UNIVERSITI PUTRA MALAYSIA
CONSTRUCTION OF A RECOMBINANT PLASMID FROM NEWCASTLE DISEASE VIRUS ANTIGEN, HAEMAGGLUTININ-NEURAMINIDASE
AND LACTOCOCCAL N-ACETYLMURAMIDASE AND ITS EXPRESSION
NUR ADEELA BINTI YASID
FBSB 2009 26
CONSTRUCTION OF A RECOMBINANT PLASMID FROM NEWCASTLE DISEASE VIRUS ANTIGEN, HAEMAGGLUTININ-NEURAMINIDASE AND
LACTOCOCCAL N-ACETYLMURAMIDASE AND ITS EXPRESSION
By
NUR ADEELA BINTI YASID
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirements of the Degree of Master of Science
February 2009
Specially dedicated to, My beloved hubby, Nuha Alia, Adam Aqil and families For their invaluable love, understanding, patience, support and care.
ii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science.
CONSTRUCTION OF A RECOMBINANT PLASMID CARRYING THE CODING SEQUENCES OF N-ACETYLMURAMIDASE AND
HAEMAGGLUTININ-NEURAMINIDASE AND ITS EXPRESSION IN CHINESE HAMSTER OVARY CELLS
By
NUR ADEELA BINTI YASID
Februari 2008 Chairman: Professor Raha Abdul Rahim, PhD Faculty: Biotechnology and Biomolecular Sciences
The anchoring of proteins to the cell surface of Lactococcus using
recombinant DNA techniques is an exciting and emerging research area that
holds great promise for a wide variety of biotechnological applications.
Presently available anchoring systems are based on recombinant bacteria
displaying proteins or peptides on the cell surface. This research is focused
on using lactococcal cell wall binding (cwb) region for display of viral epitopes
and antigenic determinants. Newcastle disease virus (NDV) is the
aetiological agent of Newcastle disease which can result in 100% morbidity
and mortality in chicken. Immune response of haemagglutinin-neuraminidase
(HN) protein antigens of NDV plays an important role in the prevention of
viral infection. A lactococcal domain coding region, acmA’ (300 bp) was
cloned into the mammalian expression vector, pcDNA3.1/His. The 1.7 kb HN
gene was then inserted at the 5’-end of the acmA’ coding region. Plasmid
extraction and restriction enzyme digestion analyses showed that cloning
iii
was carried out successfully. Sequencing results indicated that the inserts
(acmA’, HN and acmA’HN) were 99%, 97% and 98% homologous to the
published sequence of Lactococcus lactis and NDV strain AF2240. The
condition for transfection was optimised by testing various amounts of
transfected DNA (1 µg, 2 µg and 3 µg) and different charge ratio of liposome
reagent (µl): DNA (µg) in a 6-well plate. Transient expression of the fusion
protein in Chinese hamster ovary (CHO) cells was analysed by SDS-PAGE
and Western blotting. The results indicated that the best combination of DNA
used for transfection was 2 µg in 1:3 charge ratio of the liposome:DNA. This
study showed that a recombinant acmA’, HN and acmA’-HN have been
successfully constructed, transfected and expressed in CHO cells. This work
could contribute towards the development of a recombinant vaccine delivery
system for NDV.
iv
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
PENGHASILAN PLASMID REKOMBINAN MEMBAWA JUJUKAN KOD N-ACETYLMURAMIDASE DAN HAEMAGGLUTININ-NEURAMINIDASE DAN PENGEKSPRESAN DI DALAM SEL CHINESE HAMSTER OVARY
Oleh
NUR ADEELA BINTI YASID
Februari 2009
Pengerusi: Profesor Raha Abdul Rahim, PhD Fakulti: Bioteknologi dan Sains Biomolekul
Perlekatan protein pada permukaan sel Laktokokus menggunakan teknik
rekombinan DNA merupakan satu bidang penyelididkan yang menarik serta
menjanjikan harapan yang tinggi kepada pelbagai penggunaan teknologi.
Sistem perlekatan sedia ada adalah berdasarkan rekombinan bakteria yang
mempunyai protein atau peptida yang melekat pada permukaan selnya.
