal for their timely support in the process of thesis submission.
I would like to end my acknowledgement with a supplication:
“My Lord! Inspire me and bestow upon me the power and ability that I may
be grateful for Your Favours which You have bestowed on me and on my
parents, and that I may do righteous good deeds that will please You and
admit me by Your Mercy among Your righteous slaves” (Quran 27:19)
FAIZA GUL
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SUMMARY
The current study involves cloning, sequence analysis, expression, secretion, purification and
different factors influencing the secretion of ovine growth hormone (oGH) gene isolated from
local ovine breed (Lohi). On the basis of conserved sequences, two forward and one reverse
primers were designed for the amplification of oGH gene. The forward primers contained NdeI,
NcoI restriction sites whereas the reverse primer contained a BamHI site at their 5’ end. Total
RNA was isolated from pituitary gland of Lohi by using Guanidium-thiocyanate-chloroform
extraction method. cDNA was synthesized by RT-PCR using gene specific primers. Moreover,
genomic DNA was isolated from the blood sample of Lohi and was amplified by using four sets
of primers designed on the basis of conserved sequence of the ovine growth hormone (oGH)
gene. These were ligated into pTZ57R/T by the dT. dA tailing technique and used to transform
into E. coli DH5α. The sequences of the DNA obtained from multiple colonies were compared
with already published ovine GH gene sequence using multiple sequence alignment software
“Clustal W”. The sequence analysis revealed only one amino acid change when compared to
previously reported OaST (Ovis aries somatotropin) or oGH gene sequences of Indian and
Australian breeds. It showed 99% homologies with bubaline, bovine and 100 percent homology
with caprine GH genes of the local breeds. The sequence of the GH of Lohi was submitted to
"Data bank of Japan" which bears an accession number AB244790.
In the present study, we report secretion of recombinant oGH into the periplasmic space and
inner membrane of E. coli under the influence of variant signal sequences. For periplasmic
translocation the recombinant proteins were expressed under the influence of pelB leader
sequence of pET 22b vector. The effect of different factors i.e., glycerol in the medium, use of E
.coli strain BL21 DE3 and pLys S ,chemical chaperon (ZnCl2) and IPTG concentration were
studied to enhance the expression while osmotic shock conditions were also optimized and
ix
studied the effect of glycerol and ZnCl2 concentration on the release of oGH by using freeze
thaw method. Best result of 22% expressed roGH on 12% SDS-PAGE was observed at 20M
(final concentration) IPTG after 4 hrs of fermentation at 370C in LB modified medium with
50µM ZnCl2 in BL21DE3 E. coli strain. The optimized freeze thaw method including 25%
glycerol with 50µM ZnCl2 enhanced the relase of oGH upto 24% in the periplasmic space of E.
coli. The oGH thus found was further purified by FPLC and authenticated by Western blot
analysis. Although the recovery of oGH was enhanced but still there was a need to enhance the
production of accurate size (22 kDa) growth hormone which was bit higher (25 kDa) by using
pelB leader sequence.
For this purpose different signal peptides i.e., DsbA, STII and natural oGH signal peptide were
utilized. Amongst the signal sequences the DsbA signal sequence was found to exhibit the best
expression, size and secretion of oGH into the inner membrane of E. coli. We further studied the
expression of oGH and targeting to the inner membrane using signal sequence (DsbA) in E. coli
cell. Factors such as temperature, IPTG induction, expression conditions were studied and
showed diverse optical density with different media compositions. The optimum expression level
of oGH in terrific broth medium was at 25ºC on induction with 20μM IPTG in early logarithmic
phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs
revealed the translocation of oGH to the inner membrane of E. coli with DsbA signal sequence
at the N terminus of roGH. The protein was easily solublized by 40% acetonitrile with ~90%
purity and was identified by Western blot and analysis on MALDI/TOF confirmed a size of
21059Da. Relatively high soluble protein yield of 65.3gm/L of oGH was obtained. The
biological function of oGH was confirmed by HeLa cell line proliferation. It was observed that
DsbA signal sequence on the basis of its hydrophobicity gave best results of 22kDa protein in
membrane bounded form as compared to pelB and reference native signal sequence of oGH
which resulted in 25kDa oGH secreted mainly into cytoplasm.
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Despite of cost effective single step purification we encountered a problem with low yield. We
developed a novel strategy for the high yield of functional recombinant ovine growth hormone
(roGH) directed to the inner membrane of E. coli. In order to enhance the yield of soluble
fraction, bacterial cells were grown under osmotic stress (4% NaCl in terrific broth medium) and
effect of compatible solutes (sorbitol, glycine betine, glycylglycine and mannitol) were studied
on the soluble expression of roGH. Other factors; temperature, induction time, induction by
IPTG and lactose were also studied. It was observed that fermentation of roGH construct with
DsbAss was best achieved with 0.6M mannitol, 50μM ZnCl2, 50mM glycylglycine at the time of
induction with 50μM IPTG in the early logarithmic phase at OD600 ~3.10 in TB medium at 25ºC
in shaking flask culture at 150rpm. These optimized conditions resulted in very high expression
~32% of soluble roGH which was recovered by ultra centrifugation (density centrifugation) from
the inner membrane of E. coli. The unbelievably high yield, 443mg/L was obtained as compared
from previos yield. The roGH was confirmed by Western blot analysis .
Furthermore the effect of amino acid substitution in the tripartite structure of DsbA signal
sequence (DsbAss) on co-translation of recombinant oGH in E. coli was studied. Six amongst
the eight constructs were designed on the basis of increasing hydrophobicity in H domain of
DsbA signal sequence to make it more efficient for the translocation of oGH through SRP (signal
recognition particle) mechanism. For this purpose all the alanines in the hydrophobic domain of
DsbA signal sequence were replaced by Isoleucine one by one, while lysine in the N terminal
and serine in the C-terminal regions were substituted by arginine and cysteine respectively. The
substitution of arginine in the N-terminal resulted in very low expression and secretion while
cysteine substitution in the C region totally impaired the expression and secretion of the
recombinant protein. it was observed that not only the hydrophobicity but the position of amino
acid in the hydrophobic core also effects the cleavage of signal sequence from recombinant
product.
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The substitution of alanine with the isoleucine residue in H domain of DsbA signal sequence
resulted in; (a) at position 11 with respect to signal peptidase site in the H domain impaired the
correct processing of oGH protein while (b) isoleucine at position 9 resulted in correctly
processed recombinant oGH protein in the inner membrane.The results showed that the
replacement of alanine amino acid at position 11 with reference to signal peptidase site in the
hydrophobic core of the DsbA ss interferes with the binding of DsbA ss hydrophobic region to
Ffh protein of SRP. This resulted in weak or no binding of Ffh with DsbA ss and consequently
oGH protein was localised in the cytoplasmic fraction rather than membrane. Thus, the gene
mutation from alanine residue to isoleucine specifically at position 11 with respect to signal
peptidase site changed the whole mechanism of protein translocation through DsbA ss. It was
hypothesized that alanine at position number 11 with respect to the signal peptidase site is crucial
for SRP routing of recombinant proteins .
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Table of Contents
INTRODUCTION & LITERATURE REVIEW ............................................................................................ 1
1.1 Somatotropin (Growth Hormone) ..................................................................................................................................... 2
1.2 Secretion of recombinant protein in E.coli ....................................................................................................................... 3
1.3 Ovine breed of Pakistan .................................................................................................................................................... 5
1.4 Impact of our study on the economy of Pakistan ............................................................................................................. 6
1.5 Review of Literature .......................................................................................................................................................... 7
1.5.1 Structural and functional aspects of GH ........................................................................................................... 7
1.5.2 Cloning and expression of GH in bacterial systems ..................................................................................... 9
1.5.3 Secretion of growth hormone in E.coli ...................................................................................................... 12
1.5.3.1 Type I secretion systems ................................................................................................................ 14
1.5.3.2 Type II secretion Mechanism ........................................................................................................ 14
1.5.3.3 SecB-dependent pathway ............................................................................................................. 14
1.5.4 Signal sequences ............................................................................................................................................ 18
1.5.5 Expression and Purification of secreted protein in E.coli. ............................................................................... 19
1.5.6. Advantages of getting soluble proteins ......................................................................................................... 24
1.6 AIMS AND OBJECTIVES .................................................................................................................................................... 26
MATERIALS AND METHODS.................................................................................................................. 28
2.1 Sample collection and storage ................................................................................................................................ 29
2.2 Chemicals and kits ................................................................................................................................................... 29
2.3 Isolation of total RNA from pituitary sample ......................................................................................................... 30
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2.4 Formaldehyde agarose gel electrophoresis ............................................................................................................ 31
2.5 cDNA Synthesis ........................................................................................................................................................ 32
2.5.1 Primer designing.............................................................................................................................................. 32
2.5.2 Reverse transcription (RT) ............................................................................................................................... 32
2.5.3 PCR Amplification ........................................................................................................................................... 33
2.6 DNA extraction from agarose gel .................................................................................................................................... 34
2.6.1 Purification of PCR product ............................................................................................................................. 34
2.7 Cloning in pTZ57R/T vector ............................................................................................................................................. 35
2.7.1 T/A cloning Kit method.................................................................................................................................... 35
2.7.2 Preparation of competent cells and transformation....................................................................................... 37
2.8 Colony PCR ...................................................................................................................................................................... 38
2.9 Sequence analysis ............................................................................................................................................................ 38
2.9.1 Q/A prep spin miniprep kit method ................................................................................................................ 38
2.9.2 Analysis of Full-Length ST Gene....................................................................................................................... 40
2.9.2.1 Extraction of genomic DNA ............................................................................................................ 40
2.9.2.2 PCR amplification of GH gene ...................................................................................................... 42
2.9.2.3 Sequencing reaction....................................................................................................................... 42
2.10 Bioinformatics tools for sequence analysis................................................................................................................... 43
2.11 Mini-preparation of plasmid DNA ................................................................................................................................. 43
2.12 Restriction analysis of pTZ-oGH clones ......................................................................................................................... 44
2.13 Restriction analysis of pET22b (+) ................................................................................................................................. 45
2.14 Ligation and transformation in DH5α and BL21 Codon + strains ................................................................................. 45
2.15 Expression of poGH clones ............................................................................................................................................ 46
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2.16 SDS-Polyacrylamide Gel Electrophoresis (PAGE) .......................................................................................................... 47
2.17 Western transfer and immunoblot analysis ................................................................................................................. 49
2.18 Protein estimation ......................................................................................................................................................... 50
2.19 Primer designing for translocation of Ovine ST gene into periplasmic space.............................................................. 50
2.20 Subcellular fractionation of oGH ................................................................................................................................... 52
2.20.1 FPLC chromatography ................................................................................................................................... 54
2.20.2 MALDI-TOF .................................................................................................................................................... 54
2.21 Biological activity assessment assay ............................................................................................................................. 54
RESULTS ..................................................................................................................................................... 57
3.1 Genetic Analysis of oGH gene.......................................................................................................................................... 58
3.1.1 Extraction of genomic DNA ............................................................................................................................. 58
3.1.2 PCR amplification of oGH gene ....................................................................................................................... 58
3.1.3 Sequence analysis of oGH ............................................................................................................................... 59
3.1.3.1 Sequence comparison of oGH at amino acid level......................................................................... 61
3.1.3.2 Comparison of oGH gene at Nucleotide level ................................................................................ 64
3.1.4 Secondary structure analysis of oGH .............................................................................................................. 66
3.1.5 Hydropathy profile of oGH .............................................................................................................................. 67
3.1.6 Three Dimensional structure of oGH............................................................................................................... 68
3.2 cDNA synthesis , cloning and Periplasmic Expression of oGH ...................................................................................... 69
3.2.1 Isolation and purity of total RNA ..................................................................................................................... 69
3.2.2 RT-PCR amplification of cDNA ........................................................................................................................ 70
3.2.3 T/A cloning of oGH ......................................................................................................................................... 71
3.3 Expression of poGH ......................................................................................................................................................... 72
3.3.1 Restriction analysis of pTZ-oGH ...................................................................................................................... 72
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3.3.2 Cloning in pET22 b ........................................................................................................................................... 73
3.3.3 colony PCR of poGH......................................................................................................................................... 73
3.3.4 Shake flask fermentation of poGH-1 construct .............................................................................................. 75
3.4 Periplasmic expression of oGH ........................................................................................................................................ 75
3.4.1 Expression of poGH-2 ...................................................................................................................................... 76
3.4.2 Effect of different factors on the expression of oGH ...................................................................................... 77
3.4.2.1 Effect of ZnCl2................................................................................................................................. 77
3.4.2.2 Effect of IPTG concentration .......................................................................................................... 77
3.4.2.3 Effect of Ecoli Strain on expression of ovine growth hormone...................................................... 78
3.4.2.4 Optimization of somotic shock conditions ..................................................................................... 79
3.4.2.5 Effect of Glycerol ............................................................................................................................ 80
3.4.2 Purification of poGH-2..................................................................................................................................... 81
3.4.3 FPLC chromatography ..................................................................................................................................... 83
3.5 Effect of (DsbA,ST-11 & native oGH signal sequence ) on the expression & secretion of oGH .................................... 84
3.5.1 Primer designed for the constructs poGH-3,4 &5 ........................................................................................... 84
3.5.2 PCR amplification ............................................................................................................................................ 84
3.5.3 T/A cloning and construction of expression plasmid poGH-3,4,5 ................................................................... 85
3.5.4 Transformation and selection of high expression strains ............................................................................... 86
3.5.5 Expression of poGH-3,4 and 5 ......................................................................................................................... 87
3.5.5.1 Subcellular fractionation of poGH-3-5 constructs ......................................................................... 88
3.5.6 Computational analysis of signal sequences of poGH-2,3,4& 5 constructs .................................................... 91
3.6 Effect of medium composition on expression of poGH-3 ............................................................................................... 94
3.6.1 Effect of LB,TB & M9NG medium on the expression of poGH-3 ..................................................................... 94
3.6. 2 Effect of temperature on poGH3 construct ................................................................................................... 96
3.6.3 Effect of induction time and IPTG concentration on poGH3 construct........................................................... 96
3.7 Enhanced production of roGH ......................................................................................................................................... 97
3.7.1 Effect of compatible solute on the expression of poGH-3 construct .............................................................. 99
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3.7.1.1 Optimization of soluble roGH expression using compatible solutes(Glycylglycine , glycine
betaine,sorbitol and Mannitol ................................................................................................................... 99
3.7.2 Production of soluble roGH in TBC optimized medium................................................................................ 101
3.7.2.1 Effect of temperature .................................................................................................................. 102
3.7.2.2 Effect of IPTG and Lactose as an inducer .................................................................................... 103
3.7.2.3 Effect of induction time .............................................................................................................. 104
3.7.3 Subcellular fractionation of poGH-3 construct.............................................................................................. 104
3.8 Effect of amino acid alterations in DsbA signal sequence on poGH expression and secretion ................................... 107
3.8.1 PCR amplification and Cloning of pOaST varying constructs ......................................................................... 107
3.8.2 Construction of Expression plasmid poGH3-I-VIII ......................................................................................... 109
3.8.3 Expression of poGH-3-I-VIII ........................................................................................................................... 109
3.8.4 The expression of DsbA ss constructs with substitution of alanine with isoleucine in the H domain .......... 110
3.8.5 DsbA ss constructs with substitution of serine with cysteine in the C domain ............................................. 113
3.8.6 DsbA ss constructs with substitution of lysine with arginine in the N domain ............................................. 114
3.8.7 Purification of oGH from poGH-3-II construct............................................................................................... 115
3.8.8 MALDI TOF analysis of purified ovine growth hormone ............................................................................... 116
3.8.9 Biological activity assessment assay............................................................................................................. 117
3.8.10 Computational analysis of pOaST-3a-g constructs ...................................................................................... 118
DISCUSSION ............................................................................................................................................. 121
4.1 Characterization of oGH gene........................................................................................................................................ 122
4.2 periplasmic Expression of roGH..................................................................................................................................... 124
4.3 Secretion of oGH into the inner membrane of E.Coli. .................................................................................................. 128
4.4 Effect of medium composition on the expression and secretion of oGH in E.coli ....................................................... 131
4.5 Effect of mutation in DsbA signal sequence on the expression and secretion of OaST .............................................. 135
4.6 Purification and Biological activity Assessment............................................................................................................ 138
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4.7 Conclusion ...................................................................................................................................................................... 138
REFERENCES ........................................................................................................................................... 140
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TABLE OF FIGURES
FIGURE 1. SECONDARY STRUCTURE OF GROWTH HORMONE ........................................................... 2
FIGURE 2.SECRETION OF RECOMBINANT PROTEINS IN E.COLI. ......................................................... 3
FIGURE 3. (LOHI) OVINE BREED OF PAKISTAN....................................................................................... 5
FIGURE 4.RESTRICTION MAP,SEQUENCE AND MULTIPLE CLONING SITES OF PET 22B(+). ......... 46
FIGURE 5.GENOMIC DNA OF OGH ISOLATED FROM THE BLOOD SAMPLE OF LOCAL OVINE
BREED LOHI ................................................................................................................................... 58
FIGURE 6. PCR AMPLIFICATION ON 1% AGAROSE GEL. ..................................................................... 59
FIGURE 7. NUCLEOTIDE SEQUENCE OF OGH. ....................................................................................... 60
FIGURE 8.AMINO ACID SEQUENCE OF OGH .......................................................................................... 60
FIGURE 9.AMINO ACID SEQUENCE OF OGH. ......................................................................................... 61
FIGURE 10.COMPARISON OF GROWTH HORMONES OF OVINE CAPRICORN AND BUBALINE ..... 62
FIGURE 11,AMINO ACID SEQUENCE COMPARISON. ............................................................................ 62
FIGURE 12.COMPARISON OF OGH WITH DIFFERENT SPECIES OF CLASS MAMMALIA ................. 64
FIGURE 13.NUCLEOTIDE SEQUUENCE ALIGNMEN .............................................................................. 65
FIGURE 14OVINE GROWTH HORMONE. SECONDARY STRUCTURE .................................................. 66
FIGURE 15.THE HYDROPATHY PLOT OF OGH. ...................................................................................... 67
FIGURE 16.3D STRUCTURE OF OVINE GROWTH HORMONE............................................................... 68
FIGURE 17.ABSORPTION SPECTRA OF EXTRACTED RNA. .................................................................. 69
FIGURE 18.FORMALDEHYDE AGAROSE GEL OF RNA ......................................................................... 70
FIGURE 19.ANALYSIS OF THE RT-PCR. ANALYSIS OF THE RT-PCR AMPLIFIED PRODUCT
RESOLVED ON 1% AGAROSE GEL. LANE M, 1KB DNA LADDER USED AS MARKER; LANE ,
2, 3, 4 & 5 ~0.6KB AMPLIFIED PCR PRODUCTS. ......................................................................... 70
FIGURE 20.RESTRICTION MAP OF PTZ57R/T CLONING VECTOR ....................................................... 71
FIGURE 21.ANALYSIS OF COLONY PCR. ................................................................................................ 72
FIGURE 22.DOUBLE DIGESTION OF PTZ-OGH-1.. .................................................................................. 72
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FIGURE 23.CONSTRUCTION OF RECOMBINANT PLASMID POGH-1................................................... 73
FIGURE 24.COLONY PCR OF POGH-1....................................................................................................... 74
FIGURE 25.DOUBLE DIGESTION OF POGH-1. ......................................................................................... 74
FIGURE 26.SDS-PAGE ANALYSIS OF POGH-1 EXPRESSION................................................................. 75
FIGURE 27.CONSTRUCTION OF POGH-2 CONSTRUCT.......................................................................... 76
FIGURE 28.SDS-PAGE ANALYSIS OF POGH-2 EXPRESSION IN LB MEDIUM. .................................... 76
FIGURE 29.EFFECT OF ZNCL2................................................................................................................... 77
FIGURE 30,SDS-PAGE ANALYSIS OF EFFECT OF IPTG. ........................................................................ 78
FIGURE 31.EFFECT OF E.COLI STRAINS ON THE PERIPLASMIC EXPRESSION OF POGH-2. ........... 78
FIGURE 32. GRAPHICAL REPRESENTATION OF DIFFERENT OSMOTIC SHOCK CONDITIONS ON
OGH. ................................................................................................................................................. 79
FIGURE 33.SDS-PAGE ANALYSIS OF SUBCELLULAR FRACTIONS OF POGH. 2................................ 80
FIGURE 34. SDS-PAGE ANALYSIS OF POGH-2 IN LBMODIFIED MEDIUM. ........................................ 80
FIGURE 35. EFFECT OF GLYCEROL. ........................................................................................................ 81
FIGURE 36.SDS-PAGE ANALYSIS OF SUBCELLULAR FRACTIONS OF ROGH-2& WESTERN BLOT
ANALYSIS. ...................................................................................................................................... 82
FIGURE 37.FPLC PEAK OF PURIFIED OGH .............................................................................................. 83
FIGURE 38.AGAROSE GEL ANALYSIS OF PCR. ...................................................................................... 85
FIGURE 39.COLONY PCR ANALYSIS OF POGH-3-4-5. ........................................................................... 85
FIGURE 40.CONSTRUCTION OF EXPRESSION PLASMIC POGH-3,4&5 ................................................ 86
FIGURE 41.COLONY PCR ANALYSIS. ...................................................................................................... 86
FIGURE 42.DOUBLE DIGESTION OF RECOMBINANT CLONES. ........................................................... 87
FIGURE 43.SDS-PAGE ANALYSIS OF PROTEIN EXPRESSION OF CONSTRUCT POGH-3,4&5. ......... 88
FIGURE 44.SCHEMATIC REPRESENTATION OF SUBCELLULAR FRACTIONATION OF CELLS. ..... 89
FIGURE 45.SDS-PAGE ANALYSIS OF SUBCELLULAR FRACTIONATIONS OF POGH-3,4 & 5
CONSTRUCTSSDS PAGE . ............................................................................................................. 91
FIGURE 46.KYTEDOOLITTLE ANALYSIS OF HYDROPHOBICITY OF ALL FOUR SIGNAL
SEQUENCES. ................................................................................................................................... 92
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FIGURE 47.SECONDARY STRUCTURE ANALYSISOF POGH2,3,4&5.. .................................................. 93
FIGURE 48.SDS-PAGE ANALYSIS AND GRAPHICAL REPRESENTATION OF EFFECT OF MEDIUM
ON POGH-3 ...................................................................................................................................... 95
FIGURE 49.EFFECT OF TEMPERATURE,INDUCTION TIME AND IPTG CONCN. ON POGH-3. .......... 97
FIGURE 50. GROWTH OF POGH-3 IN DIFFERENT MEDIUM.................................................................. 98
FIGURE 51.GRAPHICAL REPRESENTATION OF THE EFFECT OF 2 SETS OF COMPATIBLE
SOLUTES ON THE GROWTH OF POGH-3 IN TB MEDIUM ....................................................... 100
FIGURE 52.EFFECT OF COMPATIBLE SOLUTES ON THE SOLUBLR EXPRESSION OF POGH-3 IN TB
MEDIUM . ...................................................................................................................................... 101
FIGURE 53.SDS-PAGE ANALYSIS OF OPTIMIZED COMPATIBLE SOLTE IN TB MEDIUM ON
EXPRESSION OF POGH-3............................................................................................................. 102
FIGURE 54.EFFECT OF TEMPERATURE.. ............................................................................................... 103
FIGURE 55.EFFECT OF IPTG AND LACTOSE.. ....................................................................................... 104
FIGURE 56.SUBCELLULAR FRACTIONATION OF POGH-3. ................................................................ 105
.FIGURE 57.PCR AMPLIFICATION OF POGH-3-I-VIII............................................................................ 108
FIGURE 58.COLONY PCR AND DOUBLE DIGESTION OF POGH-3-I-VIII. .......................................... 108
FIGURE 59.CONSTRUCTION OF RECOMBINANT PET FOR POGH-3-I-VIII CONSTRUCTS . ............ 109
FIGURE 60.SDS-PAGE ANALYSIS OF POGH-3-I-VIII CONSTRUCTS IN LB MEDIUM. ...................... 110
FIGURE 61.SDS-PAGE ANALYSIS OF POGH-3-II-VI&I ......................................................................... 112
.FIGURE 62.SDS-PAGE ANALYSIS OF POGH-3-III&V. .......................................................................... 113
FIGURE 63,SDS-PAGE ANALYIS OF POGH-3 VIII. ................................................................................ 114
FIGURE 64.SDS-PAGE ANALYSIS OF POGH-3-VII. ............................................................................... 114
FIGURE 65.SUBCELLULAR FRACTIONATION OF POGH-3II AND WESTERN BLOT ANALYSIS. ... 115
FIGURE 66.MALADI-TOF ANALYSIS OF PURIFIED OVINE GROWTH HORMONE ........................... 116
FIGURE 67.BIOLOGICAL ACTIVITY OF OGH IN THE PRESENCE OG HELA CELL LINES............... 117
FIGURE 68.MINNOU SERVER PREDICTION RESULTS. ........................................................................ 119
FIGURE 69.HOMOLOGY MODEL OF DSBA SS WITH ALTERED ALANINE. ...................................... 120
FIGURE 70.PHYLOGENETIC TREE OF OVINE GROWTH HORMONE ................................................. 124
xxi
FIGURE 71.MODEL REPRESENTING THE MECHANISM OF DSBA SIGNAL SEQUENCE WITH
ALTERED AMINO ACID WITH SRP MECHANISM.................................................................... 137
xxii
LIST OF TABLES
TABLE 1. SEQUENCE OF PRIMERS USED FOR CONSTRUCTION OF POGH-3-I-VIII PLASMIDS......................... 51
TABLE 2.PRIMERS DESIGNED FOR THE POGH-3,4 & 5 CONSTRUCTS ................................................................... 84
TABLE 3.HYDROPATHIES OF POGHCONSTRUCTS .................................................................................................... 92
TABLE 4.COMPOSITION OF DIFFERENT MEDIUMS USED ........................................................................................ 94
TABLE 5.EFFECT OF MEDIUM COMPOSITION ON PRODUCTION OF OGH............................................................ 96
TABLE 6. EFFECT OF COMPATIBLE SOLUTES INTB MEDIUM ON THE GROWTH OF POGH-3 ........................ 100
TABLE 7.EFFECT OF COMPATIBLE SOLUTE IN TB MEDIUM ON YIELD OF SOLUBLE OGH ........................... 106
TABLE 8.HYDROPATHY INDICES OF MODIFIED DSBA SS IN POGH-3-I-VIII CONSTRUCTS ........................... 118
xxiii
ABBREVIATIONS
APS Ammonium persulphate
oGH ovine growth hormone
BSA bovine serum albumin
CaCl2 Calcium chloride
ZnCl2 Zinc Chloride
cDNA complementary deoxy ribonucleic acid
DMEM Dulbecco’s Modified Eagle’s Medium
DMSO dimethyl sulfoxide
dNTP deoxyribonucleoside triphosphate
DTT dithiothreitol
EDTA ethylene diamine tetracetate
FBS fetal bovine serum
FPLC Fast protein liquid chromatography
GdCl Guanidinium chloride
GH growth hormone
hGH human growth hormone
HRP horse radish peroxidase
IPTG isopropylthio-β-galactoside
Kb kilo base pairs
kDa kilo dalton
LB Luria-Bertani
TB Terrific broth
xxiv
M-MuLV Molony Murine Leukemia Virus
OD Optical density
PBS phosphate buffer saline
PCR polymerase chain reaction
PEG polyethylene glycol
PMSF phenylmethyl sulfonyl fluoride
rcGH recombinant caprine growth hormone
rGH recombinant growth hormone
RNA ribonucleic acid
roGH recombinant ovine growth hormone
RT room temperature
SDS sodium dodecyl sulphate
Taq Thermus aquaticus
TEMED N, N, N’, N’ tetra methyl ethylene diamine
X-Gal 5-bromo-4-chloro-3-indolyl β-D-galactoside
ST Somatotropin
RP-HPLC Reversed phase high performance liquid chromatography
HPLC High performance liquid chromatography
TRI Trizol
FPLC Fast protein liquid chromatography
MALDI/TOF Matrix assisted laser desorption ionization/time of flight
xxv
1
Introduction & Literature Review
2
1.1 Somatotropin (Growth Hormone)
Somatotropin commonly known as Growth hormone (GH) a protein of 22 kDa, is naturally
synthesized as a pre-hormone with an extension of 26 amino acid signal peptide which directs
the release of GH into the blood stream (Paladini et al., 1983). GH consists of 190 or 191 amino
acids with two disulfide bridges.
Figure 1. Secondary structure of Growth hormone
Source;www.endotext.org
Pituitary growth hormone is a protein hormone which regulates somatic growth in most
vertebrates and has effects on various metabolic activities (Wallis, 1988). It is a polypeptide that
controls the differentiation, growth and metabolism of many cell types, and is secreted from the
hypophysis of all vertebrate species tested so far. Despite the overlapping evolutionary,
structural, immunological and biological properties, it is well-known that growth hormones
from distinct mammalian species have significant species-specific characteristics.
