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.

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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

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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.

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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

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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.

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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.

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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:

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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:

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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:

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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

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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

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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

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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

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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

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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