i CLONING AND OVEREXPRESSION OF INFLUENZA A H1N1...
Transcript of i CLONING AND OVEREXPRESSION OF INFLUENZA A H1N1...
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CLONING AND OVEREXPRESSION OF INFLUENZA A H1N1 NS1 PROTEIN IN
Escherichia coli
ONG LIH LIH
A dissertation submitted in partial fulfillment of the requirements for the award of the degree of
Master of Science (Biotechnology)
Faculty of Biosciences and Bioengineering Universiti Teknologi Malaysia
DEC 2010
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ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincere gratitude to my supervisor,
Dr. Chan Giek Far, for initiating this research project and for her continuous guidance
during the study. From this project, I have increased my own independence, creative
thinking and confidence. In addition, she has given advice, patient teaching, read and
critically commented on my draft of this thesis.
A thank you also goes out to all members of Faculty of Biosciences and
Bioengineering for guiding and giving me a warmth helping hands along throughout the
project. Thanks for their moral support, interest and constructive suggestion of this
project. There are no words to describe how much I appreciate their kindness. Last but
not least, I would like to thank my family and friends for all of their love and support.
You were there for me even when I was in difficulty and faced problems during
my research. These are deeply appreciated. Thank you again to all of you. To you I owe
this thesis.
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ABSTRACT
Influenza virus is globally pathogenic and it is usually associated with zoonotic
respiratory disease. It possesses a lipid-bounded segmented genome which encodes at
least one biochemically-distinct protein. Its subtype A can be classified according to
antigenic differences. NS1 protein is defined as nonstructural protein in the virus. It is a
known multifunctional virulence factor. It can only be detected in the infected cell. In
this study, the NS1A gene was successfully cloned into the BamHI/SacI cleaved-pET-
32c(+) vector and subsequently electro-transformed into E. coli BL21(DE3) expressing
host. There were three positive clones confirmed to contain the gene of interest by
sequencing. Protein expression in soluble and insoluble fractions was observed in E. coli
BL21(DE3). The clone 104 was selected for subsequent analysis. Better NS1A protein
expression was found at 37°C by 5mM lactose induction. Purification of the NS1A
recombinant protein from the inclusion bodies fraction was attempted by Ni-NTA
affinity chromatography and ion exchange chromatography. The physical condition
along the purification column and the biological properties of the protein itself may
perhaps result in the loss of protein and its corresponding immunogenicity. Ammonium
sulfate at 20% saturation was attempted to sufficiently concentrate and partially purify
the NS1A recombinant protein. The ammonium sulfate precipitated NS1A recombinant
protein has shown significant immno-response to the polyclonal antibody in Western
blot. The 37kDa NS1 protein was detected to react with the H1N1 NS polyclonal
antibody.
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ABSTRAK
Influenza virus merupakan patogen dalam dunia global dan ia biasanya berkaitan
dengan penyakit pernafasan zoonosis. Ia mempunyai genom tersegmentasi yang
diselaputi oleh lipid dan setiapnya mengekodkan sekurang-kurangnya satu protein yang
berbeza dalam sifat biokimia. Influenza A boleh diklasifikasikan mengikut antigennya.
NS1 protein ditakrifkan sebagai protein nonstruktural virus yang merupakan faktor
virulensi yang multifungsi. Protein ini hanya boleh dikesan dalam sel yang dijangkiti.
Dalam kajian ini, gen NS1A telah berjaya diklonkan ke dalam vektor pET-32c(+)
melalui potongan enzim pada BamHI/SacI. Plasmid rekombinan kemudian
ditransformasikan melalui denyutan elektrik ke dalam sel bakteria perumah E. coli
BL21(DE3). Terdapat tiga klon telah dikenalpastikan sebagai klon positif yang
mengandungi gen NS1A. Pengekspresan protein dalam fraksi larut dan tidak larut
diperhatikan dalam E. coli BL21(DE3). Klon 104 telah dipilih untuk analisis selanjutnya.
Pengekspresan NS1A protein didapati berkesan pada suhu 37 °C di bawah 5mM laktosa
induksi. Protein rekombinan NS1 dari fraksi tidak larut cuba ditulenkan berdasarkan
kromatografi afiniti Ni-NTA dan kromatografi penukaran ion. Namun, keadaan fizikal
sepanjang kolum penulenan dan sifat biologi daripada protein itu sendiri mungkin
menyebabkan kehilangan protein dan aktiviti keimunan. Pemendakan garam dengan
20% amonium sulfat adalah cukup untuk memendakan lalu separa menulenkan protein
NS1A. Protein rekombinan yang dimendakkan dengan amonium sulfat telah
menonjolkan keputusan positif dalam kaedah pemblotan Western dan protein NS1A
bersaiz 37 kDa telah dikesan bertindak balas dengan antibodi poliklonal NS H1N1.