3
1.2 Secretion of recombinant protein in E.coli
Various animal growth hormones such as bovine (George et al., 1985; Klein et al., 1991) ovine
(Wallis and Wallis, 1989) and porcine (Vize and Wells, 1987) have already been produced
through recombinant DNA technology. E. coli has been used extensively for the expression and
production of recombinant proteins. In E. coli., proteins are synthesized in the cytoplasm, but
many must be targeted to different destinations within the cell to perform their ultimate
functions. Towards this end, a number of active systems exist which recognize proteins destined
for various destinations and catalyze their insertion or export to these specific locations. All these
routs, cytoplasm, periplasm or inner membrane and in the culture are shown in figure below.
Figure 2.secretion of recombinant proteins in E.coli.
Source; www.slideshare.net
The production of many recombinant proteins in the bacterial cytoplasm is frequently limited by
their tendency to form inclusion bodies. These inclusion bodies can, however, in some cases ease
the isolation of the recombinant proteins but may not yield functional renatured molecules.
Correctly folded, functional recombinant proteins with a required amino terminus can be
conveniently produced by means of secretion or export into the periplasmic space where there is
4
provision of a less harsh environment compared with that of the cytoplasm. Protein misfolding in
the periplasmic space can be countered by a slower and controlled folding rate imposed by the
signal sequence and polypeptide threading through the Sec translocon.
The availability in the periplasmic space of many of the essential post-translational modification
enzymes catalyzing signal peptide processing, disulphide bridging and molecular chaperones
such as cytochrome c maturation factors can ensure generation of post-translationally modified,
bioactive heterologous bioproducts . Moreover, in vitro permeabilization of the Escherichia coli
cell wall (Kaderbhai et al., 1997) can facilitate selective discharge of the periplasmic contents
into the growth medium, easing recovery of highly pure recombinant proteins.
Several groups have reported the expression of growth hormone or its derived fusion protein in
E. coli cytoplasm (Khan et al., 1998; Wallis et al., 1995; Patra et al., 2000; Sadaf et al., 2007a).
However, the cytoplasmic production of a protein has certain disadvantages: high level
accumulation often leads to insoluble protein aggregates that can be difficult to refold and
solubilize, a refolding step is frequently required to obtain the native conformation and to form
the correct disulfide bonds (Becker and Hsiung, 1986). Alternative expression systems have been
based on the secretion of the protein into the E. coli periplasmic space, which not only allow a
greater chance to obtain the protein in a folded and soluble form but a lower load of
contaminating proteins in the periplasmic fluid makes purification process easier. Either
premature cytoplasmic protein folding or incorrect disulfide bond formation in the bacteria
periplasm are two known limitations in the overproduction of secreted proteins. Secretion
process in the periplasmic space of E. coli cells, which mimics the natural process of
somatotropic cells in the pituitary gland has been reported for the human GH (Deoliveira et al.,
1999; Soares et al., 2003). However, the secretory expression of recombinant ovine growth
hormone has not been reported to our knowledge.
5
1.3 Ovine breed of Pakistan
More than 50% of sheep are reared in the western dry mountains, western dry plateau and
northern dry mountains. Of the 31 breeds of sheep, the most important are Baltistani, Bibrik,
Cholistani, Kachhi, Kajli, Lohi and Lati, or Salt Range. The local ovine breed Lohi belongs to
family bovidae, subfamily caprinae and genus ovis. Genus ovis constitute 6species, one of them
is ovis aries commonly known as sheep. This study is specifically on the growth hormone taken
from pituatry tissues of slaughtered local ovine breed Lohi. Sheep breeds are classified into thin
tail and fat tail breeds. Lohi sheep are one of the important thin tail mutton breeds available in
the central districts of Punjab province (Pakistan). The breed exhibits an excellent capacity to
adapt to these areas. Lohi is one of the massive and highly productive breeds which comprise
some 40% of the Punjab and 15% of the national sheep population production and reproduction
performance (Economic survey of Pakistan, 1997). The Lohi is one of the best sheep breeds of
Pakistan. This breed is found in the central districts of Punjab. Its rapid body growth, coupled
with good quality meat is the main characteristics of this breed. Lohi sheep are large, having
deep and massive body, weighing on average 45-62 kg. The general body colour is white with a
.large reddish brown head having .long and drooping ears. The tail is short, thick and stumpy.
The average body weight at birth, 3, 6, 9 and 12 months of age is 3.59::!:0.69, 15±0.20,
26.5±3.56, 30.4±0.40 and 33.4±0.46 kg respectively.
Figure 3. (Lohi) ovine breed of Pakistan Livestock production research institute, Bahadur nagar, okara, Punjab.
6
The Lohi breed expressed a significant innate resistance to artificial infection of H. contortus.
Although Pakistan has been placed at tenth amongst sheep producing countries, a huge amount
of foreign exchange have to spend on the import of milk, wool and dairy products in order to
meet the increasing demands of urban population.
1.4 Impact of our study on the economy of Pakistan
The application of roGH to farm animals is of great importance for the enhanced
production of milk, meat and wool which have in turn a great role in the economy of country like
Pakistan. The enhanced milk, meat and wool production would not only reduce the import of
milk and wool products but would also encourage the production of competitive value added
quality products for exporting to the gulf states and middle eastern countries along with meeting
the local demands of milk meat and wool products. Therefore, there is tremendous scope and
certainly a compelling need for improving the growth rate of these animals.
In this context, the use of recombinant growth hormone (rGH) which is a well-known
animal productivity booster both for milk (Bauman, 1999; Walli and Samanta, 2000) and meat (
Bonneau et al., 1999) could provide an answer. Either exogenous administration of
recombinantly produced homologous GH in the immediate future or, possibly the transgenesis
and cloning approaches in the relative long-term, may provide the keys for boosting the
productivity of these animals, and help to meet the future nutritional challenges of several south
Asian countries.
Our study investigated the cloning, expression, secretion and purification of recombinant ovine
growth hormone isolated from local ovine breed Lohi .
7
1.5 Review of Literature
During the last 30 years, the gene encoding ST has been isolated and characterized from over 50
vertebrate species including cattle, sheep, goat, pig and human. In the following sections,
different aspects of ST relating to structure, function, purification, characterization, recombinant
protein production ,secretion and advantages of getting protein in soluble forms are been
reviewed.
1.5.1 Structural and functional aspects of GH
GH, in most vertebrate species is synthesized by the anterior part of pituitary gland and its
gene is composed of four introns and five exons of varying length. In mammals, only one gene
codes for ST. An exception, however, occurs in higher primates like human who have five GH-
related genes, all clustered on chromosome 17. One of these codes for pituitary ST, while the
other four for genes expressed in placenta, including two genes for placental lactogen, one for
placental lactogen-like protein and one for a GH-variant (Chen et al., 1989). A cluster of eight
GH-like genes has recently been identified in marmoset, a new world monkey (Wallis and
Wallis, 2001; Wallis and Wallis, 2002). In 1995, Wallis and Wallis demonstrated that this gene
cluster is confined to primates only and have arisen by a series of gene duplications during
evolution. However, a number of reports appeared later indicating that there are duplicate GH
genes in some caprine ruminants as well (Lacroix et al., 1996; Wallis et al., 1998). Despite the
presence of duplicate genes in primates and certain other mammals, ST is mainly secreted by the
somatotrophs and its exon-intron structure is well conserved between different taxa (Forsyth and
Wallis, 2002) Studies undertaken to elucidate the structural aspects of mammalian GHs have
shown that gene encoding GH, more specifically bST, has a primary transcript of 1793
nucleotides with five exons (I-V) interrupted by four intervening introns. The gene is mapped to
19q chromosome at 1-7qter location. Like ovine, the TATAAA sequence which is involved in
transcription initiation is also present in the 5′-flanking region of bST gene .Exon I and a part of
8
exon II code for 26 amino acids long N-terminal signal peptide, which is not retained in mature
bST (Chen et al., 1990). Presence of 28 kDa N-terminal signal sequence has been reported in
feline and other mammalian STs, as well (Castro-Peralta and Barrera-Saldana 1995; Secchi and
Borromeo, 1997). The main function ascribed to this signal sequence is to direct the maturation
of the nascent protein by a very specific protease, which recognizes and cleaves the signal
sequence, once the sorting process is completed.
While the details of this sorting and cleavage process remain unclear, it is believed that
this post-translational modification influences the protein maturation and contributes to the
structural diversity of the STs (Martoglio and Dobberstein, 1998). Ovine growth hormone gene
was isolated (Byrne, 1987) isolated and sequenced the ovine growth hormone gene. The
structure of the gene was found to be similar for other growth hormone genes, particularly the
bovine gene, which constitute five exons and 4 introns with variying sizes of 264 bp, 231 bp,
227 bp and 273 bp (Robert, 1983). The purified bovine growth hormone, consisting of a single
polypeptide chain, was found to contain NH2-terminal alanine, phenylalanine, and methionine in
nearly equal amounts. The minimum molecular weight calculated from its amino acid
composition was 20,846. It was also demonstrated that mature bST is comprised of 190 or 191
amino acids due to the presumed ambiguity in the removal of this signal peptide. The bST
amino-terminus is heterogeneous having either alanine (Ala-Phe-Pro-Ala) or the adjacent
phenylalanine (Phe-Pro-Ala) as the N-terminal residue. There is no known genetic element
responsible for the variability in this cleavage event (Bauman, 1992; 1999).
Despite the observed structural variations, biological effects of STs were found similar
amongst the different vertebrate groups. In mammals, ST is involved in a variety of metabolic
activities like protein and nucleic acids synthesis, increased secretion of insulin and glucagon
from the pancreas and lipid-mobilization (Etherton and Bauman, 1998). oGH was tested for its
effects on lipolysis of rat and ovine adipose tissue in vitro. In Australia, the gene for oGH has
been over-expressed in sheep under the control of the metallothionein promoter. The transgenic
9
animals showed improved performance in growth, body fat and wool production. More recently,
transgenic rams from this experiment have been bred and the progeny assessed (Adams et al.,
2002). Associated with the increased expression of growth hormone was a reduction in fat depth,
increase in wool yield and increase in liveweight of the progeny animals. The somatogenic and
galactopoietic effects of recombinant ovine placental lactogen (oPL) were compared with the
effects of oGH in post-weaned growing lambs and in lactating ewes. It is concluded that oPL and
oGH have similar somatogenic effects in lambs. Both hormones exhibited galactopoietic effects,
but oGH was considerably more potent than oPL ( Leibovich et al., 2000). The growth
promoting effects of oGH on both whole-body and tissue protein turnover were generally
accompanied with no change in the efficiency of deposition of newly synthesized protein
(Foster, 1991). For the same ratio size, the oGH group showed higher retentions of ingested
nitrogen. They concluded that oGH significantly enhances whole-body growth rates as a result of
the stimulatory effect on protein synthesis rates with little effect on protein degradation (Adams
et al., 2002). In birds, functions of ST, however, appear to differ from mammals. For example, in
mammals, ST plays an important role in post-natal body development, while in birds; it exhibits
no effect on growth during the post-hatch growing period (Zhao et al., 2004). Moreover, in
birds, ST signal peptide may be involved in post-translational modifications while no such
function is ascribed to mammalian ST signal peptide.
1.5.2 Cloning and expression of GH in bacterial systems
Development of successful cloning techniques led to the cloning of cDNA for GHs from a
large number of species. Molecular cloning of DNA complementary to bGH mRNA was carried
out by (Miller and co-workers, 1980). The DNA complementary to bGH mRNA coding for
cloned into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E.
coli x 1776. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal
10
peptide and confirmed the published amino acid sequence of the secreted hormone. The
nucleotide sequence of bGH mRNA showed 83.9% and 76.5% homology with rat and human
GH mRNA respectively (Miller et al., 1980). In recent research cDNAs prepared using poly
(A) mRNA from pituitaries and containing the coding sequences for bovine and porcine growth
hormones bGH and pGH were cloned in bacteria. 90% homology was found in their primary
structure. Ovine genomic library from thymus DNA was constructed by (Seeburg, 2009; Orian et
al., 1988). From this library, gene encoding oGH was isolated using the cDNA clone for bGH as
a probe. The exon-intron junctions in the oGH sequence were also determined by analogy with
the bST genomic sequence. Analysis revealed that ovine and bGH genes are 97.5 % homologous
in the coding regions. The coding sequence of oGH was found similar to the previously
published cDNA sequence for oGH (Warwick and Wallis, 1984) except for one base at position
1047 where an A was found instead of a G. This difference, however, did not result in a
difference in amino acid sequence”. mRNA from pituitary tissues of caprine was isolated and
used to construct cDNA library (Yamano et al., 1988).
After cloning, an important step was to achieve a high-level expression of these animal
GHs in bacterial systems. The high level expression of cloned gene in E. coli generally requires a
strong promoter, a properly spaced Shine-Delgarno (SD) sequence and an effective ribosome
binding site for efficient translation of the mRNA (Das, 1990).
A recombinant plasmid expression vector was constructed with six histidine residues
(His6) at the amino-terminus under the control of a T5 promoter. Upon induction with isopropyl-
β- -thiogalactopyranoside, the recombinant protein was synthesized and accumulated in the
cytoplasm in the form of inclusion bodies, at levels of approximately 18% of the total cellular
protein. The recombinant ovine growth hormone containing His tag was recovered and purified
to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special
affinity Ni2+·NTA. The purified roGH after refolding was found to be functionally active in
terms of its receptor binding and antigenicity as analyzed by radio receptor assay and radio
11
immunoassay. Yields of the purified expressed protein were found to be 32 μg/ml at a shake-
flask level ( Appa Rao et al., 1997).
oGH was cloned into plasmid poGHe101, based on pUC8 and found very high
expression (up to 25% of total cell protein) after induction. oGH was found in the form of
inclusion bodies. They described purification of oGH1 by 6 M guanidinium chloride containing
dithiothreitol. ion- exchange and gel filtration chromatography. Purified oGH1 had a Mr of 22
000, an isoelectric point of about 6·7 and an N-terminal sequence corresponding to that of oGH.
It was concluded that conclude that oGH behaved similarly to authentic bovine GH in a
radioimmunoassay, a radioreceptor assay and a weight-gain assay in hypophysectomized rats.
Thus the renatured hormone appeared to be correctly folded (Wallis and Wallis, 1990). By
feeding yeast extract along with glucose during fed-batch operation, high cell growth with very
little accumulation of acetic acid was observed. Use of yeast extract helped in maintaining high
specific cellular protein yield which resulted in high volumetric productivity of r-oGH (Panda,
1999; Ronald, 1985) got high-level expression of bovine growth hormone in Escherichia coli
resulted in the formation of distinct cytoplasmic granules that were visible with the phase-
contrast microscope, now known as inclusion bodies. They were isolated from crude cell lysates
by differential centrifugation and were further purified by a simple washing procedure that yields
nearly homogeneous bGH.
The oGH was expressed in Escherichia coli in the form of inclusion bodies using the pQE-
30 expression vector. In a simple fed-batch fermentation, 800 mg/L of recombinant ovine growth
hormone (roGH) was produced at a cell concentration of 12 g dry cell weight/L. Inclusion bodies
were isolated from cells with >95% purity by extensive washing using detergent, and the r -oGH
from the purified inclusion bodies was solubilized in 2 M Tris-HCl buffer at pH 12 containing 2
M urea. The roGH solubilized in the above conditions exhibited considerable secondary structure
as determined by circular dichroism spectra and was immunologically active. Solubilization of
the inclusion body protein with retention of native-like secondary structure gave higher yields
12
during refolding. To suppress protein aggregation, refolding was carried out in gel filtration
column. Refolding, buffer exchange, and the purification of monomeric roGH from aggregated
complex was achieved in a single step using gel filtration chromatography. More than 60% of
the initial inclusion body protein was refolded into a native-like conformation by the use of this
procedure. The refolded protein was characterized by circular dichroism, fluorescence, SDS-
PAGE, Western blotting, and radio receptor binding assay and found to be similar to native,
pituitary-derived, ovine growth hormone. (R.H.Khan,1998). High level expression of the
somatotropin gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST)
was obtained by synthesizing a codon-optimized ST gene through the introduction of silent and
non-silent mutations involving single and multiple base substitutions in +2 and subsequent
codons of BbST. The clones were expressed in the form of inclusion bodies. The inclusion
bodies represented over 20% of the E. coli cellular proteins (Sadaf et al., 2007a; Sadaf et al.,
2007b; Sadaf et al., 2008). High-level of expression~30 % of the total cell protein in a T7-
promoter based pET expression system of caprine growth hormone was achieved by making
silent base substitutions which are likely to minimize secondary structure formation at the 5'-end
of the cGH transcript. The over expressed cGH was in the form of inclusion bodies Inclusion
bodies were solubilized in 100mM Tris buffer containing 2M urea at pH 12.5. Solubilized protein
was refolded by pulsatile renaturation process in refolding buffer and purified using DEAE
sephadex anion exchange chromatography. The cGH was found to be biologically active on rat
lymphoma Nb2 cell bioassay (Khan, 2007).
1.5.3 Secretion of growth hormone in E.coli
Several groups have reported the expression of growth hormone or its derived fusion
protein in E. coli cytoplasm (Khan et al., 1998; Wallis et al., 1995; Patra et al., 2000; Sadaf et
al., 2007a). Secretion process in the periplasmic space of E. coli cells, which mimics the natural
13
process of somatotropic cells in the pituitary gland was achieved by linking the signal peptide
sequence to the human GH (Deoliveira et al., 1999; Teresa et al., 2000; Soares et al., 2003). By
fusing the human ST cDNA with an outer membrane protein A (ompA) signal peptide of E. coli,
achieved an even higher concentration (15 mg/L) of ST in the periplasm was achieved (Becker
and Hsiung, 1986). N-terminal sequence analysis of protein demonstrated that the fusion protein
was correctly processed to authentic 22 kDa human GH. Their results also showed that E. coli
periplasm provides an appropriate environment for proper folding of the expressed proteins.
In 1987, Chang and co-workers fused the human ST cDNA to E. coli heat-stable
enterotoxin II (ST-II) signal peptide and expressed it under the control of phoA promoter. E.
coli produced 15-25 µg of human ST/ml/A550, 90 % of which was exported to the periplasmic
space. Later, several research groups reported the periplasmic expression of recombinant STs in
E. coli up to a concentration of 10-25 µg ST/ml/OD).
The influence of different factors acting on Escherichia coli periplasmic expression of
recombinant hGH in shake flask cultures was investigated (Soares et al., 2003) .bacterial
vectors containing the phage lPL promoter, which is temperature activated, were utilized. Four
different signal peptides were compared: DsbA, npr, STII and one derived from the natural hGH
signal peptide, this last used as a reference. The expression level was affected by the signal
peptide and by the induction conditions, being more effective when activation started in the early
logarithmic phase which, however, exhibited remarkably different optical density (OD)
according to medium composition. Our results thus indicate that 6 hrs activation at 40±42°C,
starting with an OD (A600) of ~3 in a very rich medium, were conditions capable of providing
the maximum secretion level for a vector utilizing the DsbA signal sequence and E. coli W3110.
Multiple pathways direct protein translocation across the bacterial membranes but most of
the periplasmic pre-proteins are routed via the Sec-export-dependent pathways (Mori and Ito,
2001; Berks et al., 2000). In Gram-negative bacteria, secreted proteins have to cross the two
membranes of the cell envelope, which differ substantially in both composition and function
14
(Koebnik et al., 2000). There are five secretion pathways known as( type I, II, III, IV, and V)
but first three are most widely used (Koster et al., 2000).
1.5.3.1 Type I secretion systems
Transport proteins in one step across the two cellular membranes, without a periplasmic
intermediate (Binet et al., 1997). E. coli normally uses this pathway for the secretion of high-
molecular-weight toxins and exoenzymes. Although the type I secretion mechanism is capable of
exporting the target protein to the culture medium, it has two significant drawbacks. Firstly, the
secreted peptide remains attached to the signal sequence and therefore an additional cleavage
step is required to obtain the intact native protein (Blight and Holland, 1994). Secondly,
coexpression of the components of this system is often necessary to increase transport
capacity(Shokri et al., 2003).
1.5.3.2 Type II secretion Mechanism
The general secretory pathway is a two-step process for the extracellular secretion of
proteins mediated by periplasmic translocation (Koster et al., 2000). Three pathways can be used
for secretion across the bacterial cytoplasmic membrane: : the SecB-dependent pathway, the
signal recognition particle (SRP), and the twin-arginine translocation (TAT) pathways.
1.5.3.3 SecB-dependent pathway
Secreted proteins targeted to the SecB-dependent pathway contain an amino-terminal
signal peptide that functions as a targeting and recognition signal. These signal peptides are
usually 18–30 amino acid residues long and are composed of a positively charged amino
terminus (N-region), a central hydrophobic core (H-region), and a polar cleavage region (C-
15
region) (Choi and Lee, 2004; Fekkes and Driessen, 1999). The N-region is believed to be
involved in targeting the preprotein to the translocase and binding to the negatively charged
surface of the membrane lipid bilayer. Increasing the positive charge in this region has been
shown to enhance translocation rates, probably by increasing the interaction of the preprotein
with SecA (Fekkes and Driessen, 1999; Wang et al., 2000). The H-region varies in length from 7
to 15 amino acids. Translocation efficiency increases with the length and hydrophobicity of the
h-region, and a minimum hydrophobicity is required for function (Wang et al., 2000). Although
this pathway has been used extensively for recombinant protein production, it has one serious
drawback. This system is not able to transport folded proteins and, since transport is largely
posttranslational, the secretion of proteins that fold rapidly in the cytoplasm may not be possible.
In these cases the protein should be targeted to the SRP or the TAT pathways.
1.5.3.3.1 TAT pathway
A Sec-independent pathway was reported to be functional in E. coli (Santini et al., 1998;
Sargent et al., 1999). This pathway has been termed the TAT (twin-arginine translocation)
system because preproteins transported by it contain two consecutive and highly conserved
arginine residues in their leader peptides. The TAT pathway is capable of transporting folded
proteins across the inner membrane (Stanley et al., 2000) independently of ATP (Yahr and
Wickner, 2001) using the transmembrane PMF (DeLeeuw et al., 2002). The TAT pathway has
been used in the secretion of several recombinant proteins including antibody fragments (DeLisa
et al., 2003) glucose–fructose oxireductase(Blaudeck et al., 2001), ribose binding protein (Pradel
et al., 2003)alkaline phosphatase (Masip et al., 2004), and green fluorescent protein (Barrett et
al., 2003; De Lisa et al.,2002; Santini et al., 2001; Thomas et al., 2001).
The H-region of TAT signal peptides is usually less hydrophobic than that of Sec leader
peptides. The C-region contains the cleavage site and shows a strong bias towards basic amino
acid residues (Berks et al., 2000). It has been shown that transport via the TAT pathway is less
16
efficient (DeLisa et al., 2004) and slower than the Sec pathway with transit half-times in the
order of a few minutes (Santini et al., 1998; Sargent et al., 1998) instead of a few seconds
(Berks et al.,2000)Despite these disadvantages, the TAT pathway is capable of transporting
folded protein across the inner membrane, unlike the SecB or the SRP pathways (De Lisa et al.,
2003).
1.5.3.3.2 SRP pathway
The signal recognition particle (SRP) pathway is used by E. coli primarily for the
targeting of inner membrane proteins (Economou, 1999). This system has been exploited in the
secretion of several recombinant proteins including Mtla–OmpA fusions (Neumann-Haefelin et
al., 2000) MalF–LacZ fusions (Tian et al., 2000) maltose binding protein, chloramphenicol
acetyl transferase (Lee and Bernstein, 2001; Peterson et al., 2003) and haemoglobin protease
(Sijbrandi et al., 2003). The system consists of several proteins and one RNA molecule. SRP
recognizes its substrates by the presence of a hydrophobic signal sequence (hence the name
signal recognition particle). The presence of an N-terminal signal sequence with a highly
hydrophobic core, combined with a lack of a trigger factor binding site (Patzelt et al., 2001)
results in cotranslational binding of the nascent chain to Ffh (Beck et al., 2000).
For a productive interaction between the preprotein and Ffh, 4.5S RNA is required
(Herskovits et al., 2000). It has been suggested (Fekkes and Driessen, 1999) that the interaction
between SRP and the signal sequence is dependent on the hydrophobicity of the nascent chain
since preproteins with more hydrophobic signal sequences are translocated with higher
efficiency. It has been shown (Gu et al., 2003) that SRP binds the ribosome at a site that overlaps
the binding site of trigger factor. A discriminating process has been proposed in which SRP and
trigger factor alternate in transient binding to the ribosome until a nascent peptide emerges.
Depending on the characteristics of the nascent peptide, the binding of either SRP or trigger
17
factor is stabilised, thus determining whether the peptide is targeted to the membrane via the
SRP pathway, or posttranslationally by the SecB pathway (Gu et al., 2003). FtsY is found both
in the cytoplasm and at the membrane (Herskovits et al., 2000) and can interact with ribosomal
nascent chain–SRP complexes in the cytosol. Upon interaction with membrane lipids, the
GTPase activities of FtsY and Ffh are stimulated, thus releasing the nascent chain to the
translocation site (Nagai et al., 2003). This site may be the SecYEG translocon (Koch et al.,
1999; Valent et al., 1998; Zito and Oliver, 2003), although it has been demonstrated that
membrane insertion can occur independently of SecYEG (Cristobal et al., 1999b). Insertion of
transmembrane segments can occur in the absence of SecA (Scotti et al., 1999) while
translocation of large periplasmic loops is SecA-dependent (Neumann-Haefelin et al., 2000; Qi
and Bernstein, 1999; Tian et al., 2000). The protein YidC was also identified as a translocase-
associated component during insertion (Scotti et al., 2000). It has been proposed that this protein
facilitates the diffusion of transmembrane segments into the lipid phase (van der Laan et al.,
2001).
For recombinant protein production, SRP targeting can be achieved by engineering the
hydrophobicity of the signal sequence (Bowers et al., 2003; de Gier et al., 1998; Peterson et al.,
2003). SRP system is advantageous if for instance the target protein folds too quickly in the
cytoplasm, adopting a conformation incompatible with secretion by the SecB-dependent system
(Lee and Bernstein, 2001; Schierle et al., 2003). Various studies have shown the SRP
mechanism in E. coli with special reference to translation arrest of recombinant protein N, G and
M domains of Ffh.
The DsbA signal sequence works with SRP targeting mechanism and has been the best
choice for translocation of recombinant proteins to the inner membrane of E. coli. The extra-
cytoplasmic expression of human growth hormone under the influence of DsbA signal sequence
has been reported. However, the secretory expression of the recombinant ovine growth hormone
has not been reported to our knowledge.
18
1.5.4 Signal sequences
Proteins destined for translocation across the cytoplasmic membrane are synthesized as
precursors carrying an amino-terminal signal sequence that direct polypeptides into the secretory
pathways (Economou, 1999). Although variable in primary structures (Izard and Kendall, 1994)
signal sequences contain a conserved and ordered structure (Von Heiinr, 1999) that channels the
passenger portion into the export pathway (Thanassi and Hultgren, 2000). The amino-terminal
positively charged end, together with the central hydrophobic core, directs Sec-independent and
proton-motive force (PMF)-dependent signal peptide translocation across the membrane (van
voorst and de kruiff, 2000) and substitutions of the hydrophobic residues with charged ones
diminish or abolish export competency of signal sequences (silhavey et al., 1983). The
efficiency of preprotein translocation is independent of the structure of the cleavage region. This
region can accommodate varying hydrophobicities with the exception of bulky residues at −1, −3
positions. By reducing the signal peptide to simplified, idealized segments it has been shown
that a largely polymeric sequence with retention of the early consensus sequence and a central
hydrophobic core, MKQST(L10)−(A6), can function equivalently to the wild-type alkaline
phosphatase signal peptide (Laforet and Kendall, 1991). It has been shown that the positive
charge in the N-terminal region of signal peptide plays an important role in the function of the
eukaryotic signal peptide as well as that of prokaryote. Using signal sequence containing
additional Arginine residues, secretion levels of HLY in yeast were notably increased (Yoshinori
Tsuchiya, 2000).
The rate of inversion increases with more positive N-terminal charge and is reduced with
increasing hydrophobicity of the signal. Inversion may proceed for up to 50s, when it is
terminated by a signal-independent process. These findings provide a mechanism for the
topogenic effects of flanking charges as well as of signal hydrophobicity. It was also suggested
that translational kinetics and signal sequences act in concert to modulate the export process. The
19
expression of the natural and optimized gene sequence was compared in order to produce leech
carboxypeptidase inhibitor (LCI) in the bacterial periplasm, and evaluated export efficiency of
LCI fused to different signal sequences. The best combination of these factors acting on
translation and export was obtained when the signal sequence of DsbA was fused to an E. coli
codon-optimized mature LCI sequence (Juan et al., 2009). Proinsulin fused to the C-terminus of
the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main
catalyst of disulfide bond formation in E. coli, expected increased yields of proinsulin by intra-
or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein,
proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide
bond formation (J. Winter, 2000). The E. coli cytoplasmic protein thioredoxin 1 can be
efficiently exported to the periplasmic space by the signal sequence of the DsbA protein
(DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding
protein (MBP) (Clark, 2003). The best combination of these factors acting on translation and
export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized
mature LCI sequence (Veit goder and Martin spiess, 2003).