Kajian ini difokuskan kepada sistem laktokokal untuk perlekatan epitop virus
dan penentu antigen. Virus penyakit Newcastle (NDV), adalah agen atiologi
bagi penyakit Newcastle yang boleh menyebabkan 100% morbiditi dan
mortaliti pada ayam. Reaksi keimunan terhadap antigen protein
hemaglutinin-neuraminidase (HN) NDV memainkan peranan yang penting
dalam pencegahan jangkitan. Domain pengikatan dinding sel (cwb) dari L.
laktis, gen acmA’ (300 bp) diklonkan ke dalam vektor penzahiran mamalia,
pcDNA3.1/His (5.5 kb). Gen HN (1.7 kb) pula disubklonkan di hujung 5’-
domain pengikatan acmA’. Pada masa yang sama, gen HN diklonkan ke
dalam vektor yang kosong untuk kajian perbandingan. Pengekstrakan
v
plasmid dan analisis enzim pembatas menunjukkan pengklonan berjaya
dilakukan. Analisis jujukan DNA menunjukkan bahawa rekombinan acmA’,
HN dan acmA’HN mempunyai 99%, 97% dan 98% persamaan dengan
jujukan L. laktis dan NDV yang telah diterbitkan. Keadaan transfeksi
dioptimakan dengan mengkaji pelbagai amaun DNA (1 µg, 2 µg dan 3 µg)
dengan nisbah cas reagen liposome (µl) kepada DNA (µg) di dalam piring 6
lubang. Pengekspresan sementara telah diasai dalam sel CHO dengan SDS-
PAGE dan Pemblotan western. Keputusan menunjukkan nilai kombinasi
DNA terbaik untuk transfeksi adalah 2 µg pada nilai cas liposome kepada
DNA 1:3. Kajian ini juga menunjukkan rekombinan acmA’HN berjaya dibina,
ditransfeksi dan dizahirkan dalam sel CHO. Kajian ini boleh menyumbang
kepada penghasilan sistem rekombinan vaksin untuk NDV.
vi
ACKNOWLEDGEMENTS
I would like to express my deepest appreciation and thanks to my main
supervisor, Prof. Dr. Raha Abdul Rahim, for her never ending guidance and
encouragement throughout this project. Her patience and dedication have
inspired me to give my very best in completion of this study and the thesis.
My sincere thanks to my co-supervisor, Prof. Datin Paduka Dr. Khatijah
Yusoff for her invaluable advice and constructive criticism during the entire
progress of this project. Her suggestions and comments definitely helped in
the success of this thesis.
My heartiest appreciation goes to my beloved husband, Shahrul Ezhar, my
little daughter and son, Nuha Alia and Adam Aqil and families for their
continual support and understanding for me to proceed completion.
A special note of appreciation also goes out to the staff from Faculty of
Biotechnology and Biomolecular Sciences and everyone who have
contributed in one way or another to this study.
I am truly indebted to my lab mates, Kak Yan, Sab, Lina, Alai, Boon Hooi,
Wei Yeng, Anu, Dr. Varma, Dr. Elida, Dr. Prema, Dr. Yiap and Kak Ernie for
their helpful collaboration and discussion. Also not forgetting my friends,
Wawa, Fazu, Surini, Taznim and a bunch of others that have really helped
me in any possible way there could be. May god bless you all.
vii
I certify that an Examination Committee has met on 4.2.09 to conduct the final examination of Nur Adeela Yasid on her degree thesis entitled “Construction of a Recombinant Plasmid Carrying The Coding Sequences of N-Acetylmuramidase and Haemagglutinin-Neuraminidase and Its Expression in Chinese Hamster Ovary Cells” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Master of Science degree. Members of the Examination Committee were as follows: Raja Noor Zaliha Raja Abdul Rahman, PhD Professor, Faculty of Biotechnology and Biomolecular Studies Universiti Putra Malaysia (Chairman) Tan Wen Siang, PhD Professor, Faculty of Biotechnology and Biomolecular Studies, Universiti Putra Malaysia. (Internal Examiner) Sieo Chin Chin, PhD Dr, Faculty of Biotechnology and Biomolecular Studies, Universiti Putra Malaysia. (Internal Examiner) Mohd Nazalan Mohd Najimudin, PhD Professor, School of Biological Sciences, Universiti Sains Malaysia, Malaysia (External Examiner) BUJANG KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:
viii
This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Raha Abdul Rahim, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Khatijah Yusoff, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)
HASANAH MOHD GHAZALI, PhD Professor and Dean
School of Graduate Studies Universiti Putra Malaysia Date:
ix
DECLARATION I hereby declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently submitted for any other degree at Universiti Putra Malaysia or at any other institutions.