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TABLE OF CONTENTS
CHAPTER TITLE PAGE TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF SYMBOLS/ ABBREVIATIONS/ NOTATION/ TERMINALOGY xviii
LIST OF APPENDICES xviii
1 INTRODUCTION 1
1.1 Background of Study .................................................................. 1
1.2 Research Objectives .................................................................... 3
1.3 Research Scope ............................................................................ 4
1.4 Problem Statement ....................................................................... 4
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2 LITERATURE REVIEW 6
2.1 The Biology of Influenza Genome ................................................ 6
2.1.1 Comparative Analysis: Influenza Virus Type A, B and C ................................................................................. 8
2.2 Influenza Virus Type A: Genome Overview ............................... 11
2.2.1 Influenza Virus A: Virus-like Particles and Its Structure-Function Concordance ..................................... 11
2.2.2 Influenza Virus A: Viral Transmission and Antigenic Variants ........................................................................... 15
2.2.2.1 Influenza Virus A H1N1 Subtypes ...................... 17
2.3 Influenza Virus A NS1 Protein: Proteomics Approach ................ 18
2.3.1 NS1A Protein: Protein Structural Study .......................... 19
2.3.2 NS1A Protein: Its Innate Biophysical Characterization ... 21
2.3.2.1 Protein-RNA Interaction ..................................... 23
2.3.2.2 Protein-Protein Interaction .................................. 25
2.3.3 NS1A Protein: Cloning and Expression Profiles .............. 27
3 MATERIALS AND METHODS 32
3.1 Experimental Design .................................................................. 32
3.2 Plasmid Used in the Project ....................................................... 32
3.3 Bacterial Strains and Media Preparation ..................................... 36
3.3.1 Bacterial Culture Stocks and NS1 Synthetic Gene .......... 36
3.3.2 Culture Media Preparation ............................................. 36
3.4 Preparation of Vector and Gene Insert ....................................... 37
3.4.1 Plasmid Isolation ............................................................ 37
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3.4.2 DNA Double Restriction Digestion ................................. 37
3.4.3 Agarose Gel Electrophoresis ........................................... 38
3.4.4 Gene Extraction .............................................................. 39
3.5 Cloning of NS1 Gene into pET-32c Vector ............................... 39
3.5.1 DNA Ligation ................................................................. 39
3.5.2 Propagation of Recombinant Plasmid in E.coli BL21(DE3) Strain ........................................................... 39
3.5.2.1 Preparation of Electrocompetent E.coli ............. 39
3.5.2.2 Electroporation ................................................. 40
3.6 Clone Identification ................................................................... 40
3.6.1 Colony Screening and Selection ...................................... 41
3.6.2 Sequencing ..................................................................... 42
3.6.3 Sequencing Analyses ...................................................... 42
3.7 Overexpression of Recombinant NS1A Protein in E.coli BL21 (DE3) .............................................................................. 43
3.7.1 Induction of Target NS1A Recombinant Protein ............ 43
3.8 Protein Purification ................................................................... 43
3.8.1 Cell Disruption and Extraction ....................................... 43
3.8.1.1 Chemical Lysis Method .................................... 43
3.8.1.2 Enzymatic-Ultrasonication Method .................. 44
3.8.2 Protein Purification ........................................................ 44
3.8.2.1 Immobilized Metal Ion Affinity Chromatography (IMAC) .............................. 45
3.8.2.2 Anion Exchange Chromatography (AEC) ........ 45
3.8.2.3 Partial Purification Method: Precipitation with Ammonium Sulfate ......................................... 46
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3.9 Denaturing Gel Electrophoresis ..................................................... 46
3.9.1 Determination of NS1 Protein Immunogenicity............ 47
3.9.1.1 Western Blotting .................................................... 47
4 RESULTS AND DISCUSSION 50
4.1 NS1A Gene Cloning and Clone Identification .............................. 50
4.1.1 Sequencing Analyses ......................................................... 52
4.2 Expression Analysis of NS1A Recombinant Protein ..................... 58
4.3 Purification and Immunodetection of NS1A Fusion Protein.......... 66
4.3.1 IMAC Purification ............................................................. 66