1.5.5 Expression and Purification of secreted protein in E.coli.
The heterologous expression and production of recombinant GH from various species of
bovidae family including bovine (Klein et al., 1991), porcine (Seeburg et al., 1983) and ovine
(Rao et al., 1997) in E. coli has been reported. E. coli constitutes cytoplasm, periplasm, inner
membrane and outer membrane spaces. All proteins in E. coli are synthesized in cytoplasm and
translocated to their defined destinations. About 71, 21, 6, and 2 % of the proteins are found in
the cytoplasm, inner membrane, periplasmic space and outer membrane respectively. Several
groups have reported the expression of GH or its derived fusion protein in E. coli cytoplasm
(Wallis et al., 1995; Khan et al., 1998; Patra et al., 2000; Sadaf et al., 2007). However, the
cytoplasmic production of a protein has certain disadvantages: high level accumulation often leads
to insoluble protein aggregates that can be difficult to refold and solubilize and a refolding step is
20
frequently required to obtain the native conformation to form the correct disulfide bonds (Becker
et al., 1986). Alternative expression systems have been based on the secretion of the protein into
the E. coli periplasmic space, which not only allows a greater chance to obtain the protein in a
folded and soluble form but also lower load of contaminating proteins in the periplasmic fluid
makes purification process easier. Secretion process in the periplasmic space of E. coli cells
mimics the natural process of somatotropic cells in the pituitary gland and has been achieved by
linking the signal peptide sequence to the human GH (de oliveira et al., 1999; Teresa et al., 2000;
Soares et al., 2003). It has been reported that types of culture media and medium additives
(compatiblesolutes, chemicalchaperon) can affect the yield of recombinant protein production
(kaushik, 2003). The spectrum of compatible solutes used by microorganisms comprises only a
limited number of compounds; sugars (e.g.trehalose), polyols (e.g. glycerol and glucosylglycerol),
free amino acids (e.g. proline and glutamate) derivatives there of (e.g. proline, betaine and
ectoine) quaternary amines and their sulfonium analogues (e.g. glycine betaine, carnitine and
dimethylsulfoniopropionate) sulfate esters (e.g. choline-O-sulfate) and N-acetylated diamino acids
and small peptides. In general, compatible solutes are highly soluble molecules and do not carry
a net charge at physiological pH. In contrast to inorganic salts, they can reach high intracellular
concentrations without disturbing vital cellular functions such as DNA replication, DNA-protein
interactions and the cellular metabolic compatible solutes also serve as stabilizers of proteins and
cell components against the denaturing effects of high ionic strength (Leibly et al., 2012).
A positive effect of low molecular weight additives supplemented in the culture medium
were being observed in various studies in terms of yields of periplasmic expressed proteins
(Diamant et al., 2001). Sorbitol addition to the culture medium resulted in higher accumulation of
a functional single chain Fv (Huston et al., 1991; Hashimoto et al., 1999) glycine betaine and
sucrose were also seen to be beneficial for the folding of immunotoxin and cytochrome c550 (Ou,
2002) While L-arginine and ethanol increased the yields of human pro-insulin (Winter et al.,
2001) plasminogen activator and a single chain Fv (Huston et al., 1991). Also the supply of
21
reduced glutathione, alone or in combination with DsbC over-expression, increased the
accumulation of disulfide-dependent proteins (Zapun et al., 1995; Missiakas et al., 1995). Use of
compatible solute in the medium has shown remarkable results (Barth et al., 2000). Besides
those strategies, one of the methods for soluble expression of recombinant protein is the Dsb co-
expression system (Sandee et al., 2005).
Several groups have described the purification of recombinant STs, expressed either in E.
coli cytoplasm, periplasm or even culture medium, using either affinity chromatography or
reversed phase HPLC (RP-HPLC). The 98 % purity of human ST was achieved from the E. coli
culture medium, using RP-HPLC (Hsiung et al., 1989). RP-HPLC was used to obtain 95 % pure
oGH from E. coli (Adams et al., 1992). The recombinant oGH in this study, however, was in the
form of inclusion bodies, which were solubilized in a cationic surfactant cetyltrimethyl
ammonium chloride (CTAC) prior to application on column. The purification of an ovine ST
variant to homogeneity using ion-exchange chromatography and gel filtration, after solubilization
and renaturation of oST from inclusion bodies was reported (Wallis and Wallis, 1990). The
purification of recombinant human ST to near homogeneity using ammonium sulfate
precipitation, ion-exchange chromatography and gel-filtration on Sephacryl S-200 column.
Similarly was also studied (Igout et al., 1993). FPLC (Mono Q anion exchanger) and RP-HPLC
were also used to achieve highest purification of excreted recombinant human GH. By exploiting
metal-protein binding affinity, (Mukhija et al., 1995) described the purification of His6-tagged
human ST to virtual homogeneity in a single-step. Their strategy involved selective binding of
His6-tagged recombinant protein to Ni+2-nitrilotriacetic acid (NTA) column followed by elution
with increasing concentration of imidazole. The purified protein was obtained at a level of 30
mg/L of the culture. Using similar strategy (Appa rao et al., 1997) purified the recombinant ovine
ST to greater than 95 % homogeneity on Ni+2-NTA resin, in a single-step. Yield of the purified
protein was around 62 % of the total expressed ST and was quite significant in comparison to the
other methods of purification.
22
Single-step purification of recombinant oST using gel-filtration column was reported
(Khan et al., 1998). In a simple fed-batch fermentation, 800 mg/L of recombinant oST was
produced, but all as in the form of inclusion bodies. The inclusion bodies were isolated from E.
coli with >95 % purity by extensive washing in the presence of detergent followed by
solubilization in urea at alkaline pH. Since, sufficient purity was achieved at the stage of inclusion
bodies, gel filtration served only as a polishing step which purified the monomeric recombinant
ovine ST from aggregated complex dimers or oligomers. Instead of using conventional
chromatographic techniques, (Jeh et al., 1998) employed a somewhat different strategy for
purifying ST from E. coli cytoplasm. They found that most of the E. coli proteins contaminating
the recombinant-ST are readily precipitated with the addition of two volumes of secondary
butanol (an organic solvent), while the flounder ST remained soluble. After the secondary butanol
treatment, the purity of recombinant flounder ST was more than 98 % and the recovery yield was
around 47%.
Ni+2-chelate affinity chromatography was used as a first step while purifying His10-tagged
human ST from E. coli cytoplasm (Shin et al., 1998). Further, by employing ion-exchange
chromatography (Q-Sepharose fast flow), a purity of greater than 99 % was achieved. A five-step
purification scheme involving precipitation, gel-filtration, ion-exchange, and hydrophobic
interaction chromatography (HIC) was applied to obtain a highly purified pharmaceutical grade
recombinant human GH for clinical use (Deoliveira et al., 1999). In contrast, a single-step high
performance liquid chromatography based on size exclusion was reported to obtain human GH for
use in radioimmunoassay as standard and tracer . Although the final product was not 100 % pure,
yet it was adequate for its intended use as a chemical reagent in immunoassay. The labeling
reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of
~50%. The values were very similar to those obtained by radioiodinating the highly-purified
clinical-grade recombinant human ST which was obtained after the regular six-step purification
process.
23
A two-step procedure involving ion-exchange chromatography on DEAE-sepharose
column and gel-filtration on sephacryl S-200 was used to get purified preparation of recombinant
hGH from E. coli. Recovery of recombinant protein from ion-exchange matrix was around 65 %
while overall yield of the purified refolded GH from the E. coli inclusion bodies, was ~50 %.
(Patra et al., 2000). While a combination of hydrophobic and ion-exchange chromatographies for
the purification of bubaline and caprine GHs which expressed in E. coli cytoplasm as inclusion
bodies. Prior to application on decyl agarose column, the inclusion bodies were solubilized in
guanidinium chloride and air-oxidized to get properly folded recombinant GHs for column
applications. Nearly 90 % purity was achieved at the end of HIC purification. An additional step
of ion-exchange chromatography using fast-flow DEAE-Sepharose was then employed for
purification to near homogeneity. The overall yield of recombinant GHs at the end of two-step
purification scheme was about 40 % of the starting material and the purity was > 98
%”(Mukhopadhyay and Sahni, 2002c).
The purity of protein as judged by RP-HPLC was greater than 95 %. Purification of hGH
from the periplasmic fraction by anion exchange and size exclusion gave hGH of greater than 90%
purity. Characterization by SDS-PAGE, amino terminal analysis, trypsin mapping, and circular
dichroism demonstrated that the fusion protein was correctly processed to authentic hGH and that
the E. coli periplasm provided an appropriate environment for proper folding of hGH and disulfide
bond formation (Becker and Hsiung, 1986). High-yield purification procedure for the preparation
of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic
space was investigated. The accuracy of hGH and total protein quantification, especially in the
early steps of the process, and the maximum elimination of hGH-related forms were also studied
in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely
valuable analytical tools (Deoliveira et al., 1999). The influence of four different signal peptides
(DsbA, npr, STII and one derived from the natural hGH) acting on E. coli periplasmic expression
of hGH in shake flask cultures were described, and concluded that DsbA signal sequence gave
24
best results (Soares et al., 2003). It has been reported that types of culture media and medium
additives (compatible solutes,chemical chaperon)can affect the yield of recombinant protein
production (kaushik, 2003). The spectrum of compatible solutes used by microorganisms
comprises only a limited number of compounds; sugars (e.g. trehalose), polyols (e.g. glycerol and
glucosylglycerol), free amino acids (e.g. proline and glutamate), derivatives thereof (e.g. proline,
betaine and ectoine), quaternary amines and their sulfonium analogues (e.g. glycine betaine,
carnitine and dimethylsulfoniopropionate), sulfate esters (e.g. choline-O-sulfate), and N-acetylated
diamino acids and small peptides. In general, compatible solutes are highly soluble molecules and
do not carry a net charge at physiological pH. In contrast to inorganic salts, they can reach high
intracellular concentrations without disturbing vital cellular functions such as DNA replication,
DNA-protein interactions, and the cellular metabolic Compatible solutes also serve as stabilizers
of proteins and cell components against the denaturing effects of high ionic strength (Leibly DJ,
2012).Use of compatible solute in the medium has shown remarkable results (Barth et al. 2000).
Besides those strategies, one of the methods for soluble expression of recombinant protein is the
Dsb co-expression system (Sandee et al., 2005 ).
Periplasmic proteins are recovered from transformed gram negative bacteria by a process
comprising freezing and thawing the cells. Advantages are obtained by culturing the cells in
phosphate limiting media and by killing the cells prior to separation of periplasmic proteins . The
compatible solute supported enhanced periplasmic expression of recombinant proteins under
stress conditions. Periplasmic protein in the shock fluid was purified by combinations of metal ion
affinity and size exclusion chromatography. This was substantially stabilized in the presence of
1M hydroxyectoine after several rounds of freeze-thawing even at low protein concentration
(Barth et al., 2000).
1.5.6. Advantages of getting soluble proteins
25
Periplasmic or extracellular secretion can increase the solubility of a gene product as
exemplified by the production of bacterial PNGaseF and human granulocyte colony-stimulating
factor (Loo et al., 2002; Jeong and Lee, 2001). Obtaining a soluble protein often constitutes a
bottleneck in the production of proteins for structural studies or proteomics (Goulding and
Jeanne Perry, 2003; Pedelacq et al., 2002; Yokoyama, 2003). Product secretion can provide a
way to guarantee the N-terminal authenticity of the expressed polypeptide because it often
involves the cleavage of a signal sequence (Mergulha et al., 2000) thus avoiding the presence of
an unwanted initial methionine on a protein that does not normally contain it. This extra
methionine can reduce the biological activity and stability of the product (Liao et al., 2004) or
even elicit an immunogenic response in the case of therapeutic proteins Protein secretion can
increase the stability of cloned gene products. For instance it was shown that the half-life of
recombinant proinsulin is increased 10-fold when the protein is secreted to the periplasmic space
(Talmadge and Gilbert, 1982). Secretion was also useful in the production of penicillin amidase
from E. coli as intracellular product degradation was a severe problem (Ignatova et al., 2003).
The increased stability of gene products on the periplasm and in the culture medium probably
results from the lower levels of E. coli proteases that can be found in these locations (Gottesman,
1996; Mergulha et al., 2004).
Additionally, if the product is secreted to the culture medium cell disruption is not required
for recovery and even in the case of periplasmic translocation, a simple osmotic shock or cell
wall permeabilization can be used to obtain the product without the release of cytoplasmic
protein contaminants (Mergulha et al., 2004; Shokri et al., 2003).
Biological activity is dependent on protein folding and, particularly if disulfide bonds must
be formed, proper folding is unlikely in the reducing environment of the cytoplasm.
Additionally, the correct pair bonding of cysteines contributes to the thermodynamic stability of
the proteins (Kadokura et al., 2003; Maskos et al., 2003; Raina and Missiakas, 1997).
26
1.6 AIMS AND OBJECTIVES
The objectives we planned to be achieved were as follows:
1. Isolation of total RNA from ovine pituitary gland.
2. cDNA synthesis by reverse transcription and PCR amplification using suitable primers and cloning
into suitable expression vector.
3. Coding and non coding Sequence analysis of cloned gene and comparison with other reported
sequences.
4. Expression and secretion of oGH gene in a suitable bacterial system.
5. Effect of different factors (signal nucleotide variation, medium composition and bacterial strain,
etc) on secretion of ovine growth hormone gene.
6. Purification of oGH by anion exchange, size exclusion chromatography.
7. Biological activity assesment of recombinant oGH.
27
28
Materials and Methods
29
2.1 Sample collection and storage
Pituitary glands from the freshly slaughtered ovine were collected from a local abattoir. The
collected samples were carefully dissected to remove the anterior part of the pituitary gland (site
for the production of GH), weighed and either stored at 80C or in liquid nitrogen for further
use in RNA isolation. Approximately 10 ml blood from the subject ovine was sampled into
vaccutainer tube containing 10.5 mg ethylene diamine tetra acetate (EDTA). The blood was
mixed gently and frozen at 20C until analysis.
2.2 Chemicals and kits
All chemicals used in this study were of the highest purity grade commercially available.
Polymerase chain reactions (PCR) were performed using GC-RICH PCR amplification system of
Roche Applied Science (Mannheim, Germany). For DNA extraction and plasmid mini-
preparation, QIAquick gel extraction and QIAprep spin miniprep kits of QIAGEN Inc.(CA,
USA) were respectively used. RNeasy Mini and Midi kits used to isolate RNA from bacterial
cultures and animal tissues were also acquired from QIAGEN. RevertAidTM Moloney Murine
Leukemia Virus (M-MuLV) reverse transcriptase, T4 DNA ligase, restriction endonucleases and
DNA and protein size markers, IPTG, 6 x loading dye were either purchased from New England
Biolabs (MA, USA) or MBI Fermentas (MD, USA)
E. coli strain BL21 CodonPlus (DE3) RIPL (Stratagene, CA) was used for expression studies.
pET22b(+) expression plasmid was obtained from Novagen Inc. Trizol reagent for total RNA
isolation from pituitary gland was purchased from Invitrogen. QIAquick gel extraction for DNA
extraction from agarose gels and QIAprep Spin Miniprep kit for plasmid preparation were
procured from Qiagen. InsT/A clone PCR product cloning kit, MMLV-RTase, Taq DNA
polymerase, restriction enzymes, IPTG and T4 DNA ligase were purchased from MBI
Fermentas. For western blot analysis, rabbit anti-bovine GH and commercially bovine GH were
purchased from US Biological (USA), goat anti-rabbit IgG conjugated with alkaline phosphatase
30
was from BioRad (USA). NaCl, Trypton and yeast extract (Oxoid, England) were used in Luria -
Bertani (LB) medium for the growth of Escherichia coli strains.All other cultivation media and
bulk chemicals were purchased from Difco laboratories, or US Biological (CA, USA), unless
stated otherwise.. 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium
(NBT), used as color development substrates, were purchased from MBI Fermentas and US
Biological. The HeLa cell lines were a gift from Agha Khan University Hospital, Karachi,
Pakistan. DMEM (Dulbecco’s Modified Eagle’s Medium) mammalian cell culture medium and
fetal bovine serum (FBS) was purchased from PAA Laboratories GmbH (THE CELL
CULTURE COMPANY).
2.3 Isolation of total RNA from pituitary sample
The RNA was isolated from the pituitary tissue of freshly slaughtered young sheep breed
(Lohi) by using Guanidium- thiocyanate- chloroform extraction method (Chomczynski et al.,
1987).
Mortar and pestal were washed with DEPC treated water (diethyle Pyrocarbonate) weighted
pituitary tissue 100mg, put it in mortar and covered it with aluminium foil, kept it for 40
minutes at -80oC. Then crusedh it while covering the mortar with aluminium foil from one side,
crushed it until it became fine powder.
Added 1ml TRI reagent in it and mixed it well with the help of pestal. Poured it into eppendorff,
centrifuged at 12,000g for 10 min at 4oC. Transferred the clear supernatant to a fresh tube and
proceeded with phase separation. This step was to remove insoluble material from the
homogenate, the resulting pellet contained extracellular membrane, polysaccharides, high
molecular weight DNA, while the supernatant contained RNA.
Supernatant recovered in fresh tube was 850 ml, left it at room temperature for 5 minutes to
permit complete dissociation of nucleoprotein.
31
Added 0.28 ml chloroform for phase separation as 0.2 ml required for 0.75 TRI reagent.Left it
for 15 minutes at room temperature centrifuged it at 12,000 g for 15 minutes at 4o C. Following
centrifugation, the mixture separated into a lower red, phenal chloroform phase, interphased and
the colourless aquaus upper phase. Transferred the aquaus phase which was 600ml to the fresh
tube.
Added 1ml Isopropanal and left it at room temperature for 10 min, centrifuged it at 12,000g for
8 minutes at 4oC. Discarted the supernatant solublized the pellet in 100ml H20. Results were
taken at 280mm absorbance.
2.4 Formaldehyde agarose gel electrophoresis
Since, RNA has a high degree of secondary structure; the samples were first denatured by
treatment with formamide and then separated by electrophoresis through the formaldehyde
containing agarose gel. Formaldehyde in the gel kept the RNA in denatured state by preventing
intra-strand Watson-Crick base paring.
1.2 % formaldehyde agarose (FA) gel was prepared by mixing 0.6g agarose with 50ml 1x
FA gel buffer (20mM MOPS, 5mM sodium acetate, 1mM EDTA, pH 7.0) in an
Erlenmeyer flask. The mixture was heated to dissolve agarose, cooled to 60-70C by
gentle swirling and poured onto the gel casting tray (in a fume hood) after adding 900 l
of 12.3 M formaldehyde and 0.5 l of 10 mg/ml ethidium bromide (a dye that intercalates
into the grooves of double-stranded DNA/RNA and fluoresces in UV-light).
Comb was inserted immediately after pouring to form wells for sample loading. Gel was
polymerized at room temperature for 30-40 minutes and then equilibrated in 1x FA
running buffer (1x FA gel buffer, 2.5 M formaldehyde) for additional 30-40 minutes, prior
to electrophoresis. RNA samples were denatured by mixing 4 volumes of RNA with 1
volume 5x RNA loading buffer (0.25% bromophenol blue, 4mM EDTA, 0.9M
32
formaldehyde, 20% glycerol, 30.1% formamide and 4x FA-gel buffer), chilled on ice and
then electrophoresed at 5-7 V/cm until the bromophenol blue dye migrated approximately
2/3 of the way through the gel.
RNA bands were visualized by illuminating the gel under UV light followed by image
recording using gel documentation system.
2.5 cDNA Synthesis
2.5.1 Primer designing
cDNA was synthesized by RT-PCR. On the basis of conserved sequences, two forward and one
reverse designed primers were used in amplification of ovine growth hormone gene. Forward
primers PBGH1 (5`CAT ATG ATC CAT GGC CTT CCC AGC CAT G3`) PBGH2 (5`CAT
CAT ATG GCC TTC CCA GCC ATG 3`) and reverse primer PBGH3 (5`TAG GAT CCG CAA
CTA GAA GGC AGC 3`). The forward primers contained Nde1, Nco1 restriction sites at their
5’end whereas the reverse primer contained a BamH1 site.
2.5.2 Reverse transcription (RT)
Reverse transcription was done to synthesize cDNA. Reaction was prepared in the PCR tube by
adding 5 μl (0.1-5μg) of isolated RNA, 1 μl (200 U) of RevertAid Molony Murine Leukemia
Virus (M-MuLV) Reverse Transcriptase, 1 μl of 50 μM antisense primer [OaST], 4 μl of 5 x RT
buffer, 2 μl of 10 mM dNTPs, 1 μl of (20 U) ribonuclease inhibitor and 6 μl of RNase free water
to make up the volume up till 20 μl. The reaction was carried out in Applied Biosystems 2720
Thermo Cycler for 60 minutes at 42°C, reaction was stopped by increasing the temperature at
70°C for 10 minutes and then chilled on ice.
33
2.5.3 PCR Amplification
Polymerase chain reaction (PCR) is a repetitive bidirectional and exponential DNA synthesis by
primer extension of a nucleic acid region. Amplification of cDNA was done and reaction mixture
was prepared by adding two oligonucleotide
primers of the required gene to be amplified (50 pmol of each primer),
four deoxynucleotide triphosphates (dNTPs) each of 0.4 mM,
Taq polymerase (Fermentas #EP0402), 2.5U
10 x PCR buffer containing ammonium sulphate 5 μl
MgCl2 1.5-4 mM,
of DNA template 0.1-1 μg
plasmid DNA 50 pg-1.0 ng
was required for total reaction mixture of 50 μl.
Reaction was carried out in 0.5 ml PCR tubes in duplicates. As magnesium ions make complexes
with the primer, dNTPs and DNA template, hence concentration of magnesium ions should be
optimized. Low concentration of Mg ions results in low yield, while high concentration results in
non specific products.
Amplification was carried out in a Thermalyne DNA thermalcycler programmed as:
one cycle of initial denaturation at 94C for 3 minutes,
followed by 35 cycles of denaturation, annealing and extension (94, 55, 72C
respectively) each with a hold time of 1 minute
and a final extension at 72C for 20 minutes.
34
After amplification, the reaction product was either stored at 4C or subjected to agarose gel
electrophoresis as described by Sambrook and Russell (2001).
The PCR products were analyzed by loading 5 μl of the samples on 1 % agarose gel
electrophoresis. Gel was visualized on UV transilluminator and photographed by using gel
documentation system (Dolphin, WEALTEC, USA).
2.6 DNA extraction from agarose gel
The DNA fragments or PCR products which were needed to be analyzed were purified from
agarose gel by Vivantis GF-1 Gel DNA Recovery Kit (Cat # VSGF-GP-100). The agarose gel
portion containing the required DNA fragment or PCR product was excised by using a sterilized
sharp scalpel. The gel slice was weighed, placed in the microcentrifuge tube and dissolved in the
binding and solubilization buffer GB (1 vol. GB: 1 vol. gel). The gel slice was dissolved at 50°C
for 5-10 minutes until gel had melted completely. The dissolved gel slice was transferred to the
spin column and centrifuged at 10,000 g for 1 minute. Flow-through was discarded and 750 μl of
washing buffer was added into the column and again centrifuged at 10,000 g for 1 minute. The
residual washing buffer was removed by doing centrifugation at 10,000 g for 1 minute. Column
was then shifted and placed on a fresh 1.5 ml microcentrifuge tube. 30 μl of elution buffer51was
added on to the column and waited for 2 minutes at RT. Then centrifugation was done at 10,000
g for 1 minute and elution step was repeated to get better yield of the purified product. Hence,
total 60 μl was obtained at the end of the extraction process. The purified sample was then
visualized on 1 % agarose gel electrophoresis and stored at -20°C for further use.
2.6.1 Purification of PCR product
The PCR products were excised from the agarose gel and purified by using Vivantis GF-1 Gel
DNA Recovery Kit (Cat # VSGF-GP-100). Protocol has been mentioned earlier in section
35
2.2.2.4. The purified PCR product was again run on 1 % agarose gel to quantify its concentration
after purification and to confirm its purity.
2.7 Cloning in pTZ57R/T vector
2.7.1 T/A cloning Kit method
The amplified product was ligated into the pTZ57RT by T/A cloning technique to produce the
recombinant pTZOaST1 construct which was then transformed into E. coli DH5 .
Ligation
Autoclaved the medium, allowed to cool at 55̊ C. Then, added 50 µl of Ampicillin in 50 m l LB
medium from stock solution (50,000 mg/ 1000ml). Pipette 40 µl of 2 percent X-Gal solution and 7
µl of 20% IPTG on the centre of ampicillin plate. Used a sterile spreader to spread it over the
entire surface of the plate. Incubated the plate at 37o C until all the solution or fluid has
disappeared The plate is incubated for 3- 4 hours.
Took out the plates from incubator in which E.Coli DH5α was spreaded. Round single colonies
will be observed. From T/A kit took C_medium and added 1.5 ml of it into two test tubes 1.5 ml
each. Pre warmed culture tubes containing a required amount (1.5ml) of transformed C_ medium
at 37o C. Moved a small portion of bacterial culture (4x4 mm size for each 1.5 ml of C_medium)
of DH5α and from the overnight LB plate using on inoculating loop into the pre warmed
C_medium. Suspended the culture by gently mixing and incubating the tubes in a shaker at 37o C
for 2hrs. The colonies on LB plates can be stored at 4o C and used for inoculating fresh culture
within 10 days.
Prepared Transform Aid T- solution by mixing equal volume of T solution (A) and T solution (B).
Took 430 ml of T(A) and 430 ml of T (B) and mixed them, then put on ice. Dispensed 1.5 ml of
fresh culture into a microfuge tube (eppendorffe) and spinned at maximum speed for 1minute at
room temperature or at 4o C. Discarded the supernatant and resuspended the pellet in 300 ml
36
transform aid T solution ( mix of T(A) and T(B) ). Incubated tubes on ice for 5 min. Spin down
the cells again for 1 min at RT and remove the supernatant. Resuspended the cells in 120 ml of (T
solution mixture) and incubated on ice for 5 min.
Prepared the DNA for transformation by dispensing 1ml of supercooled DNA (10000pg) or 2.5 ml
of ligation mixture (10-20 mg of vector DNA) into a new microfuged tubes and sat them on ice
for 2 min. Added 50 µl of resuspended cells to each tube containing DNA and incubated on ice for
5 min. Plated the cells on pre warmed LB ampicillin agar plates. Incubated the plates overnight at
37o C.
For transformation used 2ml (10mg of DNA) of the ligation mixture with PCR DNA fragment.
Incubated overnight at 37o C usually result in 200- 1000 colonies per plate (about 90% of which
were white). Now prepeared again LB Ampicillin plates. Made LB medium and added ampicillin
after autoclaviing the medium and then solidifying the plates. Picked colonies from the
transformation (white colinies) and spot them on LB ampicullin plates in duplicates. Incubated at
37o C overnight.
More than 90 % colonies appeared to be white on LB agar plates (containing ampicillin 100
μg/ml, 2 % X-gal and 0.1 mM IPTG) after transformation, as genes were inserted in the
pTZ57R/T vector containing lacZ region. While blue colonies showed the absence of insert, as
lacZ is functional in E. coli and encodes β-galactosidase that hydrolyzes X-gal into a colorless
galactose and 4-chloro-3-brom-indigo giving blue color to the colony. Thus blue/white screening
was done and single white colony was picked from the plates and inoculated in 10 ml of LB
medium (100 μg/ml ampicillin) for plasmid preparation. If clear, round colonies on duplicate
plates were found on plates. It meant that gene is being inserted. To amplify the gene inserted in
the plasmid colony PCR will be done.
Plasmid extraction was done by using Vivantis GF-1 Plasmid DNA Extraction Kit (Cat # VSGF-
PL-100) as described in sectio.2 and run on 1 % agarose gel electrophoresis.
37
2.7.2 Preparation of competent cells and transformation
Picked a single bacterial colony (2-3mm in diameter) from a DH5α plate that has been incubated
for 16 to 20hrs at 37o C. Transferred the colony into 100 ml of LB broth in a 1 litre flask.
Incubated the culture for 3hrs at 37o C with vigorous agitation, monitoring the growth of the
culture. (For efficient transformation, it is essential that the number of whole cells not exceed 10-
8 cells/ml which for most strains of E_coli is equivalent to an OD600 of 0.4. To ensure that the
culture does not grow to a higher density, measured the OD600 of the culture every 15- 20min.
Began to harvest the culture when the OD600 reaches 0.35). Transferred the bacterial cells to
sterile, disposable ice cold 50ml polyprylene tubes. Cooled the cultures to 0oC by storing the
tubes on ice for 10 min. Recovered the cells by centrifugation at 2700 g (41000rpm) for 10min at
4o C. Decanted the medium. Stood the tubes in an inverted position to allow the last traces of
media to drain away.
Resuspended each pellet by gentle vortexing in 30ml of ice cold MgCl2 , CaCl2 solution (80 mM,
MgCl2, 20 mMCaCl2). Recovered the cells by centrifugation at 2700g for 10min at 4o C.
Decanted the medium from the cell pellets. Stood the tubes in an inverted position to allow the
medium to draw away. Resuspended the pellet by swirling or gentle vortexing in 2ml of ice cold
0.1 M CaCl2 for each 50ml of original culture. At this point, either use the cells directly for
transformation as dispensed into alliquots and freezed at -70o C. (The cells may be stored at 4o C
in CaCl2 solution for 24- 48 hrs. The efficiency of transformation increases 4- 6 folds during the
first 12- 24 hrs of storage and thereafter decreases to the original level). Dispensed a sample of
the competent cells (200ml) into a sterile precooled microcentrifuge tube.
