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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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To my dearest parents and siblings

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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.