4.3.2 Anion Exchange Chromatography Acts As Alternative Purification Method ......................................................... 76
4.3.3 Protein Fractionation by Ammonium Sulfate ..................... 79
5 CONCLUSION 84
5.1 The Significance of Study ............................................................ 84
5.2 Limitations and Further Study ...................................................... 86
REFERENCES 87
APPENDICES APPENDIX A ...................................................................................... 98
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LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Genomic profile of influenza virus type A, B and C. ......................... 10
2.2 Review on multifunctions of influenza A virus NS1 protein. ............. 22
2.3 Review on a series of cloning and expression work of NS1 protein. .. 29
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LIST OF FIGURES
FIGURE NO. TITLE PAGE
2.1 Structure and composition of Influenza A (Pushko, 2009). ....................... 7
2.2 Countries affected by pandemic influenza A H1N1 outbreak 18
2.3 Dimerization model of influenza A virus NS1 protein ............................. 20
3.1 A sketch showing the pET32 vector (Novagen). ..................................... 34
3.2 Multiple cloning site regions of pET-32 (Novagen). ............................... 35
4.1 Screening the transformed colonies by colony PCR. 51
4.2 BamHI and SacI restriction enzyme digested products. ........................... 52
4.3 Alignment of the clone_104 sequence and the synthetic NS1A sequence.55
4.4 Phylogentic tree of recombinant NS1A (from clone 104) produced by
BLAST pairwise alignments. ................................................................... 56
4.5 Predicted structure of NS1A recombinant protein. ................................. 57
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4.6 Batch I induction: SDS-PAGE analysis of cytoplasmic fraction
harvested from E. coli clones induced with 1mmol/L IPTG at 37°C. ..... 59
4.7 Batch I induction: SDS-PAGE analysis of inclusion bodies fraction
harvested from E. coli clones induced with 1mmol/L IPTG at 37°C. ..... 60
4.8 Batch II induction: SDS-PAGE profile of expressed NS1A fusion
protein extracted from E. coli clones after 1mmol/L IPTG induction
at 37°C. ................................................................................................. 61
4.9 IMAC purification on cytoplasmic fraction of NS1A protein expressed
in E.coli................................................................................................. 62
4.10 IMAC purification on inclusion bodies fraction of NS1A protein
expressed in E.coli. ............................................................................... 63
4.11 The deduced amino acid sequence and hydrophobicity profile of
recombinant NS1A protein. ................................................................... 65
4.12 IMAC purification on cytoplasmic fraction of NS1A recombinant
protein expressed in E. coli after 1mmol/L IPTG induction at 37°C. .... 67
4.13 IMAC purification on inclusion bodies fraction of NS1A recombinant
protein expressed in E. coli after 1mmol/L IPTG induction at 37°C. ...... 68
4.14 SDS-PAGE and western blot analysis of NS1A fusion protein. .............. 70
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4.15 IMAC purification of inclusion bodies fraction under and denaturing
condition. .............................................................................................. 72
4.16 Colloidal gold staining and Western blot analysis of NS1A fusion protein
in inclusion bodies fraction. ................................................................... 73
4.17 Analysis of crude lysate and its purified protein fractions by IMAC. ...... 75
4.18 Effect of buffer exchange with 8M urea buffer on IMAC eluates. .......... 76
4.19 Batch V expression and AEC purification on inclusion bodies fraction
of NS1A recombinant protein expressed in E.coli after 5mM lactose
induction at 37°C. ................................................................................. 78
4.20 Colloidal gold staining and Western blot analysis on AEC purification
of NS1A fusion protein from inclusion bodies fraction of batch V with
5mM lactose induction at 37°C. ............................................................ 79
4.21 Colloidal gold staining and Western blot analysis of NS1A fusion
protein after ammonium sulfate precipitation. ........................................ 81
4.22 Batch VI overexpression result and 20% ammonium precipitation on
inclusion bodies fraction of NS1A recombinant protein expressed in
E.coli after 5mM lactose induction at 37°C. .......................................... 82
4.23 Colloidal gold staining and Western blot analysis of NS1A fusion
protein after ammonium sulfate precipitation. ........................................ 83
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LIST OF SYMBOLS/ ABBREVIATIONS/ NOTATIONS/ TERMINALOGY