Added plasmid DNA (approx 0.5mg) mixed gently and left on ice for 40min. Quickly transferred
the tube to a 42o C water bath for 2 min then returned it to ice for 5min. Added LB medium
(0.8ml) to the tube, mixed and incubated it for 2 hrs at 37o C without shaking. Spread samples of
38
the transformed cells (50 -200ml) on prewarmed, dried LB plates containing the appropriate
antibiotic to select for the plasmid.
2.8 Colony PCR
The transformation resulted in the production of colonies containing the plasmids. One of
the methods to screen the colonies whether containing the desired plasmid or not is colony PCR.
For colony PCR, a single colony was picked from the LB agar plate by using a sterile tooth pick
or by pipette (P200, Gilson) tips and transferred to the PCR tube containing 30 μl of sterile
distilled water. The colony was mashed, mixed thoroughly and denatured at 95°C for 10 minutes,
25°C for 10 minutes and then the denaturation reaction was stopped by holding at 4°C. The PCR
tubes were centrifuged for 1 minute at 12,000 g and from the supernatant 5 μl were taken as a
template in the PCR reaction. PCR reaction mixture was prepared by taking two oligonucleotide
primers of the required gene present in the plasmid (50 pmol of each primer), four
deoxynucleotide triphosphates (dNTPs) each of 0.4 mM, 2.5 U Taq polymerase (Fermentas #
EP0402), 5 μl of 10 x PCR buffer containing ammonium sulphate, 5 μl of template plasmid and
MgCl2 (1.5-4 mM) was added to make total reaction mixture of 50 μl. The amplification was
done in a Thermocycler (Applied Biosystem 2720 Thermo Cycler) using similar conditions as
used to amplify the required gene.
2.9 Sequence analysis
2.9.1 Q/A prep spin miniprep kit method
This protocol is designed for purification of up to 20mg of high copy plasmid DNA from 1-5 ml
overnight cultures of Ecoli in LB medium.
39
Resuspended pelleted bacterial cells in 250ml. Buffered P1 and transferred to a microcentrifuge.
Ensured that RNase A has been added. Buffered P1. No cell clumps should be visible after
resuspension of the pellet. Added 250ml Buffer P2 and gently inverted the tube 4-6 times to mix.
Mixed gently by inverting the tube. Did not vortex as this would result in snearing of genomic
DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly
clear. Did not allow the lysus reaction to proceed for more than 5min. Added 350ml. Buffered N3
and inverted the tube immediately but gently 4-6 times. To avoid localized precipitation, mixed
the solution gently but thoroughly, immediately after addiction of buffer N3. The solution should
become cloudy.
Centrifuged for 10min at 13,000 rpm (-17, 900xg) in a table top microcentrifuge. A compact
white pellet will form. Applied the supernatant from step 4 to the Q/A prep spin column by
decanting or pipetting. Centrifuged for 30-60s. Discarded the flow through. (Optional): washed
the Q/A prep spin column by adding 0.5ml. Buffered PB and centrifuging for 30-60s.
Washed Q/A prep spin column by adding 0.75ml. Buffered PE and centrifuging for 30-60s.
Discarded the flaw through and centrifuged for an additional 1 min to remove residual wash
buffer. Important: Residual wash buffer will not be completely removed unless the flaw- through
is discarded before this additional centrifugation. Residual ethanol from buffer PE may inhibit
subsequent enzymatic reactions. Placed the Q/A prep column in a clear 1.5 ml microcentrifuge
tube. To elude DNA, added 50ml. Buffer EB (10mM Tris.Cl2, ph 8.5) or water to the centre of
each Q/A prep spin column, let stood for 1 min and centrifuged for 1 min.
Ethanol precipitation for sequencing reaction:Prepared a labeled, sterile 0.5ml microfuge tube for
each sample. Prepared fresh stop solution/ Glycogen mixture as follows (per sequencing reaction):
2µl of 3M Sodium Acetate (pH 5.2)
2µl of 100m M Na2 EDTA (pH 8.0)
1µl of 20 mg/ml of glycogen supplied with the kit.
40
To each of labeled tubes, added 5µl of the stop solution/ glycogen mixture. Transferred the
sequencing reaction to the appropriately labeled 0.5ml microfuge tube and mixed thoroughly.
Added 60ml of cold absolute ethanol from -20o C freezer and mixed thoroughly. Kept it on ice for
20 minutes. Centrifuged it on 12,000 rpm at 4o C for 20 minutes. Rinsed the pellet 2 times with
70% cold ethanol from -20o C 100µl each. For each rinsed, centrifuged immediatelyat 12,000 rpm
at 4o C for 2minutes. After centrifugation carefully remove all of the supernatant by micropipette.
Vacuumed dry for 10 minutes (or until dry). Resuspended the sample in 40µl of sample loading
solution.Stored it at -20o C.The reverse Primer used was M13/PUC reverse sequencing Primer (-
46), 24-mer 5’-d (GAGCGGATAACAATTTCACACAGG)-3’ .which had Tm: 70o C .CG
content: 46%. This primer is a synthetic single-stranded 24-mer oligonucleotide with free 5’ and
3’- hydroxyl ends. The primer anneals to the 5’- terminus of the lac Z gene such that its 5’- end
lies 46 nucleotides upstream of the ECOR l site of M13 mP18. The DNA polymerase extension
products grew in the direction that coincides with the transcription of lacZ gene. The primer was
supplied as an aqueous solution and can be used for DNA sequencing of all phages, phogennides
and plasmids carrying lacZ gene. Each lot of M13/PUC reverse sequencing Primer was assayed in
the PUC 19 DNA dideoxy sequencing reaction.
2.9.2 Analysis of Full-Length ST Gene
2.9.2.1 Extraction of genomic DNA
DNA was isolated from the blood samples of local ovine breed (Lohi) by using chloroform
phenol extraction method.
10ml blood was drawn to falcon tubes having 400ml of 0.5M EDTA, mixed gently and frozen the
samples. After few days the blood samples were taken from the freezer and proceeded the
extraction method.
TE buffer was added to all tubes up to the level of 45ml (22.5ml for 5ml blood). Samples were
shaken vigorously to resuspend all the cells evenly in TE buffer (this Hcl pH 8.00 10 m M, EDTA
pH 8.00 : 2mM). Then samples were centrifuged on table top centrifuge at 3000 rpm for 25 min at
41
25o C. Upper layer of the buffer was removed leaving 25ml in the tube ( 12.5ml in tube for 5ml of
blood) and the remaining layer was shaken vigorously to resuspend the pellet. TE buffer was
added to each tube up to (22.5) 45ml and centrifuged at 3000 rpm for 25min at 25o C. Upper layer
was poured off leaving 15ml in tubes. Pellet was shaken to resuspend evenly. TE buffer was
added to make the volume up to 45ml (22.5ml) and centrifuged at 3000 rpm for 25min at 25o C.
Again upper layer was poured off leaving 5ml of samples containing pellet. Pellets were
resuspended and volumes were roused up to (22.5) 45ml of TE buffer.
Samples were centrifuged at 3000 rpm for 25min at 25o C. After centrifugation, liquid layer were
poured off. In this way all the red blood cells were washed away. TNE buffer was added to falcon
tubes containing pellets of white blood cells according to blood volume (for 10ml blood = 6ml
buffer) ( for 5ml = 3ml buffer). Resuspended the pellet in it. 10% SDS was added to each tube
according to amount of blood (200ml SDS = 10ml blood) ( 100ml SDS = 5ml blood). Then
proteimase K (10mg/ml stock) was added according to blood volume (50ml for 10ml blood)
(25ml for 5ml blood) incubated at 37o C for 14 hours (overnight) at continuous shaking. Next day
1ml of GM Nacl (1ml for 10ml blood) (0.5ml for 5ml) was added and shaken vigourously until it
became foamy. Tubes were placed on ice for 15min. 2ml (1ml for 5ml) phenal chloroform
solution was added tilted 20 times gently and centrifuged at 3000 rpm for 20min. Upper clear
layer were taken to labeled falcon tubes (labeled with pedigrees & names). Isopropanal was added
in equal volume & centrifuged at 3000 rpm for 15min.
Supernatants were discarded & washed the DNA with 5ml (2.5ml) ethanol 70% and centrifuged
at 3000 rpm for 15min. DNA pellets were dried by inverting the tubes on the table top after
discarding the supernatant. TE buffer was added according to the amount of blood ( added 1.5ml
TE buffer (This Hcl pH 8.00: 10mM, EDTA pH 8.00: 0.1mM) for 7.5, 8, 9, 10ml blood, 1ml TE
buffer for 4, 5, 6ml blood and 20ml TE buffer for ml blood). Then the DNA samples were
incubated by placing the tubes at 37o C for overnight at continuous shaking. Next day, samples
42
were given heat shock at 70o C for 1hour to inactivate any remaining nucleases and poured to
labeled screw cap tubes.
DNA concentrations were determined by agarase gel electrophonesis or by spectrophotometery.
Working DNA concentrationn was kept at 25mg /ml & 2ml was used per PCR reaction.
2.9.2.2 PCR amplification of GH gene
On the basis of conserved sequences 4 reverse and 4 forward designed primers were used in the
amplification of growth hormone gene.
P1F(CCAGTTCACCAGACGACTCAG), P1R(TTGAAGGTGTCAGCAGCCAG)
P2F(CCCTGGACTCAGGTGGTG), P2R(GATGGTTTCGGAGAAGCAGA)
P3F(GGGACAGAGATACTCCATCCAG) P3R(CCTTCAGCTTCTCATAGACACG)
P4F(TCGCATCTCACTGCTCCTTATC ) P4R(CAGATGGCTGGCAACTAGAAC)
PCR amplification of the first strand was carried by fermentas Taq DNA polymerase in applied
biosystem ‘s2720 thermalcycler. Denaturation, annealing and extension were carried out,
respectively at 94, 60 and 72̊C with a hold time of I minute each for 35 cycles.
2.9.2.3 Sequencing reaction
Purified PCR products of DNA and cDNA were T/A cloned to pTZ57R/T vector by using dT.
dA tailing technique. Five constructs, OST1(for coding sequence analysis) and OST 2, OST 3,
OST 4, OST 5(for genomic DNA analysis) were developed. The recombinant clones were
identified by blue /white screening and confirmed by colony PCR. The nucleotide sequencing
was performed by using CEQ-800 sequencing analyzer.
43
2.10 Bioinformatics tools for sequence analysis
The nucleotide and amino acid sequences of GH were obtained by database homology
programs BLASTN and BLASTP available at www.expasy.org. The sequences were aligned and
phylogenetic tree was constructed by using neighbor joining method on MUSCLE alignment
software (http://www.phylogeny.fr/). The secondary and 3-D structures were determined by
using SWISS-MODEL (www.swissmodel.expasy.org) and the structures were visualized on
VMD version 1.8.7.
2.11 Mini-preparation of plasmid DNA
Preparation of cells
o Inoculated 2ml of rich medium (LB, YT or terrific broth) containing the appropriate
antibiotic (Ampicillum) with a single colony of transformed bacteria. Incubated the culture
overnight at 37o C with vigorous shaking. Poured 1.5ml of the culture into a microfuge
tube. Centrifuged it at maximum speed for 1 min at 4o C in a microfuge. Stored the unused
portion of the original culture at 4o C. When centrifugation was completed, removed the
medium by pupetting, leaving the bacterial pellet as dry as possible.
Lysis of cells
o Resuspended the bacterial pellet in 100ml of ice cold alkaline lysis solution I by vigorous
vortexing. Added 200 ml of freshly prepared lysis solution II to each bacterial suspension,
closed the tube tightly and mixed the content by inverting the tube rapidly 5 times. Did not
vortex, stored the tube on ice. Added 150ml of ice cold alkaline lysis solution III through
the viscous bacterial Lysate by inverting the tube several times. Stored the tube on ice for
3-5 min. Centrifuged the bacterial Lysate at maximum speed for 5 min at 4o C in a
microfuge. Transferred the supernatant to a fresh tube. Added on equal volume of phenal:
Chloroform mixed with organic and aquaus phases by vortexing and then centrifuged the
44
emulsion at maximum speed for 2 min at 4o C in a microfuge. Transferred the aquouss
upper layer to a fresh tube.
Recovery of plasmid DNA
o Precupitated nucleuc acids from the supernatant by adding 2 volumes of ethanol at room
temperature, mixed the solution by vortexing and then allowed the mixture to stand for
2min at room temperature. Cancelled the precupitated nucleuc acids by centrifugation at
maximum speed for 5min at 4o C in a microfuge. Removed the supernatant by gentle
aspiration, stood the tube in an inverted position on a papertowel to allow all of the fluid to
draw away. Used disposable pipette tip to remove any drops of fluid adhering to the walls
of the tube. Added 1ml of 70% ethanol to the pellet and inverted the closed tube several
times. Recovered the DNA by centrifugation at maximum speed for 2min at 4o C in a
microfuge. Again removed all of the supernatant by gentle pipetting. Removed any beads
of ethanol that formed on the sides of the tube. Stored the open tube at room temperature
until the ethanol had evaporated and no fluid was visible in the tube (5-10min). Dissolved
nucleuc acid in 50ml of TE (PH 8.0) store at 4o C. Vortexed the solution gently for few
secondes. Stored DNA solution at – 20o C.
2.12 Restriction analysis of pTZ-oGH clones
The plasmids were screened and confirmed by restriction analysis and reactions were prepared in
1.5 ml eppendorf tubes. Recombinant plasmid i.e. pTZoGH was digested with NcoI/BamHI and
NdeI/BamHI. The reaction was made as follows; 25 μlof recombinant plasmid (10 μg), 1 μl of
(10 U/μl) each enzyme, 10 μl of 10 x Orangebuffer [50 mM Tris-Cl (pH 7.5 at 37°C), 10 mM
MgCl2, 10 mM NaCl and 0.1 mg/mlBSA] Volume was made up to 100 μl using autoclave
distilled water and tubes were then incubated at 37°C for 16 hrs.Restriction analysis was
checked on 1 % agarose gel electrophoresisand required gene was gel excised and purified by
using Vivantis GF-1 Gel DNARecovery Kit (Cat # VSGF-GP-100) illustrated in section 2.2.2.4.
45
2.13 Restriction analysis of pET22b (+)
For restriction analysis of pET22b (+) vector, 100 μl reaction was made by adding 20 μl of
pET22b vector (4 μg), 10 μl of 10 x Red buffer, 1 μl each of (NcoI/BamHI and NdeI/BamHI
)enzyme in respective construct and volume was made up to 100 μl with distilled water. Tube
was incubated for 5 hrs at 37°C. Second restriction of pET22b was made by using 1 μl each
enzyme, (NcoI/BamHI and NdeI/BamHI ) 10μl of 10 x Orange buffer, 20 μl of pET22b (+)
vector (4 μg) and water was added to make up the volume to 100 μl. Tube was incubated at 37°C
for 16 hrs. Restrictions were confirmed by running 1 % agarose gel electrophoresis and restricted
bands were excised from the gel and gel purified by using Vivantis GF-1 Gel DNA Recovery Kit
(Cat # VSGF-GP-100) described earlier.
2.14 Ligation and transformation in DH5α and BL21 Codon + strains
Ligation of oGH gene fragments were ligated in the pET22b (+) vector .The restricted purified
fragment oGH was ligated in pET22b (+) restricted and purified]. Ligation mixture was made in
1.5 ml eppendorf tube by adding, 2 μl of 10 x ligation buffer (400 mM Tris-Cl, 100 mM MgCl2,
100 mM DTT, 5 mM ATP, pH 7.8 at 25°C), 2 μl of restricted and purified pET22b vector, 5 μl of
restricted and purified OaST product, 1 μl of T4 DNA ligase (5 U) and water was added to make
up the volume up to 20 μl. Ligation mixture was incubated at 22°C for 18hrs.
The ligated plasmid was then transformed in DH5α as stated before.
The bacterial colonies were confirmed by colony PCR and restriction analysis (using restriction
enzymes i.e. NdeI/BamHI, NcoI/BamHI ). The positive transformants were then transformed in
BL21 Codon + strain to obtain expression of the oGH gene.
46
2.15 Expression of poGH clones
The pET system is one of the most commonly used systems for cloning and expression of genes
in E. coli host. The target genes in this system are under the control of the strong bacteriophage
T7 transcription signal. The T7 RNA polymerase promoter is one of the strong promoters, when
it is fully induced it uses most of the host resources to synthesize the target protein. At the same
time the expression level can be controlled by the use of inducer. The target protein expression
can be initiated either by infecting the host cells with λCE6, a phage carrying the T7 RNA
polymerase gene or by transferring the plasmid into an expression host which carries a
chromosomal copy of T7 RNA polymerase gene under the control of lacUV5 and for this IPTG
is required as inducer. In this study the genes were cloned and expressed in pET22b (+) vector
which has a T7 lac promoter .
Figure 4.Restriction map,sequence and multiple cloning sites of pET 22b(+). Vector was used for expression of all poGH constructs
47
In this vector the lac operator sequence exists just downstream the T7 promoter region in the
plasmid and carries the natural promoter and lacI (lac repressor). The T7 RNA polymerase gene
and lacI are diverging. The lac repressor acts at the lacUV5 promoter in the host chromosome to
repress the T7 RNA polymerase gene and at the T7 lac promoter in the vector to prevent the
transcription of the target gene. The plasmid has 5493 bp size and carries an N-terminal pelB
signal sequence for potential periplasmic localization, His- Tag at C-terminal and ampicillin
resistant marker gene for selection.
For expression studies a single colony from poGH clones in BL21 Codon Plus was picked
and inoculated in 10 ml LB medium (100 μg/ml ampicillin) in 100 ml Erlenmeyer flask.
The flasks were incubated in shaking incubator (Irmeco GmbH, Germany) at 37°C with
100 rpm. Next morning one percent of cultures were refreshed in 25 ml LB medium (100
μg/ml ampicillin) and grown under same conditions as mentioned above.
The OD was checked at 600 nm after 2 1/2 hrs, when OD reached at 0.5-0.6 cells were
induced with 0.5 mM IPTG. After 4 hrs of post induction 1 ml sample was drawn from
each culture and centrifuged for 3 minutes at 12,000 g. The supernatant was discarded and
pellet was resuspended in 100 μl of lysis buffer (50 mM Tris-Cl pH 8.5 + 5 mM EDTA + 1
mM PMSF + 100 mM NaCl)
Samples were properly mixed with 3cc syringe to reduce the viscosity and denatured at
85°C for 5 minutes. Samples were then short spin and loaded on to the 12 % SDS gel, 5 μl
of protein marker (Fermentas SM0661) was also loaded for protein molecular weight
determination.
2.16 SDS-Polyacrylamide Gel Electrophoresis (PAGE)
To analyze the total cell protein and molecular size of the expressed proteins, one
dimension SDS-PAGE was done (Laemmli, 1970).
48
By adjusting the SDS-PAGE assembly (Bio-Rad), 12 % resolving gel [1.6 ml H2O, 2.0 ml of 30
% acrylamide (Bio- Rad), 1.3 ml of 1.5 M Tris (pH 8.8), 0.1 ml of 10 % SDS, 0.05 ml of 10 %
APS (freshly prepared) and 0.002 ml of N, N, N’, N’ tetra methyl ethylene diamine (TEMED)]
was prepared and poured in the assembly leaving 0.5 inch vacant at the top. Approximately 1ml of
distill water was poured on to the resolving gel to get the flat surface and to remove air bubbles.
Gel was left to polymerize for half an hour.
After polymerization, water was removed and 5 % stacking gel [3.4 ml H2O, 0.83 ml of 30 %
acrylamide (Bio-Rad), 0.63 ml of 1 M Tris (pH 6.8), 0.1 ml of 10 % SDS, 0.05 ml of 10 % APS
(freshly prepared) and 0.005 ml of N, N, N’, N’ tetra methyl ethylene diamine (TEMED)] was
prepared and poured on to the polymerized resolving gel and immediately comb was inserted so
that wells should be formed for sample loading. Gel was again left for 30 minutes for proper
polymerization, after that comb was removed carefully from the gel. The gel was assembled in a
SDS-PAGE apparatus (Mini-PROTEAN 3, BIO-RAD) and 1 x Trisglycine buffer (25 mM Tris-
Cl, 250 mM glycine and 0.1 % SDS) was poured in theelectrophoresis chamber.
Wells were washed with the above mentioned buffer and ~ 9 μl samples (5-15 μg protein) were
prepared by mixing equal volume with 2 x loading dye [100 mM Tris (pH 6.8), 20 % (v/v)
glycerol, 4 % (w/v) SDS, 200 mM dithiothrietol(DTT) and 0.2 % (w/v) bromophenol blue].
Samples were then properly mixed with the loading dye by using 3cc syringe and denatured by
boiling at 85°C for 5 minutes.
Samples were short spin and then loaded on to the gel and 5 μl of protein marker (Fermentas
SM0661) was also loaded for protein molecular weight determination. The gel was
electrophoresed at 120 volts for 1 hr and 30 minutes at RT. Electrophoresis was stopped when the
dye seemed to reach 1 mm close to the bottom of the gel.
The gel assembly was taken out of the chamber and plates were separated carefully by using
spatula. Gel was then put in a staining solution (50 mg Coomassie Brilliant Blue R250, 450 ml
methanol, 90 ml acetic acid and 450 ml H2O) for 20 minutes with gentle rotation.
49
Gel was then removed from staining solution and placed in destaining solution (50 ml methanol,
70 ml acetic acid and volume made up to 1 L) to get protein bands prominent and background
becomes transparent.
Densitometry was carried out on gels using Dolphin imager (WEALTEC, USA). The gels were
placed inside an image and illuminated with white light exposure. After that, automatic
background subtraction identical areas around the protein bands were selected for analysis. The
internal OD of all the proteins in a gel was calculated and %age of the target protein band in all
the expressed proteins was calculated by the formula as follows:
%age of expressed protein = Internal OD of the target protein x 100/
Sum of internal OD of all the proteins.
2.17 Western transfer and immunoblot analysis
The protein samples resolved by 13% SDS-PAGE were transferred to a 0.45m
nitrocellulose membrane using mini transfer-blot electrophoretic transfer cell at 50V for one
hour. The transfer buffer was 25mM Tris-Cl (pH 8.3), 192mM glycine and 20% (v/v) methanol.
To block the non-specific membrane sites, the blot was incubated in TBS-T buffer [20mM Tris
(pH 7.6), 13 mM NaCl, 0.1% (v/v) Tween 20] containing 2% (v/v) gelatin and 0.4% (v/v)
sodium azide, for 30 minutes. After blocking, the membrane was successively incubated with
rabbit anti-bovine growth hormone (1:2,000 dilutions) and IgG alkaline phosphatase conjugate
(1:6,000 dilutions) for 1 hour at room temperature with gentle shaking on a rotatory shaker. Each
successive incubation was followed by 3x10 minutes washings with TBS-T buffer. Finally, the
membrane was stained with chromogenic detection substrates [100l of 75mg/ml NBT and
150l of 25mg/ml BCIP in 25ml carbonate buffer (8.4 g/L NaHCO3, 0.203 g/L MgCl2.6H2O, pH
9.8)] until the signal was clearly visible (30-60 seconds). Chromogenic reaction was stopped by
rinsing the membrane twice with distilled water followed by image scanning of dried membrane.
50
2.18 Protein estimation
Protein concentration was determined either by absorption measurements at λ280 or dye-
binding method of Bradford (1976) using bovine serum albumin (BSA) as standard. The dye-
binding reagent was prepared by dissolving 100mg of Coomassie brilliant blue G250 in 50ml
ethanol and 100ml ortho-phosphoric acid. Volume was made up to 1liter with distilled water and
the reagent was filtered twice before use. For assay, appropriately diluted protein sample was
mixed with 5 ml dye-binding reagent and incubated at room temperature for 10-15 minutes prior
to absorbance measurements at A595. A standard curve was thereafter plotted with known
concentrations of BSA and used to calculate the concentration of protein in test samples
2.19 Primer designing for translocation of Ovine ST gene into periplasmic space
Eight constructs based on the DsbAss with amino acid variations upstream of the 5'- start codon
of OaST gene were produced. The primers (FP1-FP8) were designed as shown in Table 1.The
forward primer had NdeI site at the 5'-end, and the reverse primer had BamHI site at the 5'-end.
The wild-type growth hormone sequence in the construct pTZoGH-1 was amplified using each
of the forward and reverse primers (FP-1 to FP_8). These PCR products were then digested with
NdeI and BamHI and ligated into the NdeI/BamHI site of pET22b thus generating a series of
recombinant plasmids (poGH-3-I-VIII) as shown in Table 1. These constructs were transformed
into E. coli DH5 .and then into E. coli BL21 codon plus cells for the expression studies.
51
Table 1. sequence of primers used for construction of poGH-3-I-VIII plasmids
Primer Name *Nucleotide Sequence (5ʹ→ 3ʹ)
FP-1
CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA GCG
TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC
FP-2
CAT ATG AAA AAG ATT TGG CTG ATT CTG GCT GGT TTA GTT TTA GCG
TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC
FP-3
CAT ATG AAA AAG ATT TGG CTG ATT CTG ATT GGT TTA GTT TTA ATT
TTT AGC ATT TCG GCG GCC TTC CCA GCC ATG TCC
FP-4 CAT ATG AAA AAG ATT TGG CTG ATT CTG ATT GGT TTA GTT TTA GCG
TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC
FP-5
CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA ATT
TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC
FP-6
CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA ATT
TTT AGC ATT TCG GCG GCC TTC CCA GCC ATG TCC
FP-7
CAT ATG AGA AGG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA GCG
TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC
FP-8 CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA GCG
TTT TGT GCA TGT GCG GCC TTC CCA GCC ATG TCC
*The sequence shown in bold and italics represents the OaST cDNA sequence whereas the underlined sequence is the site for
NdeI restriction enzyme. Nucleotide variation incorporated in DsbA signal sequence are highlighted in grey
These PCR products were then digested with NdeI and BamHI and ligated at the NdeI/
BamHI site of pET22b thus generating a series of recombinant plasmid pEToGH2-11.While the
reference constructpTZ-oGH1 was double digested with Nco I and BamHI and ligated at the
NcoI/ BamHI site of pET22b thus generating a reference vector poGH-1.These constructs were
transformed into E. coli DH5 and recombinant plasmids were isolated. The reverse primer
;PBGH3 (5`TAG GAT CCG CAA CTA GAA GGC AGC 3`)was used. PCR amplification of the
first strand was carried by fermentas Taq DNA polymerase in applied biosystem ‘s2720
thermalcycler. Denaturation,annealing and extension were carried out, respectively at 94̊,60̊ and
72̊ C with a hold time of 1 minute each for 35 cycles.
52
2.20 Subcellular fractionation of oGH
E. coli BL21 codon plus was transformed with different constructs, and cells from a single
colony of each of the transformant were transferred to LB medium containing 100 g/mL
ampicillin and grown overnight at 25 C in an orbital incubator shaker. LB medium (10 mL) was
inoculated with 0.25 mL of overnight culture of each construct and induced with 1mM IPTG
when the OD600 reached 0.6. After 5h of induction, 1.5 mL culture of cells carrying each
construct was centrifuged, and the pellet was suspended in 100 L of PBS buffer (137 mM
NaCl2, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) and 100 L of reducing buffer [100
mM Tris-Cl pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200mM
dithiothreitol] was added. Total cell protein analysis of the lysate was done by SDS-PAGE using
15% gel according to the method of (Laemmli, 1970). Resolved proteins were visualized by
staining with commassie brilliant blue. The protein profile of the samples after SDS-PAGE was
analyzed densitometrically using Dolphin-ID gel analysis software (Wealtec).
Freeze thaw
cells were harvested by centrifugation at 3,700 × g for 10 min at 4°C. For all the following steps,
tubes were kept on ice. The bacterial pellet was centrifuged, and its wet weight was
determined.Cells were frozen at −196°C. After thawing, the cells were resuspended in 75 mM
Tris-HCl (pH 8)–300 mM NaCl–1 capsule of protease inhibitors/50 ml (Complete; Roche
Diagnostics, Mannheim, Germany)–5 mM dithiothreitol–10 mM EDTA–10% (vol/vol) glycerol
and were sonicated six times for 30 s at 200 W. The periplasmic fraction was recovered after
centrifugation at 21,000 × g for 30 min at 4°C and was transferred to 75 mM Tris-HCl (pH 8)–1
M NaCl–10% (vol/vol) glycerol using Hitrap desalting columns (Pharmacia).
53
Osmotic Shock method ( a )
Periplasmic osmotic shock fluid was obtained by the method of( Koshland and Botstein, 1980).
Briefly, a volume of fermentation broth corresponding to 100 A600 was harvested by
centrifugation at 3000 g for 5 min. All subsequent steps were carried out at 4°C in an ice bath.
Pellets were resuspended in 1.0 ml of ice‐cold 10 mM Tris–HCl, pH 7.5, containing 20% (w/v)
sucrose. Then, 33 µl of 0.5 M EDTA, pH 8.0, were added and incubation on ice continued for 10
min. The cells were then centrifuged and the pellet rapidly resuspended by vigorous agitation in
1.0 ml of a cold 1 mM Tris–HCl, pH 7.5, solution. The suspension was then incubated for 10
min on ice and centrifuged again for 5 min. The supernatant was removed and saved as the
periplasmic fraction, also called osmotic shock fluid.