NUR ADEELA BINTI YASID Date:
x
TABLE OF CONTENTS
Page
DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii APPROVAL viii DECLARATION x LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xviii CHAPTER
1 INTRODUCTION Introduction 1 2 LITERATURE REVIEW
2.1 Surface Display of Heterologous Proteins 5 2.1.1 Cell Wall of Gram-Positive Bacteria 5 2.1.2 Bacterial Surface Display 6
2.2 Lactococcus lactis 10 2.2.1 Cell Wall Composition of Lactococci 12 2.2.2 Autolysis of Lactococci 14 2.2.3 The N-acetylmuramidase of L. lactis 17 2.2.4 L. lactis as DNA Delivery Vehicles 20
2.3 Newcastle Disease Virus (NDV) 22 2.3.1 NDV Strain AF2240 23
2.3.2 HN Protein 25 2.4 Vaccine Development of NDV 26
2.4.1 Recombinant Vaccine against NDV 27 2.5 Mammalian Gene Expression 29 2.6 pcDNA3.1/His A Expression Vector 31
2.7 LipofectamineTM 2000 33 3 MATERIALS AND METHODS 3.1 Bacterial Strains, Plasmids, Cell Line and
Growth Media 34 3.2 Preparation of Stock Culture 35
3.3 Agarose Gel Electrophoresis 35 3.4 Miniprep DNA Extraction by Alkaline Lysis
Method 32 3.4.1 Total Genomic DNA Extraction 32
3.5 Gene Amplification by PCR 36 3.5.1 PCR Primer Design 36 3.5.2 Amplification of acmA’ Gene 38
xi
3.5.3 Amplification of HN gene 40 3.6 Purification of PCR Amplified acmA’ and HN
genes for Cloning and Sequencing 40 3.7 Preparation of Competent Cells 41
3.7.1 E. coli Top10 F’ 41 3.8 Cloning of the PCR Amplified acmA’ Gene into
pcDNA3.1/His Vector 42 3.8.1 Restriction Enzyme (RE) Digestion 42 3.8.2 Ligation 43 3.8.3 Transformation 43
3.9 Plasmid DNA Isolation 44 3.10 Recombinant Plasmid DNA Verification 45
3.11 Sequencing 45 3.12 Sub-Cloning of HN Gene into pcDNA3.1/His-
acmA’ Expression Vector 46 3.13 Cloning of PCR Amplified HN Gene into
pcDNA3.1/His 46 3.14 Mammalian Cells Culture 47
3.14.1 Propagation and Maintenance of CHO Cell Line 47
3.14.2 Cell Counting 48 3.14.3 Preparation of Stock Culture 48 3.15 Transfection of Mammalian Cells 49 3.15.1 Transient Transfection with
LipocetamineTM2000 49 3.15.2 Stable Cationic Lipid Transfection using
Geneticin® (G418) 51 3.15.3 Determination of Toxic Concentration of
Geneticin® 51 3.16 Selection of Transfected Cells 51 3.17 β-Gal Staining 52 3.18 PCR Analysis of Transfected Cells 53 3.19 Detection of Recombinant protein Expression 54 3.19.1 Detection of mRNA 54 3.19.2 Total RNA Isolation 55 3.19.3 Reverse Transcription of RNA 57 3.19.4 Preparation of Samples 57 3.19.5 SDS-Polyacrylamide Gel
Electrophoresis 58 3.19.6 Western Blotting 59
4 RESULTS 4.1 Amplification of Cell Wall Binding Domain of
acmA’ gene 61 4.2 Cloning of the Cell Wall Binding Domain of
acmA’ gene into pcDNA3.1/His Vector 63 4.2.1 Verification of Recombinant pcDNA3.1/
His-acmA’ 65 4.2.2 Sequence Analysis of Recombinant
xii
Plasmid 69 4.3 Amplification of HN gene of NDV 71
4.4 Subcloning of HN gene into pcDNA3.1/ His-acmA’ 73 4.4.1 Verification of Recombinant pcDNA3.1/
His-acmA’HN 73 4.5 Cloning of HN Gene into pcDNA3.1/His 78
4.5.1 Verification of Recombinant Plasmid pcDNA3.1/His-HN 78
4.6 Sequencing Analysis of Recombinant Plasmid, pcDNA3.1/His-acmA’HN and pcDNA3.1/
His-HN 82 4.7 Transient Expression of acmA’, acmA’HN and
HN Proteins in Mammalian Cell Line 85 4.8 Stable Cationic Lipid Transfection using
Geneticin (G 418) 86 4.9 Detection of Transfected Cells 88
4.10 Detection of acmA’ and HN mRNA in Transfected Cells using RT-PCR 90
4.11 SDS-PAGE and Western Blot Analysis 94