AEC - Anion Exchange Chromatography APS - Ammonium persulfate BSA - bovine serum albumin
bp - base pairs
CuSO4 - copper sulfate
CV - column volume
dH2O - distilled water
DNA - deoxyribonucleic acid
dNTPs - deoxynucleotide triphosphates
dsRNA - double stranded RNA
E. coli - Escherichia coli
ED - effector domain
ELISA - Enzyme-linked immunosorbent assay
EtBr - Ethidium bromide
g - gram
HA - hemagglutinin
HCl - hydrochloric acid
His - histidine
IB - inclusion bodies
IFN - interferon
IMAC - Immobilized Metal Ion Affinity Chromatography
IPTG - isopropyl-β-D-thiogalatoside
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KCl - Potassium chloride
kDa - kilo dalton
KH2PO4 - Potassium dihydrogen phosphate
kV - kilo volt
L - liter
LB - Luria-Bertani
m - mille
mmol/L; mM - milli molar
NA - neuraminidase
NaCl - Sodium chloride
NaH2PO4 - Sodium phosphate
NAOH - Sodium hydroxide
NEP - nuclear export protein
NeuAc - N-acetylneuraminic acid
NLS - nuclear localization sequence/signal
nm - nano meter
No. - nombor
OD - optical density
ORF - overlapping open reading frame
PBS - phosphate buffer saline
PCR - polymerase chain reaction
PI3K - phosphatidylinositol 3-kinase
PKR - protein kinase R
RBD - dsRNA-binding domain
RNA - ribonucleic acids
RNP - ribonucleoprotein
RT-PCR - Reverse transcriptase- polymerase chain reaction
SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEAR - Southeast Asia region
TAE - Tris-Acetate electrophoresis buffer
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TEMED - N, N, N´, N´-Tetramethyl ethylenediamine
TM - transmembrane
Tris - 2-hydroxymethyl-2-methyl-1,3-propanediol
Trp - tryptophan
µ - macro
vRNP - viral ribonucleoprotein
WHO - World Health Organization
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LIST OF APPENDICES
APPENDIX TITLE PAGE
A Electroporation result of BL21(DE3)-pET32c- 98
NS1A plasmid after overnight growth
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CHAPTER 1
INTRODUCTION
1.1 Background of Study
Influenza is generally defined as a type of infection caused by influenza virus.
This virus is highly pathogenic and it is usually considered as the causative agent of
zoonotic respiratory disease (reviewed by Pushko, 2009). In addition, it is well
documented that an avian as well as a vertebrate including human can be the
intermediate host of the influenza virus (reviewed by Cox, 1998; Gibbs et al., 2009).
This virus is often detected as a reassortant from more than one parent (reviewed by
Hampson and Mackenzie, 2006; Gibbs et al., 2009) and its infection is usually
associated with cellular alteration, apoptosis and host mortality (Schultz-Cherry et al.,
2001).
The origin of the influenza virus always raises intriguing questions for the world.
Recently, the influenza virus H1N1 outbreak is of great concern to the world. It is
believed that the influenza A strain may share circulation among the genetically-distinct
hosts (Guan et al., 1996; Rappole and Hubálek, 2006). It is not surprising that the
influenza virus subtype was detected seasonally showing co-circulation with the earlier
pandemic strains (reviewed Cox, 1998; reviewed by Hampson and Mackenzie, 2006;
Nelson et al., 2008). In 1918, the respiratory infection in swine was firstly detected
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(reviewed by Nichols and W. LeDuc, 2009) and has caused approximately over 40
million deaths in the world (reviewed by Nicholson et al., 2003 and Pushko, 2009).
Until recently, the isolation of influenza viruses has been done extensively from
a spectrum of fowls and other mammalian species including human (reviewed by
Hampson and Mackenzie, 2006). In addition, researchers nowadays are able to visualize
the viral genome in three-dimensional structure by using advanced technology. There
are many scientists concerned with the innate properties of influenza virus and most
studies are related to gene regulation, gene expression, ecology and serology of the
influenza virus. The cloning and expression experiments have generally resulted in a
better understanding of the properties of viral proteins for antigenicity analysis and
vaccine study. Subsequent advances in genetic engineering as well as protein
engineering are broadly utilized for rapid virus detection.
The influenza viruses, which can be classified into types A, B and C, are
included into the family of Orthomyxoviridae (Pringle, 1996; Bouvier and Palese, 2008).
The influenza A has shown identical pathogenic potential with influenza B and it was
extensively characterized as pandemics as well as epidemic threat (reviewed by Pushko,
2009) with a high transmission rate (Gibbs et al., 2009; reviewed by Nichols and W.
LeDuc, 2009). The influenza C characterizes an occasional spread and it is less harmful
to the human health as compared to influenza A or B (Matsuzaki et al., 2004)
Nevertheless, evidence has shown that the influenza C might be latent in the swine
(Matsuzaki et al., 2004) as well as the newborn (reviewed by Hampson and Mackenzie,
2006).