Osmotic shock method ( b ) We followed the protocol by late Dr.Mustak kaderbhai (Kaderbhai
et al., 2008).100ml culture of grown cells for 6 hours in LB medium at 25°C,150rpm were
harvested by centrifugation at 5,000g for 5minutes.Discarded the supernatant and resuspended
pellet in 20ml of STE (20%sucrose in 0.33M tris,1mM EDTA pH8.0).Left the resuspended
solution at room temperature for 10 minutes and then centrifuged at 7,500 g for 10minutes at 4°
C,discarded the supernatant while leaving behind just 250µl and resuspend pellet thoughrouly by
adding 10ml of chilled 0.5mM MgCl2.After resuspension left it on ice for 10minutes and then
centrifuged at 5,000g for 5min,at 4° C.Collected the supernatant as a shock fluid and used the
pellet for further fractions.
Sonication;Resuspended the pellet in 20ml TE (0.3M Tris, 1mM EDTA pH 8.0)and sonicated for
6 bursts of 30seconds each with interval of 3 minutes.Centrifuged the sonicated sample at 5,000
rpm for 20 minutes at 4o C. Used the supernatant as cytoplasmic fraction and pellete for further
fractionation.
Ultracentrifugation ;Washed the pellet with 5ml0.3 M sucrose and centrifuged at 11,000rpm for
10minutes with(0.3M sucrose,0.3M Tris,pH8.0).Took supernatant and ultracentrifuged it at
100,000g for 1hour,collected the pellet and resuspended it in 5ml 0.3M tris pH8.0.
54
2.20.1 FPLC chromatography
Anion exchange Resource-Q column (1.6 x 3.0 cm) was used and was pre-equilibrated with 2
column volumes of 20 mM Tris-HCl at pH 8.0. The filtered protein was then loaded and
separated using Amersham Biosciences FPLC (Amersham Pharmacia Biotech). The bound
protein was eluted with a continuous NaCl gradient (0.1-1 M) at a flow rate of 1ml/min and
dialyzed against 20 mM Tris-HCl (pH 8.0) to remove the salt.
2.20.2 MALDI-TOF
The mass spectrometry was performed on Bruker Autoflex III smart beam MALDI-TOF/TOF
(Bruker Daltonics GmbH, Fehrenheit str.-4, Bermen). 1 µg/µl of purified OaST-2 protein was
mixed with 3, 5 dimethoxy-4-hydroxycinnamic acid and 1 µl of the protein was deposited on
stainless steel target plate of the mass spectrometer. The machine was calibrated against the
standard protein BSA
2.21 Biological activity assessment assay
To analyze the biological activity of the purified roGH, HeLa cell lines were used. BSA and
commercially available bovine GH were used as negative and positive control, respectively. Prior to
the assay, HeLa cells were arrested at G0/G1 phase.Then, 30,000 HeLa cells/200 μl/well was taken;
500 μl of DMEM and 2 % fetal bovine serum (FBS) were added. BSA, bovine GH, and roGH each
of about 10 μg were added in duplicate wells (having 30,000 HeLa cells, 500 μl DMEM, and
2%FBS). The cells were then incubated for 24 h at 37 °C, and after incubation, cells were counted by
using inverted microscope (OLYMPUS IX51). By microscope, five boxes/ well were visualized and
cells of each box were counted and average number of cells in a well was calculated. The number of
cells per milliliter and in 200 μl was calculated as follows:
55
Average no. of cells x 10,000 = per ml (1000 μl)
&
No. of cells in 200 μl = average no. of cells x 10,000 x 200
1000
56
57
RESULTS
58
3.1 Genetic Analysis of oGH gene
3.1.1 Extraction of genomic DNA
The genomic DNA was isolated from the blood sample of local ovine breed Lohi by
chloroform phenol extraction method as explained in materials and method (Fig.5 ).
Figure 5.Genomic DNA of oGH isolated from the blood sample of local ovine breed lohi Lane 1, genomic DNA; lane 2, DNA Marker.
3.1.2 PCR amplification of oGH gene
The coding and non-coding regions of oGH were amplified by using five sets of gene-
specific primers as described under materials and methods. PCR amplification yielded single
products of expected length i.e. 459-, 422-, 462-, 639- and 640-bp as shown in Fig.6.
59
Figure 6. PCR amplification on 1% agarose gel.
M, 1 kb DNA ladder; Lane 1, PCR amplified product 422, lane 2, 3, 4 & 5 PCR amplified product 459, 639, 462 and 640
respectively
The best amplification of 422-,459, 462-bp long DNA fragments were achieved at an
annealing temperature of 53C while that of 640- and 639-bp DNA fragments, at 55C.
Following analysis, the amplicons were gel purified and their sequence determined.
3.1.3 Sequence analysis of oGH
The sequencing reaction was performed both in the forward and reverse directions to
resolve discrepancies, if any. Ovine growth hormone full gene sequence, capital letters (exons)
small letters (introns), mature peptide in green colour, red colour shows signal nucleotide region
and purple shows stop codon. The oGH shows 5 exons and 4 introns.as shown in Fig.7.
60
ATGATGGCTGCAggtaagctcacgaaaatcccctccattagcgtgtcctaagggggtgatgcggggggccctgccgatggatg
tgtccacagctttgggttttagggcttctgaatgtgaacatgggtatctgcacccgacatttggccaagtttgaaatattctcagtccctggagg
gaagggcaggcggggctggcaggagatcaggcgtctagctctctgggcccctccgtcgcggccctcctggtctctccctagGCCCC
CGGACCTCCCTGCTCCTGGCTTTCACCCTGCTCTGCCTGCCCTGGACTCAGGTGGTGG
GCGCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGCTGTGCTCCGGGCTCA
GCACCTGCATCAACTGGCTGCTgacaccttcaaagagtttgtaagctccccagagatgtgtcctagaggtggggaggc
aggaagggatgaatccgcaccccctccacacaatgggagggaactgaggacctcagtggtattttatccaagtaaggatgtggtcagggg
agtagaagtgggggtgtgtggggtggggagggtccgantaggcagtgaggggaaccccgcaccagttgagacctgtgtgggtgtgtcct
ccccccaggagcgcacctacttcccggaGGGACAGAGATACTCCATCCAGAACACCCAGGTTGCCT
TCTGCTTCTCCGAAACCATcccagcccccacgggcaagaatgaggcccagcagaaatcagtgagtggccacctagga
ccgaggagcaggggacctccttcatcctaagtaggctgccccagctctctgcaccgggcctggggcgtccttctccccgaggtggcagag
ggtgttggatggcagtggaggatgatggttggtggtggtggcaggaggtcctcgggcagaggccgaccttgcagggctgccccgagcc
cgcagcaccgaccaaccacccatctgccagcaggacttggagctgctTCGCATCTCACTGCTCCTTATCCAGTC
GTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGCCTGGTGTTTGG
CACCTCGGACCGTGTCTATGAGAAGCTGAAGGacctggaggaaggcatcctggccctgatgcgggtgagg
atggcattgttgggtcccttccatgctgagggccatgctcaccctctcctggcttagccaggagaacacacgtgggctgggggagagagat
ccctgctctctctctctttctagcagcccagccttgacccaggagaaacctcttccccttttgaaacctccttcctcgcccttctccaagcctata
ggggagggtggaaaatggagcgggcaggagggagccgctcctcagggcccttcggcctctctgtctctccctcccttggcaaGAGC
TGGAAGATGTTACCCCCCGGGCTGGGCAGATCCTCAAGCAGACCTATGACAAATTT
GACACAAACATGCGCAGTGATGATGCGCTGCTCAAGAACTACGGTCTGCTCTCCTGC
TTCCGGAAGGACCTGCACAAGACGGAGACGTACCTGAGGGTCATGAAGTGTCGCCG
CTTCGGGGAGGCCAGCTGCGCCTTCTAG
Figure 7. Nucleotide sequence of oGH.
Capital letters (exons) small letters (introns), mature peptide in green colour, red colour shows signal nucleotide region
and purple shows stop codon. It shows 5 exons and 4 introns
All the exons were united to get the amino acid sequence of oGH gene.which comprises of
217 amino acid in which first 26 are of signal sequence and rest of 191 are mRNA of oGH.
Figure 8.Amino acid sequence of oGH Amino acid sequence .oGH shows that it comprises of 217 amino acid.While mature hormone comprises 191 amino acids start
from AFPAM in first row.
61
(a)
(b)
Figure 9.amino acid sequence of oGH. (a) Amino acid sequence of oGH isolated from local ovine breed Lohi. (b) Nucleotide sequence of oGH.
3.1.3.1 Sequence comparison of oGH at amino acid level
Amino acid sequence of ovine growth hormone isolated from local breed (Lohi) showed
that it is comprises of 191 amino acids, calculated molecular weight of 21.85kDa while
isoelectric point was 7.86 when analyzed on( web.expasy.org/protparam/). This sequence was
than aligned with locally isolated growth hormone sequences of caprine and bubaline breeds of
Pakistan. It was analyzed that amino acid sequence of ovine Lohi breed is same for GH gene
isolated from local breed of caprine and has difference with local breed of bubaline at positions
9, 130 and 140 as shown in Fig.10.
62
Figure 10.Comparison of growth hormones of ovine capricorn and bubaline
Comparison of 3 locally isolated growth hormones of ovine, caprine and bubaline at amino acid level.
The amino acid sequence of Pakistani ovine breeds (Lohi) accession no. AB244790
showed difference with the amino acid sequence of growth hormone isolated from Australian
and Indian breeds when compared on Clustal W 1.81 for sequence alignment
(www.clustal.org/clustal2/). It showed variation of one amino acid at position 147 where
threonine is replaced with arginine as shown in Fig.11.
Figure 11,Amino acid sequence comparison. .Amino acid sequence comparison of Lohi (row 1) with Indian (row 2) accession number NM-001009315 and Australian accession
number S50877 (row3) breeds
63
Ovine growth hormone sequence was compared with the growth hormone sequence of the
species of family Bovidae and other species of class mammalian.
Homosapiens AFPTIPLSRLFDNASLRAHRLHQLAFDTYQEFEE 34
Pan AFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEE 34
Canis NAVLRAQHLHQLAADTYKEFER 22
Felis FPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 34
Equus FPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 33
Mustela AFPAMPLSSLFANAVLRAQHLHQLAADTYKDFER 34
Hippopotamus AFPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 34
Camelus AFPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 34
Ovis AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFER 34
Lohi AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFER 34
Bos AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFER 34
Capra AFPAMSLSSLFANAVLRAQHLHQLAADTFKEFER 34
Bubalus AFPAMSLSSLFANAVLRAQHLHQLAADTFKEFER 34
Monodelphis AFPAMPLSSLFANAVLRAQHLHQLVADTYKEFER 34
Crocodylus FPAMPLSNLFANAVLRAQHLYLLAAETYKEFER 33
Rana FPQMSLSNLFTNAVIRAQHLHQMVADTYRDYER 33
Ambystoma AYPAAPLSSLFNHAVARARRLHQIAMDIYTDFEG 34
Cynops AFPGVSLTNLFNNAVIRAQHLHFLAADIYQEFER 34
Amia AYPSIPLYNLFTNAVIRAEHLLQLATDIYKDFER 34
Homosapiens AYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFL 94
Pan AYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFL 94
Canis AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93
Felis AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93
Equus AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQLL 93
Mustela AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQFL 93
Hippopotamus AYIPEGQRYS-IQNTQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93
Camelus TYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93
Ovis TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93
Lohi TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93
Bos TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93
Capra TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93
Bubalus TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93
Monodelphis TYIPEAQRHS-IQSTQTAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLSPVQFL 93
Crocodylus SYIPEEQRHS-NKNSQSAFCYSETIPAPTGKDDAQQKSDMELLRFSLVLVQSWLNPVQFL 93
Rana TYIPEDQRFS-NKHSYSVYCYSETIPAPTDKDNTHQKSDIELLRFSLLLLQSWMNPIQIV 93
Ambystoma TYISDEQRQS-SRIYQAAFCCSETIPAPTGKDDAQQRSDMELLRFSLTLIRSWLTPVQFL 93
Cynops TYIPNEQRHT-SRNSQTAFCCSETIPAPTGKDDAQQRSDIELLRFSLTLIRSWLTPVQAL 93
Amia TYVPDEQRQS-SKSSPLAGCYSESIPAPTGKDEAQQRSDVELLGFSFTLIQSWISPLQTL 93
:::.. *: : : * **:**:*:.:::::*:*::*** :*: *::**: *:* :
Homosapiens RSVFA-NSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHN 152
Pan RSVFA-NSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHN 152
Canis SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRS 151
Felis SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRGGQILKQTYDKFDTNLRS 151
Equus SRVFT-NSLVFGTSDR-VYEKLRDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRS 151
Mustela SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGPILKQTYDKFDTNLRS 151
Hippopotamus SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNMRS 151
Camelus SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGQILRQTYDKFDTNLRS 151
Ovis SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDRNMRS 151
Lohi SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRS 151
Bos SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRS 151
Capra SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRS 151
Bubalus SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDGTPRAGQILKRTYDKFDTNMRS 151
Monodelphis SRVFT-NSLVFGTSDR-VYEKLRDLEEGIQALMQELEDGSSRGGLVLKTTYDKFDTNLRS 151
Crocodylus SRVFT-NSLVFGTSDR-VFEKLRDLEEGIQALMRELEDGSHRGPQILKPTYEKFDINLRN 151
Rana NRVFG-NNQVFGNIDR-VYDRLRDLDEGLHILIRELDDGNVRNYGVLTFTYDKFDVNLRS 151
Ambystoma SSVLT-NSFVFGSSDK-VYERLKDLEEGIQTLIRELDDGSPRGSSLLKLTYDNFDANQRN 151
Cynops SNVFFPNSFVFGTSER-VYERLKDLEEGIQTLIKELDDGSPRGFSLLKLTYDGFDANQRN 152
Amia SRAFS-NSLVFGTSDR-IFEKLKDLEEGIMVLMRGLDEGNPRLLGAQTLTYEKFDINLRN 151
.: *. *:. : ::: *:**:**: :: *: * **. ** * .
Homosapiens DDALLKNYGLLYCFRKDMDKVETFLRIVQCR-SVEGSCGF 190
Pan DDALLKNYGLLYCFRKDMDKVETFLRIVQCR-SVEGSCGF 190
Canis DDAL------------------------------------ 155
Felis DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191
Equus DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191
Mustela DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191
Hippopotamus DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191
Camelus DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191
Ovis DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191
64
Lohi DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191
Bos DDALLKNYGLFSCFRKDLHKTETYLRVMKCRRFGEASCAF 191
Capra DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191
Bubalus DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191
Monodelphis DEALLKNYGLLSCFKKDLHKAETYLRVMECRRFVESSCAF 191
Crocodylus EDALLKNYGLLSCFKKDLHKVETYLKLMKCRRFGESNCSI 191
Rana EEGRAKNYGLLSCFKKDMHKVETYLKVMKCRRFVESNCTF 191
Ambystoma EDALFRNYGLLSCFKKDMHKVETYLKVMKCRRILENNCTI 191
Cynops EDALFRNYGLLSCFKKDMHKVETYLKVMKCRRMLDNNCTI 191
Amia DDALMKNYGLLACFKKDMHKVKTYLKVMKCRRFVESNCTL 191
: .
Figure 12.comparison of oGH with different species of class mammalia
The sequence of the growth hormone(GH) gene or GH cDNA of Cetartiodactyla species
were downloaded from GeneBank . The Clustal W 1.81 was employed to align all the sequences
with the default option .Multiple amino acid sequence alignment of various animals (shown in
Fig. 13) was done by MUSCLE software. The amino acid sequence of growth hormone of
following species was downloaded [(Homo sapiens, accession number; AAT11509), (Pan
troglodytes, Accession number; AAL72284), (Canislupus, Accessionnumber; CAA80601),
(Felis, Accessionnumber; NP_001009337), (Hippopotamus amphibius, Accession number;
NP_001009337), (Equus caballus, Accession number; AAA21027), (Mustela vison, Accession
number; CAA42448), (Camelus bactrianus, Accession number; CAE01391),(Ovis aries,
Accession number; BAE66634), (Ovis aries (lohi), Accession number;AB24479), (Bos taurus,
Accession number; AAX0971344 ), (Capra hircus, Accession number; AAX35770), (Bubalus
bubalis, Accession number; CAA09679)]. The phylogenetic tree of multiple aligned amino acid
sequences of mammals was plotted by using neighbor joining method on
http://www.phylogeny.fr/ as shown in Fig. 13.
3.1.3.2 Comparison of oGH gene at Nucleotide level
The alignment of these three locally isolated growth hormones at nucleotide level reveal that
ovine growth hormone of Lohi is different at 16 points with locally isolated growth hormone of
Ovis aries some of them are important variation as shown by difference at amino acid level
while variation with cGH is just on 3 points and it is silent variation as shown in Fig.13.
65
ovine GCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGCTGTGCTCCGGGCTCAGCAC 60
caprine GCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGCTGTGCTCCGGGCTCAGCAC 60
bubaline GCCTTCCCAGCCATGTCCTTGTCCAGCCTGTTTGCCAACGCTGTGCTCCGGGCTCAGCAC 60
************************ ***********************************
ovine CTGCATCAACTGGCTGCTGACACCTTCAAAGAGTTTGAGCGCACCTACATCCCGGAGGGA 120
caprine CTGCATCAACTGGCTGCTGACACCTTCAAAGAGTTTGAGCGCACCTACATCCCGGAGGGA 120
bubaline CTGCATCAGCTGGCTGCTGACACCTTCAAAGAGTTTGAACGCACCTACATCCCGGAGGGA 120
******** ***************************** *********************
ovine CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTTCTCCGAAACCATCCCAGCC 180
caprine CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTTCTCTGAAACCATCCCGGCC 180
bubaline CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTTCTCCGAAACCATCCCGGCC 180
******************************************** *********** ***
ovine CCCACGGGCAAGAATGAGGCCCAGCAGAAATCAGACTTGGAGCTGCTTCGCATCTCACTG 240
caprine CCCACGGGCAAGAATGAGGCCCAGCAGAAATCAGACTTGGAGCTGCTTCGCATCTCACTG 240
bubaline CCCACAGGCAAGAACGAGGCCCAGCAGAAATCGGACTTGGAGCTGCTTCGCATCTCACTG 240
***** ******** ***************** ***************************
ovine CTCCTTATCCAGTCGTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGC 300
caprine CTCCTTATCCAGTCGTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGC 300
bubaline CTCCTCATCCAGTCGTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGC 300
***** ******************************************************
ovine CTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAAGGACCTGGAGGAAGGCATC 360
caprine CTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAAGGACCTGGAGGAAGGCATC 360
bubaline TTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAAGGACCTGGAGGAAGGCATC 360
***********************************************************
ovine CTGGCCCTGATGCGGGAGCTGGAAGATGTTACCCCCCGGGCTGGGCAGATCCTCAAGCAG 420
caprine CTGGCCCTGATGCGGGAGCTGGAAGATGTTACCCCCCGGGCTGGGCAGATCCTCAAGCAG 420
bubaline CTGGCCCTGATGCGGGAGCTGGAAGACGGCACCCCCCGGGCTGGGCAGATCCTCAAGCGG 420
************************** * **************************** *
ovine ACCTATGACAAATTTGACACAAACATGCGCAGTGATGATGCGCTGCTCAAGAACTACGGT 480
caprine ACCTATGACAAATTTGACACAAACATGCGCAGTGACGACGCGCTGCTCAAGAACTACGGT 480
bubaline ACCTATGACAAATTTGACACAAACATGCGCAGTGACGACGCGCTGCTCAAGAACTACGGT 480
*********************************** ** *********************
ovine CTGCTCTCCTGCTTCCGGAAGGACCTGCACAAGACGGAGACGTACCTGAGGGTCATGAAG 540
caprine CTGCTCTCCTGCTTCCGGAAGGACCTGCACAAGACGGAGACGTACCTGAGGGTCATGAAG 540
bubaline CTGCTCTCCTGCTTCCGGAAGGACCTGCACAAAACGGAGACGTACCTGCGGGTCATGAAG 540
******************************** *************** ***********
ovine TGTCGCCGCTTCGGGGAGGCCAGCTGTGCCTTCTAG 576
caprine TGTCGCCGCTTCGGGGAGGCCAGCTGTGCCTTCTAG 576
bubaline TGCCGCCGCTTCGGGGAGGCCAGCTGTGCCTTCTAG 576
** *********************************
Figure 13.nucleotide sequuence alignmen
Nucleotide sequence alignment of Lohi growth hormone with locally isolated growth hormone of caprine and bubalus
bubalis. The variation is shown in form of blue color for bubalus bubalis, green for LOHI and pink for caprine.
The sequence analysis of Ovine growth hormone isolated from local ovine breed Lohi suggests
that it is in complete accordance with the already published sequence of Ovine growth hormone.
The comparison also shows that it has four to five nucleotide variations which do not affect the
amino acid sequence. The length of introns and exons of growth hormone gene of local ovine
breed (Lohi) when compared with other members of the family Mammalia showed slight
variation in the length of initial intron shown in Fig.12.
66
3.1.4 Secondary structure analysis of oGH
The secondary structure of ovine growth hormone was obtained by MINNOU server.It
showed that around 60 % of amino acid residues involved in the formation of α-helices (Fig.14).
Amongst the four key α-helices, N- and C-terminal helices were longer than the other two
helices. Most of the amino acid residues involved in helix formation was hydrophobic.
Figure 14ovine growth hormone. secondary structure
The secondary structure of ovine growth hormone predicted by chou-fasman rule. H is alpha helix, E is beta sheet and C
is coil
67
3.1.5 Hydropathy profile of oGH
The hydropathy profile of ovine growth hormone was analyzed by using kyte Doolittle
hydropathy plot . It showed that 60 % of the growth hormone is hydrophobic, while the rest were
those containing either a charged or an uncharged polar side chain as shown in Fig.15.
Figure 15.The hydropathy plot of oGH. The hydropathy plot of OST using window size 9, each peak above the central line shows that part of the hormone is
hydrophobic.
68
3.1.6 Three Dimensional structure of oGH
Further, predicted three-dimensional (3-D) structure of oGH showed the presence of four
α-helices, anti-parallel and tightly packed to form a four-helix bundle a structure (Fig.16). This
peculiar structure is very similar to known structures of ovine, caprine, bovine, porcine and
human STs.
Figure 16.3D structure of ovine growth hormone. 3D structure of ovine growth hormone was predicted by using Phyre server and taking human growth hormone as a basic
reference source. 1, 2, 3 & 4 depicts the helix and N & C terminals.
N
C
69
3.2 cDNA synthesis , cloning and Periplasmic Expression of oGH
3.2.1 Isolation and purity of total RNA
Total RNA was extracted from the anterior pituitary tissue of local ovine breed (Lohi) by
guanidium thiocyanate chloroform extraction method (Chomezynski and Sacchi, 1987). The
concentration of extracted RNA was found to be 1.94µg/mg of the pituitary tissue when
measured at λ260. The A260/A280 ratio for isolated RNA was found to be 1.82 (Fig.17), which
indicates sufficiently good purity of extracted RNA (Sambrook and Russell, 2001).
Figure 17.Absorption spectra of extracted RNA. Absorption spectra of extracted RNA from pituitary of local ovine "Lohi" at λ220 - λ300.
70
The extracted RNA was further analyzed on denaturing 1.2% formaldehyde agarose gel. Two
prominent bands of 18S and 28S ribosomal RNA could be seen on the gel indicating that the
extracted RNA is intact and has suffered no major degradations (Fig. 18).
Figure 18.Formaldehyde agarose gel of RNA Formaldehyde agarose gel of total RNA isolated from ovine pituitary tissue.
3.2.2 RT-PCR amplification of cDNA
The purified RNA was subjected to reverse transcription in order to get cDNA which was
amplified by PCR using gene specific primers as described in Materials and Methods. The RT-
PCR yielded a single product of approximately 0.6 kb which was expected size of OaST gene
(Fig. 19)
Figure 19.Analysis of the RT-PCR. Analysis of the RT-PCR amplified product resolved on 1% agarose gel. Lane M,
1kb DNA ladder used as marker; lane , 2, 3, 4 & 5 ~0.6kb amplified PCR products.
71
3.2.3 T/A cloning of oGH
The gel purified PCR products were cloned by using InsTAcloneTM PCR Product Cloning kit.
pTZ57R/T vector was designed for cloning of Taq DNA polymerase amplified PCR products, as the
enzyme adds up extra adenine residues to the 3’end of the PCR products. These single stranded A-
overhangs are required for the base pairing with the 5’-T overhangs in the pTZ57R/T vectors .(Fig.
20).
Figure 20.Restriction map of pTZ57R/T cloning vector .
The amplified oGH cDNA was purified, T/A cloned in pTZ57R/T vector and recombinant
plasmid (pTZ-oGH) thus obtained was used to transform in E. coli strain DH5α. The
recombinant clones were identified by blue/white screening, as vector is genetically marked
with LacZ gene. Several white colonies along with blue colonies appeared on LB-agar plates
supplemented with ampicillin, X-gal and IPTG. These white colonies were further analyzed by
colony PCR using gene specific primers. Six white colonies were picked up from the plate and
five of them gave positive result while one proved as false colony as shown in Fig.22. The four
72
recombinant pTZ-oGH colonies were selected for plasmid preparation and then subjected to
sequence analysis.
Figure 21.Analysis of colony PCR. Analysis of positive transformants by colony PCR. lane 1, DNA marker; lane 2, 3, 5, 6, 7 products of different colony
PCR reactions; Lane 4, colony no 4 indicates negative clone
3.3 Expression of poGH
3.3.1 Restriction analysis of pTZ-oGH
The single colony of recombinant pTZ-oGH construct confirmed by sequencing was used for the
further analysis. For this purpose the pTZ-oGH was amplified and double digested by Nde I and
BamH I restriction enzymes (Fig.22).
Figure 22.Double digestion of pTZ-oGH-1..
Double digestion of pTZ-oGH-1.M, DNA size markers;Lane1,plasmid pTZ-oGH-1after double digestion with
NdeI/BamHIrestriction endonucleases
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3.3.2 Cloning in pET22 b
The amplified product was gel purified and cloned between Nde I and BamH I sites of pET-
22b(+) using restriction enzyme digestion and ligase mediated cloning. This generated an
expression plasmid designated as, poGH-1 (Fig.23.). The construct contained the native
sequence of OST mRNA and was predicted to encode a 191 amino acid oGH (MW ~22 kDa) in
frame with the translational initiator codon under the control of T7lac promoter.
Figure 23.Construction of recombinant plasmid poGH-1. Construction of recombinant plasmid poGH-1 by cloning a 0.6 kb long oGH cDNA in pET-22b(+) expression vector.
pT7lac, T7lac promoter; rbs, ribosome binding site; ori, origin of replication; fi ori, F1 origin of replication; lacI, Lac
repressor gene; ampr, ampicillin resistance gene.
3.3.3 colony PCR of poGH
poGH-expression plasmid was transformed into E. coli DH5α (cloning host) for vector
propagation and clone selection. Efficiency of transformation reaction was very high; almost 100
% of the screened colonies were positive for the insert as confirmed by colony PCR and/or
restriction digestion. The results obtained from a representative plasmid are presented as Fig. 25.
When resolved on 1 % agarose gel, colony PCR amplification products yielded a single band of
~0.6 kb length (Fig.24)
74
Figure 24.Colony PCR of poGH-1 Colony PCR,lane M,marker;lane 1,2,,3,4 recombinant clone s of poGH-1
In order to confirm the in frame cloning of oGH in poGH-1 construct the clone was double
digested again with NdeI and BamHI and hence showed 573kb band of oGHand 5.4kb band of
pET expression vector when analyzed on 1% agarose gel (Fig.25) which showed successful
cloning of oGH in pET expression vector. Four colonies were picked and spotted on the plate
.The desired oGH band of approximately 0.6kb was confirmed with the colony PCR of poGH -1
clone.
Figure 25.Double digestion of poGH-1.
M, DNA size markers;Lane1,plasmid poGH-1 after double digestion with NdeI/BamHIrestriction endonucleases
75
3.3.4 Shake flask fermentation of poGH-1 construct
The E. coli BL21 were transformed with recombinant plasmid poGH-1. The transformants in set
of four were grown in LB medium at 37 ̊C, induced with 0.2mM IPTG when growth of the cell
reached 0.6 at OD600. The cells were collected from each transformants and were treated with
lysis buffer to check total cell protein as explained in material and methods. The SDS-PAGE
analysis of total cell protein of poGH-1 construct revealed no visible expression (Fig. 26)
Figure 26.SDS-PAGE analysis of poGH-1 expression.. SDS-PAGE analyses of poGH-01 plasmid ..M is commercially available bovine growth hormone used as a marker .lane
1,induce pOaST-1 plasmids total cell protein after 4 hrs of 0.5mM induction of IPTG in LB medium
3.4 Periplasmic expression of oGH
The very low levels of expression of GH gene have been treated with several strategies
(secondary structure changes, bicistrone methods, changes at N terminal of GH gene and lot
more). We tried to use leader sequence of pET vector in order to get expression of oGH gene.
For this purpose a plasmid was constructed with oGH gene at NcoI and BamHI sites of pET 22b
vector so that to attach leader sequence at N terminal site of oGH gene as shown in Fig. 27.