5 GENERAL DISCUSSION 97 6 SUMMARY AND CONCLUSION 107
REFERENCES 109 APPENDIX 122 BIODATA OF THE STUDENT 123
xiii
LIST OF TABLES
Table Page
2.1 The Specificities of Different Peptidoglycan Hydrolases 16
3.1 Characteristics of Primers for PCR 39
3.3 Characteristics of Restriction Enzymes 42
3.4 Ratio of DNA (µg) : LipofectamineTM 2000 (µl) 50
3.5 Determination of toxic concentration of Geneticin (G 418) 87
xiv
LIST OF FIGURES
Figure Page
2.1 Cell wall of Gram-positive bacteria 7
2.2 Cell wall composition of Gram-positive bacteria 13
2.3 A general presentation of the lactococcal peptidoglycan structure
15
2.4 Diagrammatic presentation of three domains in acmA’ gene of Lactococcus lactis MG1363
19
2.5 Newcastle disease virus structure and genomic organization
24
2.6 Diagrammatic presentation the features of pcDNA3.1/His vectors
32
4.1 Agarose gel electrophoresis of genomic extraction of L. lactis ssp. lactis (MG1363)
62
4.2 Agarose gel electrophoresis of PCR product of acmA’ gene
62
4.3 Plasmid extraction of pcDNA3.1/His 64
4.4 Agarose gel electrophoresis of putative recombinant plasmid pcDNA3.1/His-acmA’
66
4.5 Verification of pcDNA3.1/His-acmA’ by Restriction enzyme analysis
66
4.6 Verification of pcDNA3.1/His-acmA’ by PCR 67
4.7 The map of pcDNA3.1/His-acmA’ construct 68
4.8 The sequencing result of pcDNA3.1/His-acmA’ 70
4.9 Agarose gel electrophoresis of the HN gene of NDV strain AF2240
72
4.10 Agarose gel electrophoresis of putative recombinant plasmid of pcDNA3.1/His-acmA’HN
74
4.11 Verification of pcDNA3.1/His-acmA’HN by RE analysis 74
xv
4.12 Verification of pcDNA3.1/His-acmA’HN by PCR 77
4.13 The map of pcDNA3.1/His-acmA’HN construct 73
4.14 Agarose gel electrophoresis of putative recombinant plasmid of pcDNA3.1/His-HN
79
4.15 Verification of pcDNA3.1/His-HN by RE analysis 79
4.16 Verification of pcDNA3.1/His-HN by PCR 80
4.17 The map of pcDNA3.1/His-HN construct 81
4.18 The sequencing result of pcDNA3.1/His-acmA’HN 83
4.19 The sequencing result of pcDNA3.1/His-HN 84
4.20a Agarose gel electrophoresis of pcDNA3.1/HislacZ 86
4.20b Verification of pcDNA3.1/HislacZ by RE analysis 86
4.21a Agarose gel electrophoresis of PCR analysis of transfected pcDNA3.1/His-acmA’ in CHO cells
89
4.21b Agarose gel electrophoresis of PCR analysis of transfected pcDNA3.1/His-acmA’HN in CHO cells
89
4.22 Amplification of acmA’ and HN genes by RT-PCR 91
4.23 The sequencing result of acmA’, detected in transfected cell and ampolified bu using RT-PCR
92
4.24 The sequencing result of HN gene, detected in transfected cell and ampolified bu using RT-PCR
93
4.25 SDS-PAGE and Western blot analysis of CHO cells transfected with recombinant plasmid, pcDNA3.1/His-acmA’ at 48 h post-transfection
95
4.26 SDS-PAGE and Western blot analysis of CHO cells transfected with recombinant plasmids, pcDNA3.1/His-acmA’HN and pcDNA3.1/His-HN at 48 h post-transfection
96
xvi
LIST OF ABBREVIATIONS
Aa amino acid
AcmA N-acetylmuramidase
Amp Ampicillin
anti-His antibody against His tag
Bp Basepair
BLAST basic local alignment search tool
Β Beta
CO2 carbon dioxide
CaCl2 calcium chloride
dH2O distilled water
DMSO Dimethylsulfoxide
DNA deoxyribonucleic acid
dNTP deoxyribonucleotides triphosphates
DTT Dithiothreitol
E. coli Escherichia coli
EDTA ethylenediamine tetraacetate
EtBr ethidium bromide
F fusion protein
GRAS generally regarded as safe
H Hour
His Histidine