Influenza A virions are normally found in spherical shape with 80 to 120nm in
diameter (Donatelli et al. 2003; reviewed by Pushko, 2009). However, its size may
reach 300nm in length for filamentous form (Suri, 2007). This progeny virus particle is
unconquered. This virion is known to govern its genetically-distinct proteins either in
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extracellular or intracellular activity. Its membrane-bounded proteins, detected on or in
the coated virion, involved not only in the viral replication but also ribonucleoprotein
(RNP) assembly, thereby suggesting they are deployed to help in viral regulation during
viral infection when the influenza virus is resisting the ongoing host immune response
(reviewed by Cox, 1998; Bouvier and Palese, 2008).
In influenza A virus, the NS1A protein is encoded by the shortest viral RNA
segment. This protein is specifically assembled by at least 230 amino acids. It is a
multifunctional protein, involving significantly in the protein-RNA (Qiu and Krug, 1994)
and protein-protein interaction (Xia et al., 2009). The NS1A protein plays an important
role not only in the antiviral response but also in the post transcriptional activity in its
host (Lin et al., 2007). Further, Zohari et al. (2008) by studying the phylogenetic
relationship of NS1A gene isolated from genetically distinct infected cells, demonstrated
that the NS1A protein could undergo evolutionary divergence occasionally.
1.2 Research Objectives
This research presented here focused on three main objectives. First, it aimed to
clone the targeted gene, NS1A gene. Next, overexpression of the recombinant protein
was attempted in the E.coli strain. Then, the partially purified recombinant protein was
further determined through immunodetection. The specific objectives of this study were:
i. To clone influenza A NS1 gene in pET-32c(+) vector
ii. To over express influenza A NS1 recombinant protein in E. coli BL21(DE3)
iii. To partially purify influenza A NS1 recombinant protein
iv. To determine the immunogenicity of influenza A NS1 recombinant protein
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1.3 Research Scope
The research was divided into four main parts which were cloning,
overexpression, purification and immunogenicity analysis. The NS1 recombinant
protein of influenza A H1N1 was successfully cloned into pET-32c(+) vector and this
led to subsequent expression of the recombinant protein in E. coli BL21 (DE3) strain. In
this project, a series of protein separation and purification process were used to purify
NS1A recombinant protein. Furthermore, the protein immunoblotting was briefly
performed to detect the targeted NS1 protein from the separated protein.
1.4 Problem Statement
The influenza virus spreads globally in the biosphere and it may lead to critical
causalities during an outbreak. Since the Spanish influenza in 1918, the record has
indicated significantly its potential circulation around the world and successively caused
the Asian influenza in 1957, Hong Kong influenza in 1968 (reviewed by Cox, 1998 and
Pushko, 2009) and the recent pandemic influenza 2009. In an update to influenza
situation in Southeast Asia region (SEAR) by World Health Organization (WHO), up to
5th of August 2010, the pandemic H1N1 2009 has caused severe outbreaks, killing 3%
of the population in Southeast Asia alone. The epidemiological summary has indicated
that Ukraine which is the second largest country of Eastern Europe and India which
possesses the largest population in Southern Asia, were detected to be still active in
influenza A H1N1 virus.
Accumulated studies have revealed that the NS1A protein could give rise to a
higher virulence (reviewed by Cox, 1998; Nicholson et al., 2003). Yet, most of the
researchers have widely studied the viral surface glycoproteins, hemagglutinin (HA) and
neuraminidase (NA), which are considered to involve significantly in the virus
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classification and antigenic shift accompanied with genomic reassortment (reviewed by
Cox, 1998; Wagner et al., 2001).
Besides that, this nonstructural protein is not synthesized within the virion itself
(reviewed by Cox, 1998) but it expresses abundantly in the nucleus of the newly-
infected cell (Li et al., 1998). Investigation done by Birch and his colleagues (1997) has
revealed that both the healthy cell and the vaccinated cell are able to inactivate or
attenuate the NS1A protein. In addition, the expression of the NS1 protein was
attempted in the prokaryotic bacteria, yeast and mammalian cells for protein interaction
study. The characteristics of expressing vector (Ma et al., 2009), codon-tRNA
correlation (Gouy and Gautier, 1982; Ikemura, 1985) as well as the toxicity of NS1
protein (Ward et al., 1994) could affect the protein expression.
Nowadays, the cloning and expression work is ubiquitous in biotechnology field.
Nevertheless, the cloning and expression of NS1 protein is still new, elementary and not
very in-depth exploration particularly in the Asian region. However, it can be
considered as the fundamental source for future research. This nonstructural protein can
be further investigated by studying the protein characterization, their innate properties,
the protein binding mechanism, protein topology and others. Much remained to be
learnt about this protein.