76
Figure 27.construction of poGH-2 construct
3.4.1 Expression of poGH-2
When the leader sequence of pET22b was used in reference construct (poGH-1) and
expressed in E. coli BL21, the construct (poGH-2) showed visible expression of oGH but at
25kDa. The expected size of ovine growth hormone calculated from its amino acid sequence is
approximately 22kDa, while the band appeared on SDS-PAGE showed extra 3kDa.
Figure 28.SDS-PAGE analysis of poGh-2 expression in LB medium. SDS PAGE analysis of poGH-2 expression in LB medium.. Lane 1, induced post-2colony no1; lane 2, induced poGH-2colony no2;
lane 3, induced poGH-2colony no1; Lane 4, uninduced poGH-2colony no2; Lane 5, induced poGH -2 colony no2; lane M,Marker.
pelB leader
77
3.4.2 Effect of different factors on the expression of oGH
3.4.2.1 Effect of ZnCl2
The ZnCl2 is being used to reduce the proteolytic degradation of periplasmic protein.The
different combination of ZnCl2 were used in the pre-culture in order to see its effect on the
production of recombinant oGH protein . For this purpose 0.1mM,0.5mM,1mM,5mM , 10mM
and 50mM concentrations of ZnCl2 were used and it was observed that by increasing the amount
of ZnCl2 from 0.1 to 1mM the cell growth enhances while increasing it upto 50 mM reduces
the cell growth. The best selected concentration was observed at 0.5 mM as shown in graph.
Figure 29.effect of ZnCl2. A graph representing the effect of ZnCl2 on the protein content of expressed cells in LBmedium
3.4.2.2 Effect of IPTG concentration
The IPTG as an inducer was used in the shake flask fermentation of roGH-2 construct
with above optimized conditions. a study of the effect of IPTG concentration (10uM to 1mM)
showed that beyond 20uM there was no increase in the expression levels (Fig. 30).We observed
a constant expression of roGH by adding IPTG 20uM,40,60,80uM,0.1mM,0.5,1mM A
progressive decrease in expression levels was observed below 20uM IPTG concentration as
shown in fig.
78
( a ) ( b )
Figure 30,SDS-PAGE analysis of effect of IPTG. SDS-PAGE analysis of effect of IPTG concentration on the expression of oGH-2(a).:lane M ;marker,lane C,control pET22b ,lane
U,uninduced,lane 1-&,IPTG concn 20,40,60,80,100,1000 and 2000uM (b) effect of IPTG concentrattion less than 10uM on the
expression;lane M,markaer,U uninduced,lane 1-3,IPTG concn 2,5 and 10 uM respectively
3.4.2.3 Effect of Ecoli Strain on expression of ovine growth hormone
OGH1-pET22b construct was transformed into BL21 DE3 and P Lysis strains of E.coli. The
protein expression was observed on 15% SDS-PAGE.It was observed that BL21-DE3 results in
better expression of roGH as shown in fig.We observed the subcellular fractions in both strains
as well .The cytoplasmic fraction in the case of Plysis appeared to have more roGH as compared
to BL21 DE3,but the periplasmic fraction in both strains appeared to be same 10% as shown in
fig 31.
Figure 31.Effect of E.Coli strains on the periplasmic expression of poGH-2. SDS-PAGE analysis of expressed protein of poGH-2(lane1-4,expression in BL21DE3 strain & lane6-8 exspression in PLysis) Lane 1, sonicated sample for cytoplasmic fraction cf; lane 2,shock fluid for periplasmic fraction pf; lane 3,induced poGH-2 ; lane 4,un- induced
pET 22b; lane 5,SDS protein marker, lane 6, cytoplasmic fraction cf , lane7 shock fluid for periplasmic fraction pf, lane,8 induced
poGH-2,
79
3.4.2.4 Optimization of somotic shock conditions
The construct poGH-2 was used for further analysis.As this constitute PelB leader sequence
of PET22b which translocate recombinant protein into the periplasmic space. The destination of
recombinant protein was checked by the analysis of periplasmic and cytoplasmic fractions. For
this purpose 10ml sample was taken after the above optimized fermentation conditions in LB
medium. Already optimized osmotic conditions for the release of oGH into the periplasmic space
were used. I used 3ml each for the each osmotic shock procedure in seperate falcon tubes. These
were proceeded for subcellular fractionations as explained in the material and methods. The
protein content in the periplasmic and cytoplasmic samples was analyzed by Bradford method
explained earlier.The samples were loaded on 15% SDS-PAGE and analysed the result(fig 32 ).
The graphical representation shown (fig 32 a ,b and c) that we obtained the best release of oGH
in osmotic shock when treated with our optimized freeze thaw method as the release was
3.12ug/ml as compared to other protocols where it was achieved much lower.
Figure 32. graphical representation of effect of different osmotic shock conditions on oGH.
.
The SDS-PAGE analysis of above three osmotic shock procedures showed the clear difference
on the release of oGH in periplasmic fraction.Fig a and b showed negligible oGH when treated
80
with osmotic shock conditions stated in graphs a and b (fig 33 ) while the periplasmic and
cytoplasmic fraction in freeze thaw method showed a visible 20% molecular weight band.
Figure 33.SDS-PAGE analysis of subcellular fractions of poGH. 2 SDS-PAGE analysis of cytoplasmic and periplasmic fraction of roGH by using different osmotic shock methods .Cf is
cytoplasmic fraction,Pf is periplasmic fraction and Tcp is total cell protein.(a) osmotic shock conditions described
by(Koshland ,1980) and its effect on release of oGH( b ) osmotic shock conditions narrated by( Ramakrishnan et al., 2010) and its effect on release of oGH( c ) Freeze thaw conditions explained by (Barth et al., 2000) and its effect on
release of oGH.
3.4.2.5 Effect of Glycerol
We studied the effect of glycerol addition in the growth medium and in the osmotic shock
procedure. In LB medium the addition of glycerol enhanced the expression level of oGH from
18% to 22% when observed a on SDS-PAGE .
Figure 34. SDS-PAGE analysis of poGH-2 in LBmodified medium. Lane 1, M; lane 2,uninduced poGH-2; lane 3 induced poGH-2 after 3 hrs of induction, Lane 4, induced poGH-2 after 4
hrs of induction ;lane 5, uninduced poGH-2;lane 6, induced poGH-2 after 6 hrs of induction ;lane 7, induced poGH-2
after 8 hrs of induction
81
As freeze thaw method resulted in best release of oGH (20%) as shown (fig 32& 33). We further
optimized its condition in order to enhance the roGH release in periplasmic space. For this
purpose we used 9 different falcon tubes with 5ml each sample of fermented cells in above stated
optimized conditions.The glycerol was added in a range of (10,15,20,25,30,35,40,45,50%).The
protein content after osmotic shock was calculated by Bradford method.It was observed that the
protein content was highest 110ug/ml when treated with 25% glycerol in freeze thaw
method.The release of oGh in the specific sample was analysed on SDS-PAge and observed
24%band as shown in fig 35.
Figure 35. effect of Glycerol. A graphical representation of the effect of glycerol on the release of oGH into the shock fluid
3.4.2 Purification of poGH-2
The oGHT band was also confirmed by western blot by using specific antibody. The gene linked
with pelB signal peptide is destined for the secretion into periplasmic space of E. coli. In order to
see the fate of this expressed oGHgene with an extra 3kDa, total cell protein was proceeded for
cytoplasmic and periplasmic fractions as explained in the materials and methods. The fractions
were analyzed on SDS-PAGE and showed 25kDa oGH gene band both in cytoplasmic and
periplasmic fraction. Fig. 36 a. The use of glycerol in the osmotic shock procedure enhanced the
release of oGH in shock fluid as shown in fig below .we recovered 24% roGH from the shock
fluid which was aunthenticated by western blot analysis ( fig 36 b )
82
( a ) ( b ) Figure 36.SDS-PAGE analysis of subcellular fractions of roGH-2& western blot analysis. (a)SDS-PAGe analysis of periplasmic and cytoplasmic fraction of roGH by using optimized freeze thaw method ,Tcp is total cell protein,Pf is periplasmic fraction,Cf is cytoplasmic fraction.(b) western blot analysis of roGH
83
3.4.3 FPLC chromatography
The 80% purified ovine growth hormone was further purified by anion exchange,Q sepharose
FPLC chromatography. For this purpose 2M NaCl2 gradient was established and the peak was
observed at 0.5M concentration .The tube were collected with absorbance at 280 attached with
FPLC equipment. The purified product resulted in single sharp peak as shown in figure 37.
Pooled fractions were run on 12 % SDS-PAGE to visualize the purified protein. Further, they were
dialyzed against 20 mM Tris-Cl (pH 8.3) to remove salt traces
resQ004( FAiza ) 001:10_UV1_280nm resQ004( FAiza ) 001:10_UV2_0nm resQ004( FAiza ) 001:10_UV3_0nm resQ004( FAiza ) 001:10_Conc resQ004( FAiza ) 001:10_Flow resQ004( FAiza ) 001:10_Fractions resQ004( FAiza ) 001:10_Inject resQ004( FAiza ) 001:10_Logbook
0
500
1000
1500
mAU
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 ml
A1 A2 A3 A4 Waste A1 Waste A2 A3 A4 A5 A6 A7 A8 A9 A10 A12 A14 B1 Waste
Figure 37.FPLC peak of purified oGH
84
3.5 Effect of (DsbA,ST-II & native oGH signal sequence ) on the expression &
secretion of oGH
On the basis of inefficiency of leader sequence to translocate exact size (22kDa)
recombinant ovine growth hormone, a new strategy of using different signal sequences in pET
22b expression vector was applied. For this purpose three different signal sequences (DsbA,
ST11, native signal sequence of ovine growth hormone gene) were introduced in pET22b
expression vector in place of pelB leader sequence as described in materials and methods
.
3.5.1 Primer designed for the constructs poGH-3,4 &5
The following primers were designed for the introduction of signal sequence prior to N
terminus of OaST gene.The reverse primer was same as previously used with BamH1 siteas
explained in materials and methods
Table 2.primers designed for the poGH-3,4 & 5 constructs
3.5.2 PCR amplification
The DNA purified previously was amplified with the primers as described above and got
the PCR products of approximately 0.6kb when observed on agarose gel as shown in (Fig.38)
primer Primer sequence Features
pF-2 CATATGCATGCCCCCGGACCTCCCTGCTCCTGGCTTTCA
oGH signal
sequence
pF-3 CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGT
TTAGCGCATCGGCGGCCTTCCCAGCCATGTCC
DsbA signal
sequence
pF-4 CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGT
TTAGCGCATCGGCGGCCTTCCCAGCCATGTCC ST-11,signal
sequence
85
Figure 38.Agarose gel analysis of PCR. .Agarose gel analysis of amplified PCR products of primers ,pF2,pF3,pF4. Lane M, DNA marker; lane 2 PCR product of
primer set 2; lane 3, PCR product of primer set 3; lane 4, PCR product of primer set 3
3.5.3 T/A cloning and construction of expression plasmid poGH-3,4,5
These PCR products were than T/A cloned to pTZ57RT vector as explained in material
and methods. The recombinant clones were confirmed by colony PCR as shown in (Fig.39)
Figure 39.Colony PCR analysis of poGH-3-4-5. Agarose gel analysis of Colony PCR of pTZoGH-3,4 and 5 .M,marker; Lane 1, pTZ-oGH-3construct; Lane 3, pTZ-oGH-4 construct; lane 4, pTZ-oGH-5 construct
The amplified products were gel purified and cloned between Nde I and BamH I sites of
pET-22b(+) using restriction enzyme digestion and ligase mediated cloning. This generated
series of expression plasmid designated as, pOaST-3,4 and 5 (Fig.40). The construct contained
86
the native sequence of oGH mRNA with the signal sequence of DsbA, native signal sequence of
ovine growth hormone and signal sequence of ST-II and was predicted to encode a 191 amino
acid oGH (MW ~22 kDa) in frame with the translational initiator codon under the control of
T7lac promoter.
Figure 40.construction of expression plasmic poGH-3,4&5
3.5.4 Transformation and selection of high expression strains
All poGH-series expression plasmids were transformed into E. coli DH5α (cloning host)
for vector propagation and clone selection. Efficiency of transformation reaction was very high;
almost 100 % of the screened colonies were positive for the insert as confirmed by colony PCR
and by restriction digestion.
Figure 41.colony PCR analysis. Colony PCR of recombinant clones. lane M, DNA marker, lane 1, poGH-3 construct, lane 2,poGH-4 construct;lane 3, poGH-5 construct.
87
The results obtained from a representative plasmid are presented (Fig. 34). When resolved
on 1 % agarose gel, colony PCR amplification products yielded a single band of ~0.6 kb length,
while restriction digestion products generated two bands corresponding to 5.5 kb vector and 0.6
kb insert DNA (Fig. 42). The data thus confirmed the presence of insert in all poGH-series
plasmids.
Figure 42.Double digestion of recombinant clones. Agarose gel analysis showing double digestion of recombinant clones, M, DNA marker, lane 2, uncut plasmid pET22b
with insert, , lane 3,4 & 5, double digested pOST-3,4,5 constructs respectively.
3.5.5 Expression of poGH-3,4 and 5
E. coli transformed with poGH-series vectors (poGH-3 to -5) were grown in LB-ampicillin
broth and induced with 0.5 mM IPTG at an OD600 of 0.6. After 4 hours of induction, equal
amounts of cells (based on OD600 values) were lysed and the protein expression was analyzed by
15% SDS-PAGE as shown in Fig. 43.
88
Figure 43.SDS-PAGE analysis of protein expression of construct poGH-3,4&5. SDS PAGE analysis of total cell proteins of poGHconstructs. lane 1, poGH-5; lane 2 uninduced poGHT-5; lane 3, poGH-3; lane 4,
poGH-4; M , protein marker.
3.5.5.1 Subcellular fractionation of poGH-3-5 constructs
The fate of the expressed oGH was analysed by subcellular fractionation of the fermented
cells.The scheme of subcellular fractionation was as explained in materials and method.To
devise an appropriate strategy for downstream processing, the relative distribution of the
expressed oGH was examined in soluble and insoluble fractions. Cells expressing oGH were
lysed and then centrifuged as described under materials and methods. The supernatant and pellet
fractions thus obtained were analyzed by 15 % SDS-PAGE. When the cells were lysed under
native conditions, virtually all the expressed oGH was found in the supernatant representing the
soluble fraction while no or very little traces were found associated with the pellet .The total
cells were analyzed for the periplasmic, cytoplasmic and membrane fractions of the cell as
explained in materials and method. The subcellular fractionation was proceeded as shown in
following flow chart.
89
Figure 44.Schematic representation of subcellular fractionation of cells.
The destination of the expressed roGH was analyzed by subcellular fractionation of the
bacterial cells of each poGH3-5construct. The scheme of subcellular fractionation as shown in
Fig. 44 was devised for downstream processing and relative distribution of the expressed roGH
in soluble and insoluble fractions. Cells expressing roGH in each construct (poGH3-5)was lysed,
centrifuged and then TCP, P (Periplasmic), C (Cytoplasmic) and MF (Membrane fraction) were
analyzed on 12% SDS-PAGE (Fig. 45a,b,c,d).
90
The results of subcellular fractions of all four constructs showed variable outcomes; in construct
poGH1, roGH was expressed at 25kD though expected molecular weight for GH was 22kD.
However, western blot analysis of the gel showed the authenticity of roGH (data not shown). It
was assessed that the additional 3 kD was due to attached signal peptide as the approximate
molecular weight of pelB leader sequence is ~3 kD. During the secretion process, this signal
sequence did not get cleaved from the roGH. The sub-cellular fractions of this construct showed
that half of the roGH was found in soluble form as in the periplasmic fraction while half was
detected in the cytoplasmic fraction and no trace was found in membrane fraction (Fig. 44a).
Similar results were observed for constructs poGH2 and poGH4 showing roGH of 25 kD with
the attached signal sequence.
However, construct poGH2 showed roGH protein in the cytoplasmic fraction while very little
amount was found in periplasmic fraction(Fig. 44b). Construct poGH4 showed no trace of roGH,
suggesting lack of expression and/or protein degradation(Fig. 44d).Importantly, the subcellular
fractionation of the construct poGH3 showed complete translocation of roGH into the inner
membrane (MF) of E. coli. Here the molecular weight of the expressed roGH was 22kD and was
in complete accordance with the molecular weight of the other GH reported (Paladini et al.,
1983). . Furthermore, it was observed that none or very minute traces of roGH were found in the
C or P fractions as shown in Fig. 44C. Construct poGH3 was used for optimization studies and
the roGH production.
91
Figure 45.SDS-PAGE analysis of subcellular fractionations of poGH-3,4 & 5 constructsSDS PAGE . 12% of sub-cellular fractionation of poGH1-4 constructs. Rectangle box showing expression in all the constructs. (A) SDS PAGE
analysis of subcellular fractions of poGH-5. Lane MF, Membrane fraction; lane C, Cytoplasmic fraction; lane P, Periplasmic fraction; lane TCP, Total cell protein. (B) SDS PAGE analysis of subcellular fractions of poGH-4. Lane C, Cytoplasmic fraction; lane P,
Periplasmic fraction; lane TCP, Total cell protein; lane MF, Membrane fraction. (C) SDS PAGE analysis of subcellular fractions of
poGH-3. Lane MF, Membrane fraction; lane C, Cytoplasmic fraction; lane P, Periplasmic fraction; lane TCP, Total cell protein. (D)
SDS PAGE analysis of subcellular fractions of poGH-2. Lane MF, Membrane fraction; lane C, Cytoplasmic fraction; lane TCP, Total cell protein; lane P, Periplasmic fraction; lane M, Protein marker.
3.5.6 Computational analysis of signal sequences of poGH-2,3,4& 5 constructs
In order to understand the reason of varying behavior of these four signal sequences. Each
signal sequence used in this study; DsbA ss ,ovine growth hormone signal sequence, STII and
pelB leader sequence were compared on the basis of probability of signal sequence by signalp3.0
server (results attached in appendix), hydropathy plot by kytedoolittle method and their
secondary structure by polyview prediction server. The data showed that all of them are good
structured signal sequences. However the hydropathies of these signal sequences gave variable
results as shown below (Fig 46).
92
Figure 46.Kytedoolittle analysis of hydrophobicity of all four signal sequences. (a) pET signal sequence (b) DsbA signal sequence (c) ST11 signal sequence (d) ovine growth hormone signal sequence.
Table 3.Hydropathies of poGHconstructs
Constructs
(signal
sequence)
Nucleotide Sequence (5'-3') Hydropa
thy
poGH2 (pelB) CATATGAAATACCTGCTGCCGACCGCTGCTG
CTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCG
ATGGCCATGGCCTTCCCAGCCATGTCC
1.157
poGH3 (DsbA) CATATGAAAAAGATTTGGCTGGCGCTGGCTG
GTTTAGTTTTAGCGTTTAGCGCATCGGCGGCC
TTCCCAGCCATGTCC
1.389
poGH4 (oGH) CATATGCATGCCCCCGGACCTCCCTGCTCCT
GGCTTTCA
1.840
poGH5 (STII) CATATGAAAAAGATTTGGCTGGCGCTGGCTG
GTTTAGTTTTAGCGTTTAGCGCATCGGCGGCC
TTCCCAGCCATGTCC
0.986
The above table shows that there is an optimal range of hydropathy value, above or below which
the signal sequence does not function properly .The secondry structure of these signal sequence
(DsbA, ST-11, pelB and ovine growth hormone signal sequence)were also analysed.They
93
showed that they have varying charges in their N terminal and C terminal regions. The presence
of Beta sheet especially in case of ST-11 signal sequence was a prominent difference among
these signal sequences.
Figure 47.Secondary structure analysisof poGH2,3,4&5.. Secondary structure of all four signal sequences. (a) pET signal sequence (b) DsbA signal sequence (c) ST11 signal sequence (d) ovine growth hormone signal sequence.
The construct with DsbA signal sequence (poGH-3) was chosen best among all four signal
sequences used in this study as it resulted in the accurate size (22 kDa) of recombinant ovine
growth hormone as compared to rest of the rest of the constructs.
94
3.6 Effect of medium composition on expression of poGH-3
3.6.1 Effect of LB,TB & M9NG medium on the expression of poGH-3
On the basis of our results the poGH-3 construct was selected for the further studies. The first
objective was to enhance the production of Soluble recombinant oGH. For this purpose we
studied 9 different medium compositions in 2 sets as listed in table.The first one constitue effect
of LB,M9NG and TB medium while second set constitute seven mediums based on different
carbon and nitrogen source(LB, LB modified, GM-1, GM-2, UM, TB, TBC) to analyze their
effect on the bacterial growth and expression of roGH .All the experiments were proceeded in
shake flask fermentation. The composition of these mediums were as follows ( Table .4)
Table 4.composition of different mediums used
Culture Medium Composition
TB 2.4% yeast extract, 1.2% trypton, 0.4% glycerol, 2.31%
KH2PO4, 12.54% K2HPO4
M9NG
0.5% NaCl, 1% NZ-amine Type A, 0.5% glycerol, 0.05%
glucose, 25mM NH4Cl, 25mM KH2PO4, 50mM Na2HPO4,
2mM MgSO4 and trace metals mix (0.004mM CaCl2,
0.0004mM each of CuCl2, NiCl2, Na2MoO4, H3BO3, 0.002mM each of ZnSO4, MnCl2 and 0.01mM FeCl3)
LB 1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, pH 7.2
LB-1 1% Tryptone, 0.5% Yeast extract, 1% NaCl, pH 6.8
GM-1 0.1% (NH4)2SO4, 1% Tryptone, 0.5% Yeast extract, 0.05%
Glucose, 0.5% Glycerol, 0.05M KH2PO4 dibasic, pH 7.6
GM-2 0.1% (NH4)2SO4, 1% Tryptone, 0.5% Yeast extract, 0.05% Glucose, 1% Glycerol, 0.07M KH2PO4 dibasic, 0.1M KH2PO4
monobasic, pH 7.0
UM 0.1% (NH4)2SO4, 1% Urea, 0.5% Yeast extract, 0.05% Glucose, 1% Glycerol, 0.1M KH2PO4 dibasic, 0.1M KH2PO4
monobasic, pH 7.0
TBC 2.4% Yeast extract, 1.2% Tryptone, 0.4% Glycerol, 2.31% KH2PO4, 12.54% K2HPO4, 0.5mM Mannitol,4% NaCl and
0.5M Glycylglycine, ZnCl2 0.5mM
We proceeded these experiments in (2 sets 1;LB,TB & M9NG and second set LB-1GM-1GM-
2,UM &TBC on roGH production.The construct poGH3 transformed in BL21 Codon Plus (DE3)
95
RIPL cells was grown in First set ( TB, LB, and M9NG media ) for enhanced production of
roGH. The post-induction cell growth was monitored for up to 14hrs in LB, TB and M9NG
media. The (250ml) fermentations were carried out at 37°C, induction by 1mM IPTG in the
logarithmic phase and aeration of 5 times in 1L Erlenmayer shake flask. All fermentations were
performed at least in triplicates and the results presented were the averages. The cells were
harvested at pre and at 2hrs post-induction for each fermentation. All the total cellular protein
samples were processed for analysis in 12% SDS-PAGE and the expression in each medium was
observed as shown in Fig. 47a, b & c. It was found that expression level of roGH was enhanced
up to 18% in TB medium while in LB and M9NG media 10% expression.The growth pattern in
each medium was observed to be different as in LB, M9NG and TB media the maximum cell
growth reached up to OD600 1.2, 2.3 and 5.6, respectively (Fig. 48 d). Moreover, it was
observed that in TB medium, 65.3mg/L of roGH was obtained as compared to 13mg/l in LB and
16mg/l in M9NG (Table 2).
Figure 48.SDS-PAGE analysis and graphical representation of effect of medium on poGH-3 SDS PAGE (12%) showing effect of different media composition (TB, LB, M9NG) on poGH3 construct. (A) SDS PAGE analysis of poGH3 expression in TB medium. Lane M, Protein marker; lane U, un-induced poGH3 construct; lane 1, induced poGH3 sample at
10hrs post induction; lane 2, induced poGH3 sample at 12hrs post induction; lane 3, induced poGH3 sample at 14hrs post induction.
(B) SDS PAGE analysis of poGH3 expression in LB medium. Lane 1, induced poGH3 sample at 12hrs post induction; lane 2, induced
poGH3 sample at 14hrs post induction. (C) SDS PAGE analysis of poGH3 expressed in M9NG medium. Lane 1, induced poGH3 sample at 12hrs post induction; lane 2, induced poGH3 sample at 14hrs post induction. (D) Effect of OD600 on cell growth (hrs) of
poGH3 construct fermented in TB, LB and M9NG media.
96
Table 5.Effect of medium composition on production of oGH
Medium Maximum
OD600
Dry cell mass
(g/L)
Total cell
proteina
(mg/L)
roGH
(%age of total
protein)
roGH
(mg/L)
TB 5.6 2.7 390 18 65.3
LB 1.2 1.3 135 10 13
M9NG 2.3 1.6 167 10 16
Protein concentration was determined by absorbance measurements at A280.
These results suggested further optimization of fermentation conditions in TB medium such as
the effect of lowering temperature, concentration of IPTG and induction time in cell growth
cycle.
3.6. 2 Effect of temperature on poGH3 construct
As periplasmic protein processing occurs better at low temperature as stated by Novagen;
variable range of temperatures i.e. 20, 25, 28, 30, 35 and 370 C in shake flask cultures were
applied. It was observed that cell growth was maximum when fermentation was carried out at
25ºC, it reached up to OD600 5.6 while at 28ºC and 20ºC final OD600 reached up to 5 and 4.8,
respectively suggesting that the optimum temperature is between these two ranges. However,
fermentation at elevated temperatures of 30ºC, 35ºC and 37ºC resulted in reduced cell growth
and recombinant protein with OD600 of 4.1, 3.6 and 2.8 respectively and almost half in the case
of 37ºC grown culture. In conclusion, the best soluble expression of roGH was obtained at 25ºC as
shown in (Fig. 48,A).
3.6.3 Effect of induction time and IPTG concentration on poGH3 construct
The expression of soluble recombinant proteins enhances with lowering amount of inducer
(Novagen) and in the current study, IPTG concentrations ranging from 10μM to 1mM were used
for expression level of roGH and analysed in 12% SDS-PAGE. The roGH was best expressed
with 18% protein in total cell protein at 20μM IPTG while at 1mM IPTG, 14% expression was
observed (Fig. 49 B)The induction time with 20μM. IPTG was also studied .It was found that
97
induction at absorbance 0.5, 1.0 and 1.5, resulted in maximum cell growth of 2.8, 3.5 and 4.2
respectively at OD600. Induction at OD600 2.0 resulted in the best maximum cell growth i.e. 5.6
as shown in Fig. 49C. The induction at later stages like 2.5, 3.0, 3.5 and 4.0 resulted in decrease
cell growth which eventually turned at OD600 1.8 (Fig. 49C). These results showed that
induction time is best in the initial log phase of the cell cycle.
Figure 49.effect of temperature,induction time and IPTG concn. on poGH-3. Graphs showing the cell growth of poGH3 construct in TB medium at variable temperatures, induction time and IPTG concentration.
(A) Effect of temperature on cell growth of poGH3 construct fermented in terrific broth medium. (B) Effect of IPTG concentration on
cell growth of poGH3 construct fermented in terrific broth medium. (C) Effect of IPTG induction time on cell growth of poGH3
construct fermented in terrific broth medium.
3.7 Enhanced production of roGH
After achieving above optimized expression we further studied more medium inorder to
enhance the soluble production of roGH.There was still need to enhance the yield of this soluble
growth hormone. For this purpose we used combination of seven mediums based on different
carbon and nitrogen source(LB, LB modified, GM-1, GM-2, UM, and, TBC) to analyze their
effect on the bacterial growth and expression of roGH .All the experiments were proceeded in
shake flask fermentation. The composition of these mediums were as follows ( Table .4)For this
98
purpose all the mediums were inoculated with roGH as explained in materials and methods. The
samples were taken after every 2 hrs from each medium flask until it reached the final OD600.
The results were analyzed on 12% SDS-PAGE (data not shown). The graphical representation of
results showed that TBC resulted in the best bacterial growth of 10.4 while rest of the mediums
reached up to maximum 5.6 (Figure 50). In TBC medium we followed the same concentrations
of all the medium components as stated by (Barth et al, 2000) From this it was decided to study
the effect of different components on TBC medium for the enhanced production of roGH and on
this basis following parameters were studied; effect of different compatible solutes on the
bacterial growth, chemical chaperon for the stability of recombinant proteins, temperature,
inducer concentration and the time of induction. In order to understand the behavior of the
compatible solute, constant concentration of NaCl (4%) was used.
Figure 50. Growth of poGH-3 in different medium.
The T.B medium supplemented with compatible solute was used for the further analysis. For this
purpose all the variables which can effect the production and expression of recombinant OaST
were analysed. For this purpose the effect of ZnCl2, temperature, induction time, effect of IPTG
or lactose as inducer and variable combinations of compatible solute.