HRP horseradish peroxidise
Kb kilobase pair
xvii
kDa Kilo Daltons
LAB lactic acid bacteria
LB luria bertani
L. lactis Lactococcus lactis
M Molarity
mA Milliampere
MCS multiple cloning site
Mg Milligram
MgCl2 magnesium chloride
Min Minute
Ml Millilitre
mM Millimolar
mRNA messenger RNA
NaCl sodium chloride
N-terminal the amino-terminal (NH2) of a polypeptide
nm Nanometer
NDV Newcastle disease virus
ºC degrees centrigrade
OD optical density
ORF open reading frame
PBS phosphate buffered saline
PCR polymerase chain reaction
pH potential of Hydrogen
PVDF polyvinylidene difluoride
RE restriction enzyme
xviii
xix
RNA ribonucleic acid
rpm revolutions per minute
RT room temperature
RT-PCR reverse-transcriptase PCR
s Second
SDS-PAGE Sodium dodecyl polyacrylamide gel electrophoresis
sp. Species
subsp. Subspecies
TAE tris-acetate EDTA
Taq thermus aquaticus
TEMED Tetramethyl-ethylene diamine
U Unit
UV Ultraviolet
v Volt
μg microgram
μl microlitre
μm micromolar
v/v volume per volume
w/v weight per volume
% Percentage
x g centrifugal force
X-gal 5-bromo-4-chloro-3-indol-β-D-galactosidase
CHAPTER 1
INTRODUCTION
Heterologous surface display of proteins in bacteria has become an increasingly
important study. Cell-surface display allows peptides and proteins to be
displayed on the surface of bacterial cells by fusing them with anchoring motifs
such as Leu-Pro-Xaa-Thr-Gly- (LPXTG), bacterial fimbriae and S-layer proteins
(Lee et al., 2000; Sang et al., 2003). The most common application for bacterial
surface display is the development of live bacterial vaccine delivery systems,
since cell surface display of heterologous antigenic determinants has been
considered advantageous in order to induce antigen-specific antibody
responses when live recombinant cells were used for immunisation (van
Roosmalen et al., 2005).
Many studies have been carried out to evaluate the effect of antigen-presenting
systems by Gram-positive bacteria. This is because Gram-positive bacteria
have certain properties that potentially make them more suitable for bacterial
surface display applications. First, the surface receptors of Gram-positive
bacteria seem to be more permissive for the insertion of extended sequences of
foreign proteins (Fischetti et al., 1993) compared to the different Gram-negative
systems. Another advantage is the translocation proud in which only a single
membrane is required to achieve proper surface exposure of the heterologous
peptides. In addition, the thicker cell wall of Gram-positive bacteria is more rigid
and allows various laboratory procedures to be carried out without extensive cell
lysis. Expression systems for the mouth commensal bacterium Streptococcus
gordinii (Pozzi et al., 1992) and the nonpathogenic bacterium Staphylococcus
xylosus (Hansson et al., 1992) have been developed based on the fibrillar M6
protein from Streptococcus pyogenes and protein A from Staphylococcus
aureus, respectively. Staphycoccal protein A (SpA) has often been used as a
model system to study the anchoring mechanisms of surface proteins in Gram-
positive bacteria (Ton-That et al., 1997; Marraffini et al., 2004). It is because the
sorting signals of SpA include LPXTG motif contains a cleavage site for sortase
(SrtA). The hydrophobic domain is a membrane-spanning region. The charged
tail serves as a retention signal to prevent secretion of the polypeptide chain into
the surrounding medium.