99
3.7.1 Effect of compatible solute on the expression of poGH-3 construct
As per our results (Fig.50) we decided to use T.B with varying combinations of compatible
solute in order to further enhance the production of recombinant oGH. The compatible solute
which was previously used was namely ( glycerol, sorbitol, glycine betaine, and hydroxyectoine)
during the production phase. In this study we have used 2 different sets (Glycylglycine ,glycine
betaine) and (sorbitol and Mannitol) of compatible solutes in production phase of recombinant
ovine growth hormone construct poGH-3.
3.7.1.1 Optimization of soluble roGH expression using compatible solutes(Glycylglycine ,
glycine betaine,sorbitol and Mannitol
In the present work we used different concentrations of each compatible solutes ( sorbitol,
mannitol, glycylglycine and glycine betaine) and compared their effect on the final absorbance
of the growing culture. It was observed that all these components increase the final OD of the
growing culture. We found that glycylglycine results in higher final density of growing culture
as compared to glycine betaine and so mannitol gives the better results as compared to sorbitol as
shown in graphs. We further studied the effect of glycylglycine and mannitol on the soluble
expression of roGH. The concentration of Glycylglycine effects the cell growth as by increasing
the concentration of Glycylglycine from 10 to 50mM. The cell growth also reached at the
maximum OD600 of 13.0. Mannitol also enhanced the cell growth and found the maximum
growth at 13.5 with optimized concentration of 0.6M as shown in Table 6. The results were also
observed on 12%SDS-PAGE separately for each of them (data not shown) and graphically
summarized in (Figure 51).
100
Table 6. Effect of compatible solutes inTB medium on the growth of poGH-3
The optimized concentration of Glycylglycine (50mM) and Mannitol (0.6M) enhanced the
percentage expression of roGH. The subcellular fractionation [periplasmic (P), cytoplasmic (C),
membrane fraction (MF)] of recombinant cells grown in above optimized conditions of TB were
proceeded as explained in materials and methods and were analyzed on 12% SDS-PAGE (Figure
52 a, b, c ). Figure 51 (a) shows the expression of roGH in TB medium supplemented with no
glycylglycine and Mannitol .we observed a good expression of roGH in total cell protein but we
could nt get the good yield of soluble roGH from membrane fraction . As we found some part of
the recombinant protein in periplasmic fraction and very diminished in the membrane fraction
while didn't receive any protein in the cytoplasmic fraction.
Figure 51.Graphical representation of the effect of 2 sets of compatible solutes on the growth of poGH-3 in TB medium
Glycylglycine Glycine betaine Mannitol Sorbitol
Conc(mM) OD600 14hrs post-
induction
Conc.(mM) OD600 14hrs post-
induction
Conc. M OD600 14hrs post-
induction
Conc. M OD600 14hrs post-
induction
10 8.2 10 6.8 0.1 7.1 0.1 7.2 20 9.3 20 7.4 0.2 8.4 0.2 8.2 30 10 30 8.7 0.3 10.1 0.3 8.9 40 11.5 40 9.2 0.4 11.9 0.4 9.8 50 13 50 9.4 0.5 12.6 0.5 10.7 60 12.6 60 9.8 0.6 13.5 0.6 11.5 70 11.8 70 10.2 0.7 13.1 0.7 12.4 80 11 80 9.8 0.8 12.7 0.8 12.6
101
However, Figure 52 (b) shows TB medium supplemented with Mannitol but no glycylglycine
and the total cell expression of roGH was observed to be good and at least half of the
recombinant protein in membrane fraction was found which was then easily solubilized by use of
40% acetonitrile. Moreover, some protein was also observed in periplasmic and cytoplasmic
fractions. While Figure 52 (c) shows TB medium supplemented with the optimized concentration
of glycylglycine and a very good expression of roGH as total cell protein was observed with
almost all of it transferred to the membrane fraction and no visible fraction was observed in
periplasmic or cytoplasmic fractions..On the basis of above observation we found that
glycylglycine and mannitol both effects the soluble expression of recombinant
( a ) ( b ) ( c )
Figure 52.effect of compatible solutes on the solublr expression of poGH-3 in TB medium . (a) shows the expression of roGH in TB medium supplemented with no glycylglycine and Mannitol.( b ) shows TB medium supplemented with Mannitol but no glycylglycine.(c) shows TB medium supplemented with the optimized concentration of
glycylglycine
3.7.2 Production of soluble roGH in TBC optimized medium
From the above observations we selected the optimized concentrations of Glycylglycine (50mM),
Mannitol (0.5M) and used it with optimized conditions of temperature (25ºC), IPTG (20μM),
induction at early logarithmic phase i.e. OD600 ~3.0 with 0.5mM of ZnCl2 and 4% NaCl in TB
medium for the batch of shake flask fermentation. Continued growth of bacterial cells was observed
up to 20 hrs post-induction. For expression studies 1ml sample was collected from the culture after
every 2hrs as explained in the materials and methods and samples were analyzed on 12% SDS-
PAGE. The optimized compatible solutes in TB medium enhanced the expression and solubility of
roGH with ~32% expression at 18hrs post-induction as shown in (Figure 53). The analysis on 15%
SDS-PAGE shows 32% expression of recombinant oGH.
102
Figure 53.SDS-PAGE analysis of optimized compatible solte in TB medium on expression of poGH-3. SDS PAGE analysis of poGH-3 expressed in compatible solute supported terrific broth medium. Lane M, prestained protein
marker;lane 2,un induced poGH-3;lane 3,induced poGH-3 after 4 hrs of induction ; lane 3,induced poGH-3 after 4 hrs of induction;
lane 4,induced poGH-3 after 8 hrs of induction; lane 5 ,induced poGH-3 after 10 hrs of induction; lane 6,induced poGH-3 after 12 hrs of induction ; lane 7,induced poGH-3 after 14hrs of induction ; lane 8,induced poGH-3 after 16 hrs of induction ; lane 9,induced
poGH-3 after 18 hrs of induction lane 10,induced poGH-3 after 20 hrs of induction .lane,11,western blot of recombinant oGH
3.7.2.1 Effect of temperature
As periplasmic protein process better at low temperature as stated by Novagen. The range
of temperatures 20̊C,25̊C,28̊C,30 ̊C and 37̊C in shake flask culture were applied .It was observed
that at low temperature conditions 20̊C, 25̊C and 28̊C the cell growth was very slow in the
beginnig till it reached upto 3.0 at O.D600 at which medium is supplemented with compatible
solute and 0.1mMIPTG ,the cell growth speeds up and continued growing after 16-20 hours of
induction. while by increasing the temperature from 28 to 30 and 37̊C the cell growth takes 5 hrs
to reach upto 3 at O.D600 and induction was given, the cell growth enhances quickly but it lasts
upto 8-9hours after induction. At lower temperature 25̊C the O.D600 of cell growth reached upto
17.2 while increasing the temperature 37̊C it reached upto 10.2 as shown in the graph (figure 54).
103
Figure 54.Effect of temperature.. Effect of temperature on cell growth of poGH-3 construct fermented in terrific broth medium supplemented with compatible solute
.
3.7.2.2 Effect of IPTG and Lactose as an inducer
The lactose and IPTG as an inducer were used seperately in the shake flask fermentation
of poGH-3 construct with above optimized conditions. a study of the effect of IPTG
concentration (10µm to 0.1mM) showed that beyond 20µM, there was no significance increase
in the expression levels (Fig. 55). A progressive decrease in expression levels was observed
following addition of IPTG till a final concentration of 0.5mM. Further increase in inducer
concentration up to 0.1 mM, however, did not result in any improvement in poGH-3 production.
20µM IPTG, therefore, was found optimal for maximal expression of poGH-3. Lactose-based
auto-induction strategy was employed to poGH-3 construct in terrific broth medium
supplemented with compatible solute. In this methodology, inducer (lactose) is added right at the
beginning of inoculation but the induction is completely repressed due to the presence of glucose
in the cultivation medium. Upon glucose depletion, induction and hence the production of
recombinant protein starts, automatically. This is advantageous, as unlike IPTG induction,
culture growth is not required to be monitored prior to induction.
104
Figure 55.effect of IPTG and lactose.. (a)SDS-PAGE analysis of poGH-3 construct in terrific broth medium with different concentrations of IPTG (b) SDS-PAGE analysis
of poGH-3 construct in terrific broth medium with lactose as an inducer
3.7.2.3 Effect of induction time
When induced at OD600 0.5-1.0,the cell growth enhanced quickly but it dropped after
reaching 4.3 at O.D600 . While by increasing the induction stage 1.5 ,2,2.5 and 3 the cell growth
enhances quickly after induction and it continued growing after 14-18 hrs of induction .As
compatible solutes has 4% NaCl2 which gives stress to the medium so its better if induction is
given in early logarithmic phase. The best induction of recombinant E. coli by IPTG, therefore,
was obtained at logarithmic phase, i.e., between OD600 of 2.5 to 3.
3.7.3 Subcellular fractionation of poGH-3 construct
The fate of the expressed was analysed by subcellular fractionation of the fermented
cells.The scheme of subcellular fractionation was as explained in materials and method .To
devise an appropriate strategy for downstream processing, the relative distribution of the
expressed oGH was examined in soluble and insoluble fractions. Cells expressing oGH were
lysed and then centrifuged as described under materials and methods. The supernatant and pellet
fractions thus obtained were analyzed by 15 % SDS-PAGE. When the cells were lysed under
native conditions, virtually all the expressed oGH was found in the supernatant representing the
105
soluble fraction while no or very little traces were found associated with the pellet .The total
cells were analyzed for the periplasmic, cytoplasmic and membrane fractions of the cell as
explained in materials and method. The subcellular fractionation was proceeded as shown in the
flow chart.The above strategy was used for the analysis of poGH-3 construct expressed under
optimized fermentation conditions of T.B medium supplemented with compatible solute. The
oGH was found in the membrane bounded form which was than solubilized with 40% of
acetonitrile. The recombianant oGH was also confirmed by western blot as shown in Fig.56.
Figure 56.Subcellular fractionation of poGH-3. SDS PAGE analysis of sub cellular fractionation of pOaST-3 construct expressed in T.B medium supplememnted with compatible
solute. lane 1, western blot of OaST. lane 2, soluble fraction . lane 3, membrane fraction..lane 4, periplasmic expression 5, cytoplasmic protein. lane, lane 6, total cell protein.
We studied the effect of sorbitol plus glycine betaine as compatible solute in the terrific broth on the
expression and final yield of recombinant oGH.we found that TB with above combination of
compatible solute produces a final yield of 189mg/L which is 3 times more than the yield obtained
from simple terrific broth medium.The results were extraordinarily high when we used Glycylglycine
plus mannitol in terrific broth medium.The glycylglycine and mannitol proved to be the better option
than sorbitol and glycine betaine as they resulted in final yield of 443mg/L which is about 9 times
higher when using TB simply without compatible solute.as shown in table
106
Table 7.Effect of compatible solute in TB medium on yield of soluble oGH
Medium Maximum
OD600
Dry cell
mass
(g/L)
Total cell
proteina
(mg/L)
roGH
(%age of
total
protein)
roGH
(mg/L)
TB
5.6
2.7
389
18
65.3
TB with
(Sorbitol and
Glycine
betaine)
10.4
4.1
1020
18
189
TB with (glycylglycine
and mannitol
17
6.4
1380
32
443
107
3.8 Effect of amino acid alterations in DsbA signal sequence on poGH
expression and secretion
The role of amino acid substitution in the tripartite structure of DsbA signal sequence in
targeting recombinant ovine growth hormone to the inner membrane of Escherichia coli cell was
investigated. Construct’s were designed by altering amino acids in the H, C and N domain of
DsbA signal sequence (DsbA ss) and for this purpose alanine, serine and lysine were replaced by
isoleucine, cysteine and arginine residues respectively.
3.8.1 PCR amplification and Cloning of pOaST varying constructs
We designed basically three types of mutant DsbA constructs
1. Mutant construct of DsbA with varying hydrophobic region
2. Mutant construct of DsbA with varying N terminal region
3. Mutant constructs of DsbA with varying C terminal region
The primer were designed for each construct as explained in materials and method. These
forward primers with one reverse primer PBGH3 (5`TAG GAT CCG CAA CTA GAA GGC
AGC 3`) were used for the PCR amplification. The wild-type growth hormone sequence in the
construct pTZoGH-1 was amplified using each of the forward and the reverse primers (Fp1-
Fp8)as shown in the (Table 7). The oGH gene was PCR amplified and analysed on 1% agarose
gel as shown in figure. All the primers resulted in 573bp oGH amplified product.
108
.Figure 57.PCR amplification of poGH-3-I-VIII. lane M; marker, lane 1-7 poGH-3 a-g constructs
These PCR products were then T/A cloned into pTZ57RT vector. The recombinant
colonies were chosen and confirmed by colony PCR analysis.
( a ) ( b )
Figure 58.colony PCR and double digestion of poGH-3-I-VIII.
(a) colony PCR of poGH-3a-g constructs.Lane 1-7 colony PCR of pOaST3a-g constructs.(b)Agarose gel analysis of double digested
T/A clone recombinant pTZ-oGH-3a-g
These (pTZoGH-3-I-VIII) series of recombinant plasmids were digested with NdeI and
BamHI (figure )and ligated at the NdeI/ BamHI site of pET22b thus generating a series of
recombinant plasmids ( poGH-3-I-VIII).
109
3.8.2 Construction of Expression plasmid poGH3-I-VIII
Figure 59.Construction of recombinant pET for poGH-3-I-VIII constructs .
3.8.3 Expression of poGH-3-I-VIII
oGH expression analysis. The recombinant plasmids of poGH-3 were transferred into E. coli
BL21 CodonPlus (DE3) RIPL strain. The transformants were grown in LB medium at 25 C and
induced with 20µM IPTG when OD600 reached 0.6. Lysates of E. coli cells of the various
transformants, after IPTG induction for 6 hrs, cells were treated with lysis buffer, and the total
cell protein was analyzed by SDS-PAGE. Equal amounts of cells (based on OD600 values) were
lysed from each culture and the protein expression was analyzed by SDS-PAGE. The expected
molecular mass of oGH is ~22 kDa, variations were observed both in mass and expression levels
pEToGH
NdeI
lacI
f1 origin
BamH1
pT7-lac
CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCG
CATATGAAAAAGATTTGGCTGATTCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCG
CATATGAAAAAGATTTGGCTGATTCTGATTGGTTTAGTTTTAGCGTTTAGCGCATCG
CATATGAAAAAGATTTGGCTGATTCTGATTGGTTTAGTTTTAATTTTTAGCATTTCG
CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAATTTTTAGCATTTCG
CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTTGTGCATGT
CATATGAGAAGGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCG
CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAATTTTTAGCGCATCG
oGH-3-I-VIII
oGH
110
when the total protein in lysates of the E. coli, transformed with different poGH constructs, were
compared (Fig. 60).
Figure 60.SDS-PAGE analysis of poGH-3-I-VIII constructs in LB medium. lane 1, oGH -3I; lane 2 , oGH-3II; lane3, oGH -3-IV; lane 4, OaST -3VI; lane 5, OaST -3VII, lane 6, OaST-3V; lane 7, OaST-3III; lane 8, OaST-3VIII; Pre-stained protein molecular markers, lane 9.
The new constructs with variations in DsbA signal peptide gave variable results. The
poGH-3 constructs III, V and VIII resulted in 25kDa of ovine growth hormone and rest of the
constructs resulted in 22kDa of band on 15% SDS-PAGE as shown in figure 60. Since, the
approximate size of 18 amino acids long DsbAss is ~2 kDa, the higher molecular mass of oGH
in these constructs is likely to be the result of incomplete processing of DsbAss.
In the case of poGH-3-IV construct, size of expressed oGH was 22 kDa but the expression level
was barely detectable on SDS-gel and therefore it was confirmed by western blot analysis (data
not shown).
3.8.4 The expression of DsbA ss constructs with substitution of alanine with
isoleucine in the H domain
The DsbAss has four Ala residues in its H- and near H-domain region, which are present at
position I, IV, IX and XI with respect to the signal peptidase cleavage site. In the present study
these alanine residues were replaced by Isoleucine in poGH-3-II to -VI constructs and the impact
of substitutions was observed on oGH expression and export in E. coli. When analysed by SDS-
1 2 3 4 5 6 7 8 9
25kDa 22kDa
kDa
250
130 100
70
55
35 25
15
111
PAGE, expression levels of oGH in these constructs ranged from undetectable (poGH-3-IV) to
up to 25 % (poGH-3-V) of the total E. coli cellular proteins. Variations in molecular mass were
also observed in the case of poGH-3-III and -V (~25 kDa) and poGH-3-II, -IV and -VI proteins
(~22 kDa).
In order to understand the behavior of expressed oGHs, the hydropathy index of each
DsbAss mutant construct was analyzed using the Swiss ExPASy Protparam tool (Table 2).
poGH-3-II and -V having the same hydropathy indices but variable molecular weight of
expressed oGH were the most interesting constructs. The subcellular fractionation of proteins
from the cells transformed with these constructs showed that in case of poGH-3-II, DsbAss
directs oGH-II into the inner-membrane of E. coli (Fig. 61). While in the case of poGH-3-V,
more than 75 % of the expressed protein remains in the cytoplasmic fraction with no traces in the
membrane fraction. Thus, Ala13 of DsbAss when substituted with Ile13 somehow hampered the
export and processing of oGH in E. coli. This suggests that it is not the hydropathy but the nature
of amino acid substitution at specific position, which influences the mechanism of protein
translocation.
In the present study, oGH-3-II, the one which is highly expressed (up to 20 % of the total E.
coli cellular proteins) with complete processing of DsbAss, was purified by employing FPLC
chromatography for further confirmation of size using the MALDI-TOF mass spectrometry. The
mass of purified oGH-II-2 was almost the same as the theoretically calculated mass of mature
oGH (data not shown), reflecting the complete processing of DsbAss.
112
(a) (b)
(c)
Figure 61.SDS-PAGE analysis of poGH-3-II-VI&I SDS PAGE analysis of subcellular fractions of construct poGH-3-I,II,VI ,in terrific broth medium.lane1,(a) SDS PAGE analysis of
subcellular fractions of construct poST-3-I;lane,M, marker;lane 2,total cell protein; lane 3,periplasmic fraction;lane 4, membrane fraction ;lane 5, cytoplasmic fraction. ,(b) SDS PAGE analysis of subcellular fractions of construct poST-3-ii;lane,1 ,membrane
fraction; lane 2, cytoplasmic fraction; lane 3,periplasmic fraction;lane 4, total cell protein; lane,M, marker. ,(c) SDS PAGE analysis of
subcellular fractions of construct poST-3-Vi;lane,1 , total cell protein; lane 2, cytoplasmic fraction; lane 3,periplasmic fraction;lane 4,
membrane fraction;lane,5,soluble fraction; lane,M, marker.
While the fractions poGH-3,III and V resulted in 25kDa protein found in both cytoplasmic and
periplasmic spaces as shown in( Fig 62).
113
(a) (b)
.Figure 62.SDS-PAGE analysis of poGH-3-III&V. SDS-PAGE analysis of sub cellular fractionation of pOST-3,III and V constructs. (a)subcellular fractions of pOaST-3-d;Lane 1 membrane fraction,M ,marker,lane-3 cytoplasmic fraction,lane 4,periplasmic fraction.,lane 5, total cell protein of pOST-3,b
construct,.(b)subcellular fraction of pOaST-3c; lane 1,membrane fraction , lane 2,periplasmic , lane 3,total cell protein ,lane
4,cytoplasmic fraction, lane 4 ,SDS marker.
The plasmid constructs poGH-3, III and V showed variation in the sub cellular localization
of the recombinant growth hormone. These constructs showed extra 3kDa size in the expected
band and it was very slightly translocated into cytoplasmic and periplasmic space while most of
it is being lost.
3.8.5 DsbA ss constructs with substitution of serine with cysteine in the C domain
In this construct two serine residues at C domain of DsbA ss were substituted with two
cysteine residues. The replacement of serine by cysteine residue in clone ( pOaST-3VIII)
affected the translocation process and the expressed recombinant ovine growth hormone was
found in the cytoplasmic fraction with 25kDa molecular weight when analyzed by SDS-PAGE
and Western blot analysis. This result is in accordance with the already reported importance of
polar C terminus which is essential for recognition of signal peptidase. The change of serine to
cysteine residue impaired the signal peptidase activity( Fig.63).
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Figure 63,SDS-PAGE analyis of poGH-3 VIII.
SDS_PAGE analysis of poGH-3, VIII constructs. Lane 1 SDS marker, lane 2 total cell
protein,lane3,cytoplasmicfraction.lane 4,periplasmicfraction.lane 5,membrane fraction
.
3.8.6 DsbA ss constructs with substitution of lysine with arginine in the N domain
The two lysine residues were substituted by arginine in the N domain in (poGH-3VII) and
the expression and subcellular localization of recombinant ovine growth hormone was analyzed
by SDS-PAGE and Western blot as described for the rest of the constructs. The ovine growth
hormone of 22kDa was found in the membrane fraction and no traces were found in cytoplasmic
fraction and periplasmic fraction as shown in fig 64.
Figure 64.SDS-PAGE analysis of poGH-3-VII. SDS-PAGE analysis of pOST-3-Vii construct.lane 1 SDS marker,lane 2 ,total cell protein,lane 3,periplasmic fraction,lane 4 cytoplasmic fraction,lane 5,membrane fraction
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3.8.7 Purification of oGH from poGH-3-II construct
Ovine growth hormone was purified by simple procedure of subcellular fractionation and
it was found in membrane bounded form. This membrane bounded growth hormone was
solubilzed by 40% acetonitrile and was observed on SDS gel. Western blot analysis of
membrane bounded growth hormone confirmed it as growth hormone (Fig.65)and MALDI TOF
analysis proved its molecular weight(Fig.66).
Figure 65.Subcellular fractionation of poGH-3II and western blot analysis. SDS PAGE analysis of sub cellular fractionation of pOaST-3-II construct expressed in T.B medium supplememnted with compatible
solute. lane 1,uninduced pOaST-3 construct.lane 2,total cell protein,lane 3,periplasmic fraction,lane 4,cytoplasmic fraction,lane
5,membrane fraction.lane 6, soluble fraction.lane 7 western blot of OaST.
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The purification of OaST from membrane bounded fraction is just single step of solubilization as
shown in Table.9
3.8.8 MALDI TOF analysis of purified ovine growth hormone
The purified product from FPLC was then applied on MALDITOF analysis and for that
0.1microlitre of the purified product was used .The MLDITOF analysis showed that purified
ovine growth hormone is of 21,059kDa of mass which is the approximately very near to the
actual 21759kDa mass of ovine growth hormone. A single sharp peak was observed at 0.5M
concentration. The identity of the purified oGH was further confirmed by MALDI-TOF analysis
as a single peak with a mass of 21,059.
Figure 66.MALADI-TOF analysis of purified ovine growth hormone
.
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3.8.9 Biological activity assessment assay
Cell-based proliferation assay was used to assess the biological activity of the purified
recombinant ovine growth hormone from oGH-3-II construct. The HeLa cells incubated with
BSA and purified recombinant ovine growth hormone were counted after 24 hrs by using
hemocytometer and it was found that cell growth in BSA was 40,000 +/- cells and growth was up
to 90,000 +/- cells in the presence of purified recombinant ovine GH. The biological activity of
oGH was checked in duplicate and the average no. of cells was determined by applying the
formula given in materials and methods. It was observed that in the presence of oGH, the growth
of the cells was found to be three-fold higher than the control
( a ) ( b )
Figure 67.Biological activity of oGH in the presence og Hela cell lines..
(a). HeLa cells in the presence of BSA (10 ug).(b), HeLa cells in the presence of ovine GH (10 ug)
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3.8.10 Computational analysis of pOaST-3-I-VIII constructs
In order to understand the behavior of these construct hydropathies and the secondary
structure of these construct was studied. On the basis of hydrophobicity all constructs were with
higher hydrophobicity than original DsbA signal sequence when calculated by Swiss Expasy
Protparam.
Table 8.Hydropathy indices of modified DsbA ss in poGH-3-I-VIII constructs
PoGH-
3
Construct
DsbA signal sequence* Description
Hydr
opath
y index
Approx.MW of oGH
PoGH-
3-I
N-terminal C-terminal
KKIWLALAGLVLAFSASA
H-domain Signalpeptidase
18 amino acids long native
DsbA signal sequence having
N-terminal, H- and C-terminal
domains incorporated at the N-
terminus of oGH sequence
1.389 22
poGH-
3-II KKIWLALIGLVLAFSASA
Modified DsbA with 1 Ala
changed to Ile 1.539 22
poGH-
3 -III KKIWLILIGLVLIFSISA
Modified DsbA with 4 Ala
changed to Ile 1.989 25
poGH-
3-IV KKIWLILIGLVLAFSASA
Modified DsbA with 2 Ala
changed to Ile 1.689 22
poGH-
3-V KKIWLILAGLVLAFSASA
Modified DsbA with 1 Ala
changed to Ile 1.539 25
poGH-
3-VI KKIWLALAGLVLIFSISA
Modified DsbA with 2 Ala
changed to Ile 1.689 22
poGH-
3-VII RRIWLALAGLVLAFSASA
Modified DsbA with 2 Lys
changed to Arg 1.321 22
poGH-3-VIII
KKIWLALAGLVLIFCICA Modified DsbA with 2 Ser changed to Cys
1.531 25
*The amino acid residues modified in native DsbA signal sequence are highlighted in grey.
The varying hydrophobicity didn’t gave the answer as the constructs poGH-3-II and
poGH-3-V with same hydropathies gave two different results .The construct (poGH-3-II)
appeared with the exact molecular weight of ovine growth hormone of 22kDa and construct
( poGH-3-V )with the additional 3 kDa. Both these constructs with the difference of alnine at
position 9 and Isoleucine at position 11 resulted in two different kind of expression levels(Table
8).One with the exact 22 kDa of oGH and the other resulted in 25kDa as shown in the figures52
and 53 respectively.
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We hypothesized that the secondary structure of the mutant DsbA signal sequence effect the
proper translocation of recombinant protein attached with it. By analysis of the secondary
structure of these mutant signal sequences it was observed that the mutation at position 11 in
DsbA signal sequence brings breakage in the alpha helix structure of hydrophobic
region(Fig.68).The homology model of DsbA signal sequence shows the alpha helix structure in
the first part of hydrophobic portion of DsbA signal sequence as shown in (Fig.69) .It is already
established fact that helix breaker in cleavable signal sequences prevents recognition by SRP,
and it appears that besides hydrophobicity the α-helix propensity of the hydrophobic core of the
signal sequence helps to determine the targeting pathway (Adams et al., 2002 ).
Figure 68.Minnou Server prediction results.
is α and other helices, is coil and is β-strand or bridge. is hydrophobicity,yellow is
hydrophobic, pale green is amphipathic, pink is polar and dark brown is charged
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Figure 69.Homology model of DsbA ss with altered alanine.
THe homology modeling and molecular docking of the altered amino acids in DsbA signal
sequence can answer all the questions of varying behaviour of DsbA by changing amino acid
alinaine at position 11.This can give good base for further work in this direction for new research.
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Discussion
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4.1 Characterization of oGH gene
The gene encoding oGH was isolated from the ovine pituitary tissue by RT-PCR methodology
and its sequence was determined. Like most other mammalian GHs (Vize and Wells, 1987;
Wallis and Wallis, 1989) oGH cDNA was found to contain 573 nucleotides and an intact ORF
coding for 191 amino acids with an in-frame stop codon (Fig.7 ). The percentage of G+C and
A+T nucleotides was 59.34 and 40.66 % respectively, which differs from 42 % GC contents of
vertebrate DNAs in general .The sequence of GH of local ovine breed (Lohi) of Pakistan was
submitted to the Gene bank with Accession numbers GQ45268 and AB24479 for genomic and
coding sequence of ovine growth hormone respectively. The genomic sequence details about the
exons, introns and regulatory sequence of the GH deduced that it constitutes 5 exons and 4
introns (Fig.7 )
While analyzing GHs of other vertebrate species, it was observed that a single residue (V130)
makes the ovine GH different from the bGH. The change i.e., valine (V130) versus glycine (G130)
in the latter, is interesting because G130 residue is widely conserved in the GHs of different
vertebrate (Fig.10 ) (Mukhopadhyay and Sahni, 2002b).It was reported that caprine GH also
differs from bGH by a single residue, i.e., glycine (G9) in bGH while serine (S9) in caprine GH.
Similarly, (Castro and Barrera, 1995) observed a single amino acid variation at position 155,
making the feline GH different from the canine and porcine GHs. The change (P155) like G130 in
ovine GH, was again in the highly conserved region.
Homology analysis revealed that the mature oGH exhibits only 67% similarity with human GH.
However, high homologies (98.5-99.5 %) were being observed between oGH and the reported
bovine, ovine, caprine and giraffe GHs (Mukhopadhay and Sahni, 2002). Due to high degree of
sequence identity between vertebrate GHs, similarities in three-dimensional structure were also
anticipated. Analysis of 3-D structure revealed that oGH exhibits a typical topology of cytokine
superfamily (Fig.16). The structure has all the salient features including four antiparallel α-
helices forming four-helix bundle structure, as described (deVos et al., 1992). In oGH like
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human GH more than 50% amino acid residues were involved in the helix formation which are
largely hydrophobic.
The sequence analysis of GH isolated from local ovine breed (Lohi) revealed the difference of
one amino acid when it was compared with the Indian and Australian ovine breeds. It showed
variation of one amino acid at position 147 where threonine is replaced with arginine as shown
in Fig.11 . The coding sequence comparison with local isolated caprine GH showed 100%
homology at amino acid level The sequence of oGH isolated from (Lohi) showed no variation at
amino acid level with the sequence of caprine growth hormone .The only variation which it has
are silent mutations (Fig 13). while its comparison with other species of same family Bovidae
showed 95-99% homology. It was also recognized that this difference increases when compared
with other families of class Mammalia (Hominidae, Canidae, Felidae, Equidae, Hippopotamidae,
Camelidae, Didelphidae) as shown in (Fig 12 & 13). The difference enhanced at inter class level
and the comparison showed that the N terminal of GH is highly variable, central region is less
conserved while C terminal is highly conserved. The phylogram of oGH showed that members of
Bovidae species are evolutionary more allied than other animals. The percentage homology of lohi
was 99, 98, 97, 97, 93, 91, 91, 90, 89, 76, 74, 69, 67, 50, 50, 49, 43 and 45 % with cow, deer, goat,
sheep, rabbit, panda, dog, pig, guinea pig, human, monkey, rat, mouse, domestic pigeon, chicken,
frog, goldfish and catfish respectively.As shown in fig below.
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Figure 70.Phylogenetic tree of ovine growth hormone
4.2 periplasmic Expression of roGH
The confirmed clones of recombinant ovine GH tested by sequencing were used for the
expression studies. For this purpose T7 promoter based expression vector pET22b was used and
E. coli strain BL21 codon plus . The oGH gene in the first construct poGH-1 was expressed with
NdeI/BamHII sites of pET22b and found negligible expression of native oGH in E. coli . As
shown ( Fig.26) the oGH expression with the clone poGH-1, carrying native gene was less than
0.5 % of the total E. coli cellular proteins. The observation was in good agreement with the
previous reports where bovine (Schoner et al., 1984), ovine (Puri et al., 1999), caprine
(Mukhopadhyay and Sahni, 2002a,c; Khan et al., 2007) and porcine GHs (Vize and Wells, 1987)
with native N-termini expressed poorly in E. coli systems. The high-level expression of the
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cloned gene in E. coli generally requires a strong promoter, a properly spaced Shine-Delgarno
(SD) sequence and an effective ribosome binding site for efficient translation of the mRNA (Das,
1990). The GHs of several vertebrate species however expressed poorly in E. coli regardless of
the promoter strength, the SD-sequence, host strains and culture conditions (Schoner et al., 1984;
George et al., 1985; Hsiung and MacKellar, 1987). Very low levels of expression resulted when
bubaline and caprine ST cDNAs were directly placed under the E. coli (trc) or phage (T7)
promoters (Mukhopadhyay and Sahni, 2002). The approximate expression levels were less than
0.1 % of the intracellular E. coli proteins, respectively. also had trouble while expressing ovine
GH in E. coli under the regulation of phage T5 promoter had a trouble too (Puri et al., 1999).
The gene encoding bGH was also found to be expressed poorly in E. coli (Tomich et al., 1989).
According to (Paik et al., 2006) GH coding sequence has some inherent properties that inhibit its
expression in E. coli. The suggested explanations for this behavior includes: two putative
secondary structures at the beginning of the coding region (Tomich et al., 1989) a basic pI value
of the bGH as described (Saito et al., 1987) and the existence of number of non-preferred
codons present in the bGH gene (Seeburg et al., 1983; George et al., 1985). The results of
(Schoner et al., 1984) however showed that the native bGH codons are not a barrier to efficient
translation.
Several different strategies were adopted to improve the expression levels of animals GHs in E.
coli. Expression levels of animal GHs could also be improved by making use of a two-cistronic
expression system. A synthetic two-cistronic expression system was constructed for high-level
expression of bGH and human GH in E. coli (Schoner et al., 1986). Puri et al., (1999) while
expressing the ovine GH in E. coli observed that the expression levels of GH are greatly affected
by nature of codon following the ATG start site. Presence of GCC (Ala) codon at position next
to ATG completely blocked the expression of ovine GH, whereas its removal or replacement
with Fusion of heterologous proteins with a short stretch of histidine residues was also found to
improve the expression levels of cloned genes in E. coli. The cDNAs encoding human and ovine
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STs were cloned with the His6 tag and expressed in E. coli under the control of T5 promoter
(Mukhija et al., 1995; Appa Rao et al., 1997). The GGC (Gly) codon restored the GH
expression, though, with different levels. High-level expression (representing ~ 20 % of the
soluble E. coli proteins) of buffalo and goat-GHs was also achieved as a fusion with glutathione-
s-transferase (gGH) partner, under the control of Trc promoter (Mukhopadhyay and Sahni,
2002). High-level expression could also be obtained by using procedures known to optimize both
gene transcription and mRNA translation (Hsiung and MacKellar, 1987). we tried to solve this
issue by utilizing PelB leader sequence of pET22b in construct poGH-2. The N terminal of ovine
GH gene was linked to PelB leader sequence of pET22b. As a result we analyzed good level
expression of about 18% of oGH but with an additional 3 kDa in its molecular weight on SDS-
PAGE (Fig 28). We further confirmed the expressed protein to be as GH by Western blot
analysis(data not shown). GH consists of 190 or 191 amino acids with two disulfide bridges. Of
particular interest for the expression of disulfide bonded proteins is a family of pET vectors
containing the N-terminal pelB secretion signal, which directs synthesized polypeptides to the E.
coli periplasm(Yoon et al., 2010) . Disulfide oxidoreductases and isomerases located in the E.
coli periplasm catalyze the formation of disulfide bonds enabling the accumulation of properly
folded, soluble protein making the periplasm an ideal compartment for expression of certain
therapeutic proteins .
Here we studied different factors effecting the release of oGH in periplasmic space by using
PelB leader sequence of PET vectore at the N terminus of oGH gene. We studied the effects of
IPTG concentration ,chemical chaperon (ZnCl2) and glycerol in the LB medium and also studied
the effect of ZnCl2 and Glycerol in the osmotic shock procedure.Glycerol is the cheapest carbon
source and is used widely to enhance the expression of recombinant protein in E.coli.We
replaced the carbon source of LB medium with glycerol with the same percentage and found that
it enhanced the expression of roGH from 18% to 22% as analysed on SDS-PAGE (fig,34)
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It has earlier been reported that a reduction of the IPTG concentration has a positive effect on
periplasmic yield of soluble protein expressed from a lac promoter( Kipriyanov et al., 1997).
The lac promoter is highly inducible and overexpression of the recombinant oGH is evident
even at IPTG concentrations as low as 20 µM(Fig 30 ). Similarly high levels of expression are
obtained at final IPTG concentrations of 60,80, 100, 1000 µM. Un-induced cultures typically
display some evidence of background (“leaky”) expression, which is well characterized for T7
promoter-based vector systems (Pushkar Malakar, 2015). IPTG is a costly chemical and here we
showed that IPTG concentrations as low as 20 µM are sufficient for high levels of roGH protein
expression. Following induction, the recombinant oGH can be recovered from the periplasm.
E. coli cells are easily lysed by several methods and for most laboratory set-ups sonication and
freeze/thaw cycles are the method of choice (Berrow et al., 2006). Mechanical and physical cell
disruption methods have been assessed for the release of periplasmic proteins. Mechanical
methods such as high pressure homogenizer (Balasundaram et al., 2008) hydrodynamic
cavitation (Balasundaram et al., 2006), bead mill (Bakir, 1997) are not selective in releasing the
individual periplasmic proteins. On the other hand, physical methods such as osmotic shock
could be considered as a mild treatment with low operation cost which also has the ability to
release periplasmic proteins with very high selectivity. we compared different methods of
somotic shocks including freeze thaw method.We found that molar concentration of Tris
buffer,sucrose concentration and time of incubations along with concentration of chelating agent
i.e EDTA effects the release of recombinant oGH into periplasmic space.We found 20% release
of oGH by using freeze thaw method for the recovery of oGH as compared to 12 and 10 % yield
by other (Koshland, 1980; Ramakrishnan et al., 2010) osmotic shock procedures( Fig.32&33
).We optimized the freeze thaw method for the better yield of roGH from shock fluid.For this
purpose different factors were studied i.e. glycerol concentration,use of chemical chaperon
(ZnCl2 )concentration and incubation time.It was observed that by increasing glycerol
concentration from 10% to 25% in the shock procedure enhanced the released of oGH when
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analysed by Bradford method and SDS-PAGE( Fig, 35& 36a) The oGH was further
aunthenticated by western blot analysis ( Fig 35 b).Optimization of osmotic shock procedure for
the specific study has been conducted by several researchers ( Chen et al., 2004; Rastgar et al.,
2007) .They claimed that the pre treatment of cells with divalent cations of calcium and
magnesium prior to the introduction of hypertonic solution would have chelated the
lipopolysaccharide and increased the recovery of creatinase from 60% to 75 on the other hand,
the addition of magnesium chloride in hypotonic solution reduced the cytoplasmic contaminants
in the medium by reducing the chelation of plasma membrane .These findings helped us to study
the effect of ZnCl2 in the medium and also in shock fluid which enhanced the release of oGH in
periplasmic space as shown in graph(Fig 29). Chaperones are known to enhance expression
yields as they facilitate folding, prevent aggregation, reactivate aggregates and reduce protein
degradation (Ying et al., 2004).The roGH thus released was 80% pure and for further
purification FPLC chromatoghraphy resulted in 95% pure roGH (Fig 37 ) .
4.3 Secretion of oGH into the inner membrane of E.Coli.
Secretory production of recombinant proteins in E. coli has been particularly useful for the
production of pharmaceutical proteins as compared to cytoplasmic production. Targeting a
protein of interest to the periplasmic space or the culture medium enables downstream
processing at a reduced process cost. Isolation and purification of the over-expressed products
can be simplified and rapid due to reduced contamination of various cellular components and
hence reduce proteolytic degradation by intracellular proteases. Correct folding of eukaryotic
proteins containing multiple disulfide bonds is also likely to occur in the reducing environment
of the periplasmic space. Secretory process allows removal of the amino-terminal signal
sequence from the recombinant on reaching the destination and appearance of mature protein and
naturally occurring sequences contain no N-terminal methionine residue.
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we utilized PelB leader sequence of pET22b in construct poGH-2. The N terminal of ovine GH
gene was linked to PelB leader sequence of pET22b. As a result we analyzed good level
expression of about 18% of oGH but with an additional 3 kDa in its molecular weight on SDS-
PAGE (Fig 28). We optimized the conditions for poGH-2 construct got enhanced yield in the
periplasmic fraction too(36 a& b ) but still the yield was very low as periplasmic space of E.coli
constitute just 6% of the cell.
In order to get the correct size (22kD) roGH with high yield. We expressed roGH in extra
cytoplasmic space and for this purpose three roGH constructs were designed with different signal
sequences; oGH, DsbA and STII. T7 promoter based expression vector pET22b(+) and E. coli
strain BL21 codon plus were utilized. Among the constructs, constructs poGH4 and 5 with signal
sequences (oGH and ST-II) respectively showed roGH expression at 25kDa while in construct
poGH3 with DsbA signal sequence, roGH was found to be 22kDa ( Fig 43 ). In order to
understand the reason of varying behaviour of these all signal sequences( including pelB) they
were analyzed based on probability of signal sequence by signal p3.0 server
(http://www.cbs.dtu.dk/services/signalP/), hydropathy plot by kyte-doolittle method and
secondary structure by PolyView prediction server (http://polyview.cchmc.org) (Porollo et al.,
2004). The hydropathies of all the signal sequences gave variable results with an optimal range
of hydropathy value above or below at which the signal sequence does not function properly
(Table 3). The secondary structure of these signal sequence were also analyzed (Fig. 47 A, B, C
& D). The hydropathy value for these signal sequences varied from least value of 0.986 for ST-II
signal sequence up to highest value of 1.840 for oGH signal sequence. The hydropathy profile of
oGH showed that it is more than 60% hydrophobic (data not shown). The amino acid sequence
of DsbA, STII, pelB and oGH signal sequence showed that they have varying charges in their N
and C terminal regions. The N terminal of pelB leader sequence constitutes KYL in which K
(lysine) was basic, Y (tyrosine) and L (leucine) were hydrophobic neutral. Whereas, C terminal
consisted of AMA, in which A (alanine) was neutral hydrophobic and M (methionine) was polar
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hydrophobic amino acid. However, N terminal of STII signal sequence had KKNI sequence,
where K was basic hydrophilic with positive charge, N (asparagine) hydrophilic neutral, I
(isoleucine) hydrophobic neutral and at C terminal A (alanine) and tyrosine were hydrophobic
neutral. However, DsbA signal sequence constituted of KKI at N terminal and ASA at C
terminal where S (serine) was polar hydrophilic. Based on the above stated facts we can explain
the reason behind inefficient translocation of native oGH, STII and pelB signal sequences. Thus,
a very high hydrophobicity (1.8) of oGH signal sequence in construct poGH2 blocked export, as
oGH itself is very hydrophobic protein. The secondary structure of STII (Fig. 47,C) showed that
it constitutes beta sheets and had aromatic tyrosine residue at cleavage region which blocked
export. The hydrophobicity of the signal sequence has been a dominant structure for proper
functioning of the signal sequence (Goder and Spiess, 2003). A wide survey performed by
(Beckwith and coworkers, 2005) identified a strong correlation between hydrophobicity of the
leader peptide and export mechanism (Huber et al., 2005). The DsbA signal sequence when
compared with npr, ST-II, PhoA signal sequences gave best periplasmic expression in E. coli
(Soares et al. 2003). In bacteria the naturally occurring cleavable DsbA signal sequence
promotes SRP-based protein export, which is attributed to its apparent hydrophobicity that is
greater than that of two other signal sequences (pelB and STII) which do not promote SRP-
dependent export. DsbA signal sequence is thought to direct the fused thioredoxin protein to the
co-translational SRP pathway by acting as an inhibitor of folding, thus allowing effective
posttranslational export (Schierle et al., 2003). Similarly, the secretion of the human GH to the
periplasm has been reported earlier (Soares et al. 2003; Becker and Hsiung 1986) using DsbA
signal sequence. Moreover, the theoretical analysis of 22 different signal peptides by using
bioinformatics tools also proved DsbA as the best signal sequence for the soluble expression of
human GH (Zamani et al., 2015). In this study we found complete translocation of roGH to the
inner membrane of E. coli (E.coli constitutes three biological boundaries: the inner membrane,
the cell wall and the outer membrane). The inner membrane separates the cell into two
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compartments; cytoplasmic space contained within the inner membrane and periplasmic space
between the inner and outer membranes. In E. coli, proteins are synthesized in the cytoplasm and
are targeted to different destinations within the cell. A number of systems have been studied for
translocation of recombinant proteins. The information about primary and secondary structure of
target protein also affects its translocation (Zamani et al., 2015) as in GH because it is highly
hydrophobic possesses alpha helical structures that resemble helical bundle class of inner
membrane proteins (IMPs). It is known that DsbA is an SRP signal sequence (Schierle et al.,
2003) and SRP pathway is primarily used by E. coli for targeting the IMPs. The combination of
SRP based DsbA signal sequence and GH structure explains its translocation into the inner
membrane.
4.4 Effect of medium composition on the expression and secretion of oGH in
E.coli
When the expression of the recombinant protein is low and cannot be increased by the proposed
mechanisms, then the volumetric yield of desired protein can be augmented by growing the
culture to higher densities. This can be achieved by changing a few parameters, like medium
composition and providing better aeration by vigorous agitation (Cui et al., 2006; Blommel et al.,
2007).
Different mediums have been applied to enhance the expression of recombinant proteins. The
poGH-3 was selected as the best construct for the expression of recombinant ovine GH as it
gave a protein of accurate size at 22kDa but with an expression level of 14% (Fig 43). Therefore,
different medium compositions were used in order to enhance the expression level of poGH-3.
We demonstrated that by using different media compositions, simple additives and changing
temperatures could easily enhance production of recombinant protein roGH. We compared seven
different mediums and found a remarkable variation in the final density of growth culture
inoculated with roGH( Fig 50). We found that cell growth stops at a relatively low density by
using LB(as shown in( fig 48). This happens because LB constitute very less amounts of carbon
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source and divalent cations (Sezonov et al.,2007). Not surprisingly increasing the amount of
peptone or yeast extract leads to little bit higher cell densities. Also divalent cation
supplementation (MgSO4 in the millimolar range) results in higher cell growth( as shown in
fig.48 ). Terrific Broth and SB (Super Broth) media recipes have been shown to be superior to
LB for reaching higher cell densities(studier, 2005) .TB constitute mainly Glycerol that aids in
forming a solvent shell around a protein molecule as a protein stabilizer, and increases the
viscosity of a solution for prevention of protein association (Swartz, 2001). It was observed that
expression level was much enhanced up to 22% in terrific broth medium supplemented with
compatible solute whereas it was up to 12% in LB and M9NG medium. A positive effect of low
molecular weight additives (chemical chaperones) supplemented in the culture medium were
being observed in various studies in terms of yields of periplasmic expressed proteins. Sorbitol
addition to the culture medium resulted in higher accumulation of a functional scFv , glycine
betaine and sucrose were beneficial for the folding of immunotoxin and cytochrome c550(
Swartz, 2001; Barth et al., 2000) . The problem of production of sufficient amounts of pure and
fully active recombinant immunotoxins, however, still remains an obstacle for clinical
application.
Thre strategy was seen and explained ( Barth et al., 2000) as a new approach for the production
of sufficient amount of pure and fully active recombinant immunotoxins in an optimized
periplasmic expression under osmotic stress. Barth and his coworkers used protein-stabilizing
compatible solutes ( glycerol, sorbitol, glycine betaine, and hydroxyectoine) during the
production phase and in the course of purification and storage to optimize the functionality and
stability of the proteins.
In this study we compared 2 set of osmolytes .1; glycylglycine and glycine betaine (both are
glycine with different side chains) and 2;Sorbitol and mannitol ( isomers) on the production
enhancement of roGH ( Fig 51 a & b). The general mechanism for stabilization with osmolytes is
believed to be through changing the protein hydration by exclusion from the hydration layer of
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the protein The resulting change in the protein hydration increases the energy needed to denature
proteins. Only compatible osmolytes, i.e., molecules which could interact favorably with protein
side chains and stabilize them against inactivation and which could potentially contribute to ATP
generation by the cell, proved to be effective solubilizers of the overexpressed proteins and
inhibited formation of inclusion bodies (Jain et al., 2008).It has been previously reported that
when sorbitol with glycyl betaine ,it enhanced the expression and solubl purification e (Barth et
al., 2000). The mechanism by which Sorbitol can be transported across E. coli via the sorbitol-
specific phosphoenol-pyruvate phospho-transferase system in the form of sorbitol-6-phosphate
(Sussman et al., 1971). Sorbitol-6-phosphate can enter glycolysis by the action of sorbitol-6-
phosphate dehydrogenase, which converts it to fructose-6-phosphate, a key intermediate of
glycolysis. However ,the mechanism of glycylglycine-mediated enhanced solubilization remains
to be understood. E. coli is known to possess specific transporters for dipetides and
oligopeptides. These in turn are of particular advantage to the bacteria, which thrive in the
peptide-rich gut lumen environment (Lengeler, 1975). Another possibility is the direct
interaction of glycylglycine with the expressed protein by acting as a chemical chaperone (Ou,
2002). Glycylglycine transport behaves similar to other shock-sensitive transport systems
requiring ATP for its transport (Cowell, 1974). In the presence of higher concentrations of
glycylglycine in the media, the bacteria probably ends up spending considerable energy in active
glycylglycine transport, thus slowing down the overall metabolic rate including the rate of
translation. This probably allows more time for the expressed proteins to be folded correctly.It is
also reported that higher concentration of glycine added in medium also enhance the periplasmic
yield of recombinant protein( kaderbhai et al., 1997).
We observed that mannitol can much enhance the production of roGH as compared to sorbitol as
shown in graph and it was also observed that mannitol also enhanced the soluble recovery of
roGH as clearly shown in fig our result is in complete accordance with (Leibly et al., 2012)
which states that. naturally occurring osmolyte mannitol can effectively aid in the stability and
134
solubility of recombinant proteins.The second set of comparative study was between
glycylglycine and glycine betaine both are derivatives of glycine.We found glycylglycine as
much better additive in the TB medium for enhance soluble production of recombinant roGH as
compared to glycine betaine as shown in (fig 51 ).
There are other factors like temperature ,concentration of inducer and induction time which
were also optimized. studies showed that rate of expression and culture temperature can affect
the proper folding of recombinant proteins and inclusion body formation( Kaushik et al.,
2003).Reducing culture temperature usually leads to slower growth of bacteria, slower rate of
protein production and lower aggregation of target protein(Clark et al., 2004). For recombinant
oGH, as shown in ( fig 54 ), culture at 25°C resulted in more expressed protein than 37°C. This
finding has shown less aggregation of recombinant protein in lower temperatures (M.rezaei et
al., 2013). It has been shown that the level of recombinant protein expression is affected by
inducer concentration. Generally most of recombinant proteins tend to be aggregated at high
concentrations of IPTG, while some other proteins are less sensitive to aggregation due to their
inherent higher solubility ( Shivcharan et al., 2013).
Based upon our findings, 20µM IPTG resulted in the highest amount of recombinant protein.
After optimizing all the above factors we recommended 0.6M mannitol,50mM
glycylglycine.4%Nacl2 in terrific broth medium with 20µM IPTG ( inducer) concentration at
25C ,at 150rpm shaking rate for the best yield of soluble recombinant oGH ( Fig 57 ).we
compared the yield by using sorbitol and glycyl-betaine as a known compatible solute used for
the soluble protein production(Barth et al., 2000) and our designed mannitol plus glycylglycine
and we found that it results in more than double concentration of soluble roGH as shown in(
table 6 .
135
4.5 Effect of mutation in DsbA signal sequence on the expression and secretion
of OaST
In order to enhance the expression and secretion level of oGH we thought to design new
constructs based on the mutation in the tripartite structure of DsbA signal sequence. In bacteria,
the naturally occurring cleavable DsbA signal sequence promotes SRP-based protein export and
is attributed to its apparent hydrophobicity that is greater than that of two other signal sequences
(PelB and ST11) which do not promote SRP-dependent export. DsbA signal sequence is thought
to direct the fused thioredoxin protein to the co-translational SRP pathway by acting as an
inhibitor of folding, thus allowing effective posttranslational export; or (ii) DsbA signal sequence
is able to direct more rapid engagement of the protein with the secretory machinery (Schierle,
2003). Similarly, the secretion of the human growth hormone to the periplasm was reported
earlier (soares et al., 2003) using DsbA signal sequence. The effect of signal peptide changes on
the expression and secretion of bovine growth hormone was also investigated by (Klein et al.,
1992) but they failed to find any significant influence of the signal sequence. However, most of
the work on recombinant growth hormone is still being carried out for cytoplasmic expression to
study refolding procedures and effect of different media or hosts systems (Patra et al., 2000).
In the present study we studied the effect of change of amino acid in all three parts of DsbA
signal sequence on the expression and translocation of recombinant oGH protein. The change of
amino acid in N terminal (poGH-3VIII construct) and C terminal (poGH-3-VII construct) did not
show any deviance from already reported facts. As in case of poGH-3g construct the substitution
of arginine did not enhance the expression of OaST while the substitution of cystein in poGH-3f
changed the polar nature of C terminal and affected the cleavage of signal peptide (Tsumoto et
al., 2010).
The substitution of each alanine with Isoleucine in the hydrophobic domain of DsbA signal
sequence showed the importance of specific position of alanine in the H domain of DsbA signal
sequence.The DsbAsignal sequence directs export via the SRP mechanism (Gerstein et al., 2005;
136
Nobuyuki et al., 2005) and recognizes its substrate by the presence of a hydrophobic signal
sequence which interacts with Ffh and 4.5sRNA ( Herskovits et al., 2000; Gerstein et al., 2005).
The N and G region of bacterial SRP binds to the hydrophobic part of the signal sequence while
M binds to SRP and 4.5sRNA (Robert et al., 2002).
In the present study, we show that the amino acid substitution in the hydrophobic (H) part of
DsbAss determines the translocation of the precursor protein. It has been proven that Gly
residues in the H region of GspB signal sequence affect the routing of a recombinant protein by
the sec pathway (Barbara et al., 2007). We investigated Ala in the H domain of DsbA signal
sequence and observed that Ala at position 11 with reference to signal peptidase site is necessary
for SRP routing of recombinant OaST to the inner membrane of E. coli.
The poGH-3I,II,IV and 3VII showed 22kDa while pOaST-3III and 3V and VIII resulted in 25
kDa band of recombinant oGH on SDS-PAGE (Fig.51) while constuct poGH-3VII resulted in
negligible expression. The hydropathy analysis of these substitution in H domain (Table 8)
showed same hydropathy of 1.539 for constructs pOaST-3II, -3V and 1.689 for constructs
pOaST-IV and VI. The hydropathy did not affect the translocation but the substitution at
specific position changed the translocation. The DsbA signal sequence has 4 alanine in its H
domain which are at position -1,-4,-9 and -11 with respect to signal peptidase site (Table 7). It
was observed that substitution of alanine with Isoleucine at position -11 changes the whole
mechanism of translocation.
On the basis of our findings we suggest a model for SRP routing of the recombinant OaST
protein with an appended DsbA signal sequence. The Ffh part of SRP constitutes N, G and M
domain of Ffh induces the conformational change in the nascent hydrophobic site of the signal
sequence ( Robert et al., 2002). This means that a specific protein conformation in the
hydrophobic part of the signal sequence affects binding with the N and G domain of the Ffh (
Manuvera et al., 2010). The Ala at position -11 in the H domain with respect to signal peptidase
site of DsbA signal sequence is important for the binding of N, G domain of Ffh in SRP
137
mechanism as replacement by Ile at this position resulted in the localization of recombinant oGH
majorly in the cytoplasmic fraction without cleavage of signal peptide as explained with the
poGH-3V construct . We suggest that there is amino acid specificity in the H domain of DsbA
signal sequence which is essential for its binding with the SRP as described in the model (Figure
71).The model explains the substitution of Isoleucine instead of alanine at position 11 with
respect to signal peptidase site effects the translocation of oGH protein through SRP mechanism.
(a)
(b)
Figure 71.Model representing the mechanism of DsbA signal sequence with altered amino acid with SRP mechanism.
SRP with Ffh , 4 . 5 s RNA
OaSt Ffh
OaSt
Ribosome DsbA with Ile at -11 position
Inner membrane
DsbA ss
4 . 5 sRNA
R i b o s o m e
Inner membrane
SRP
OaSt Ribosome
DsbA ss with Ile at - 9 position
R i b o s o m e SRP
Ffh
SRP with Ffh , 4 . 5 s RNA
4 . 5 sRNA
posi
tio
138
4.6 Purification and Biological activity Assessment
The poGH-3 construct with the best expression of 32% by compatible solute medium was used
further for the purification and biological activity assessment. In order to solubilize the
membrane bounded oGH a simple procedure of adding acetonitrile v/v was used and it was
observed that 40% acetonitrile was considered to be the best concentration for the solubility of
oGH or extraction of oGH from membrane. For this purpose ultra-centrifugation was used and
protein was recovered from the inner membrane of E. coli( kaderbhai et.al.2008) To our
knowledge this was the first study describing the oGH extraction from the inner membrane of E.
coli while all the previous studies have shown different techniques like freeze thaw, osmotic
shock etc. to obtain a protein in soluble form . The soluble oGH obtain was 90% pure.
The mass was determined by MALDI TOF analysis which showed oGH of 21059 which was a
little bit less than calculated value i.e. 21086 of ovine ST . This little variation in the molecular
weight didn’t affect the biological activity of the ovine GH.There are different cell lines that are
being used to assess the biological activity of the GH, while in the current study we used HeLa
cell lines. An enhancement or proliferation of HeLa cells was being observed in the presence of
recombinant oGH though in the presence of BSA (control) no cell proliferation was detected.
Hence, this result proved that recombinant ovine GH was biologically active.
4.7 Conclusion
In this study we have described the GH sequence of locally isolated ovine breed Lohi and its
comparison with the other Bovidae species. we report a simple system for production of
recombinant ovine growth hormone directed to the E. coli periplasm via the pET based
expression platform to yield soluble, properly folded oGH . The amount of glycerol and ZnCl2 in
the medium and shock fluid enhanced the yield of oGH from periplasmic space. The oGH
isolated by optimised shock methods and FPLC binds the GH receptor with high affinity. This
139
system will be useful for the average research laboratory wishing to produce material to study
oGH biology.
Further we studied the high yield of expression and purification of recombinant oGH from the
inner membrane of E.coli by using DsbA signal sequence.We found that recombinant protein
yields can be increased significantly by supplementing the TB medium with 0.6M mannitol and
50mMglycylglycine in the presence of 4% NaCl .
Further the effect of amino acid substitution in the tripartite structure of DsbAss emphasized the
alanine at position 11 is of most importance in the translocation mechanism of DsbA signal
sequence.
We suggest for further studies
Application of the compatible solute conditions in fermentor in order to get higher yields
of soluble ovine growth homone
To apply poGH-3 construct in mammalian host cells can also enhance the production of
oGH in soluble form .
Homology modeling of all the DsbA based constructs should be done.
140
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