There is an interest in developing an alternative vaccine carrier system based
on harmless and nonpathogenic Gram-positive bacteria, such as Lactococcus
lactis. The bacterium has a long history of safe use in food fermentation
(Sorensen et al., 2000; Hansen, 2002) and possesses both GRAS (generally
regarded as safe) and food grade status. It is able to survive in the
gastrointestinal tract of humans and other animals for 2-3 days, but it does not
invade or colonise the mucosal surface, nor does it evoke an immune response
against itself. The fact that it is non-colonizing, non-invasive and has low innate
immunogenic makes it a desirable model as a recombinant vaccine delivery
system.
2
However, this model has one major drawback in the delivery of eukaryotic
epitopes from the bacterial system. For example, lactococcal processing does
not assure post-translational modifications such as glycosylations, and disulfide
bond formation may be limited due to the lack of disulfide isomerase. This
results in a very low quantity of the active form of the protein of interest, due to a
low rate of passive formation of disulfide bonds as observed with IL-2 in L. lactis
(Bermudez-Humaran et al., 2003a) and ovine omega interferon (Bermudez-
Humaran et al., 2003b).
Previous research has shown the capability of targeting cell wall binding protein
to L. Lactis to the surface of bacterial cells (Steen et al., 2003; van Roosmalen
et al., 2006; Moorthy and Ramasamy, 2007). A strategy to use recombinant cell
wall of L. lactis as an antigen carrier and display of proteins or peptides in an
eukaryotic expression system has been developed to try and overcome the
above problem. The cell wall anchor motif (protein anchor) of the L. lactis cell
wall hydrolase AcmA is a versatile tool for the display of heterologous proteins
on the surface of Gram-positive bacteria. It consists of three domains. The N-
terminal signal sequence is followed by an active domain and C-terminal
membrane anchor. In the C-terminal part of the protein, three 44-amino acid
repeated regions are present, and these are separated by intervening
sequences. A system displaying heterologous proteins to the C-terminus of
AcmA has been established by Raha et al. (2005) and Okano et al. (2008). They
successfully expressed the protein in E. coli and L. lactis, and also
demonstrated the display of target protein on L. lactis.
3
Haemagglutinin-neuraminidase (HN) gene encodes for a surface glycoproteins
of Newcastle disease virus (NDV) possesses both haemagglutinin (HA) and
neuraminidase (NA) activities. It also has an undefined role in fusion as well as
the major antigenic determinant of the virus (Bousse et al., 2004). In this study,
a constructs were developed containing the HN recombinant protein that can be
expressed together with the anchor motif in an animal culture system. Since the
expression of AcmA protein in mammalian cells has not been demonstrated so
far, the main objective of this work was to determine the ability of a bacterial cell
wall binding protein to fuse with HN gene and express in a mammalian cell
system. This system would allow for the expression of viral epitopes that needs
post-translational system to be displayed on the surface of L. lactis.
The objectives of this study were:
a. To isolate and amplify the acmA’ gene of L. lactis and HN gene of NDV
strain AF2240;
b. To construct specialized vectors for expression of recombinant HN
proteins; and
c. To study the expression of the above genes in Chinese hamster ovary
(CHO) cells.
4
CHAPTER 2
LITERATURE REVIEW
2.1 SURFACE DISPLAY OF HETEROLOGOUS PROTEINS
2.1.1 Cell Wall of Gram-Positive Bacteria
Gram-positive bacteria are simple cells. On the basis of morphological criteria,
three distinct cellular compartments can be distinguished: the cytosol, a single
cytoplasmic membrane and the surrounding cell. The cell envelope of lactic acid
bacteria consists of a cytoplasmic membrane and a cell wall. The cell wall
protects the underlying protoplast, resists turgor and maintains the shape of the
cell. Structured protein layers (S-layer) or polysaccharide Gram-positive bacteria
also serves as an attachment site for proteins. Protein attachment involves
either the covalent binding of LPXTG-carrying proteins to the peptidoglycan
pentaglycine cross-bridge or the noncovalent binding of protein to the
peptidoglycan. It requires a C-terminal sorting signal which an LPXTG motif, a
hydrophobic domain interacting with the cytoplasmic membrane, a charged tail
preventing secretion and a sortase, which catalyses the transpeptidation
reaction (Schneewind et al., 1992; Mesnage et al., 2000). In general, the cell
wall of Gram-positive bacteria is composed of a thick layer of peptidoglycan,
accessory polysaccharides, teichoic acid, lipoteichoic acid (LTA) and proteins
5
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