Studies on cell growth, plasmid stability and protein expression by recombinant Escherichia coli.

STUDIES ON CELL GROWTH, PLASMID STABILITY AND PROTEIN EXPRESSION BY RECOMBINANT ESC

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by

ASHWANI MATHUR

Department of Biochemical Engineering and Biotechnology

Submitted

In fuiffilment ofthe requirements ofthe degree of Doctor of Philosophy

to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI HAUZ KHAS, NEW DELHI 110016, INDIA

AUGUST 2008

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CERTIFICATE

This is to certify that the thesis entitled, "Studies on ぐ乞 I Growth，円asmid Stabil如andi物tein E;甲ression 勿Recombinant Escherichia co/i" being submitted by Mr. Ashwani Mathur to the Indian Institute of Technology Delhi, for the award of the degree of Doctor of Philosophy in Biochemical Engineering and Biotechnology is a bonaffide record of original research work carried out by him under my supervision in conformity with the rules and regulations ofthe institute.

The results presented in this thesis have not been submitted, in part or full, to any other university or institute for the award of any degree or diploma.

Prof. Subhash Chand Professor

Biochemical Engineering and Biotechnology Indian Institute of Technology Delhi

New Delhi-i 10016, India

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Research provides one an opportunity to pit one's wits with the seminal principle underlying this creation. It also furnishes the opportunity of meeting people at their best.

The deeper my research grew, the more I had the pleasure to meet people who loved their work and delighted in sharing in with other. I am taking this opportunity to add a'few heartfelt words to all those who made it possible especially to my supervisor, Prof.

Subhash Chand, Professor, Department of Biotechnology, Indian Institute of Technology, Delhi.

With great reverence, I express my gratitude and respect to Prof Subhash Chand, for his fruitful and constructive guidance in the most erudite manner. His unbounded affection, inspirational guidance, unfailing enthusiasm, and tireless interest make me remain indebted to him. I am extremely thankful for his keen interest and perpetual encouragement offered all through the pursuance ofthis work.

I would like to take the opportunity to thank SRC members Dr. S.N.

Mukhopadhyay (SRC, Chairperson), Dr. P.K. Roy Chaudhary and Dr. T.R. Rao (External expert) for their valuable suggestions and encouragement during this period.

I ac細owledge Dr. Raj Kamal Bhatnagar, Group Leader, IR Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi for providing me with bacterial strains and vectors and permitting me to carry out some of the experimental work in his laboratory.

I would like to express my thanks to Dr. S. Yasuda, Former Head, E. coli Genetic Stock Centre, Japan, for providing me with plasmid vectors.

I have deep and sincere sense of gratitude towards our Head of the Department, Dr. A.K. Srivastava for providing me all in-house research facilities and an excellent working environment in the department.

I thankfully acknowledge Council of Scientiffic and Industrial Research (CSIR), Government of India, for providing me ffinancial assistanceship during this period.

I appreciate and wish to express my heartfelt thanks to my seniors Dr. Ritu Verma, Dr. Ruchi Shukla, Dr. Milli Prabhakar and Dr. Rupali Walia for their support. I would also like to thank all my colleagues in the department for their support during this period.

I express my sincere thanks towards Mrs. Renu Sethi for her all time support in the research work. With great pleasure, I express my sincere thanks to departmental staff for providing me help in all possible ways.

Finally, I owe my family, my father, mother, brother, in-laws and my beloved wife for providing me strength and conffidence to attain this academic level.

(Ashw 轟

ABSTRACT

Recombinant protein expression in Escherichia coli is always an issue of concern because of problems such as protein aggregation, lack of protein secretion and plasmid instability. Present study shows the effect of four different expression vectors (pET29(b), pET32(b), pET43(b) and pCS22) on expression, localization and secretion of a model recombinant protein (u-amylase). An analysis of vector characteristics on cell growth and protein expression was observed. The reduction in speciffic growth rate of recombinant bacteria as compared to untransformed host can be attributed to higher metabolic load. Metabolic load on the host cell was analyzed in terms of oxygen uptake rate (OUR) by recombinant hosts in both uninduced and induced conditions and was found to increase with increase in plasmid size. Effect of metabolic load on bacterial size was also compared and no signifficant difference in the size ofhosts in both uninduced and induced conditions was observed.

Localization of the enzyme protein indicated its distribution as intracellular, periplasmic and extracellular fflactions. Activity was analyzed using in-gel assay ( zymogram) in different cellular fflactions. Transmission electron microscopy (TEM) showed intracellular aggregation of recombinant protein expressed in chemically (IPTG) induced recombinant cells as inclusion bodies while no such aggregation was seen with host transformed with temperature inducible pCS22 vector. Total activity of a-amylase under uncontrolled and controlled dissolved oxygen conditions was highest in host transformed with pCS22 vector with values of 56.8 and 80. 1 U/ml respectively.

An analysis of plasmid instability by different recombinant system was studied and results were analyzed based on available mathematical models. It was observed that segregational instability (R) shown by recombinant host increased with increase in plasmid size and ranged between 1.4 x lO-3 per generation (jCS22-amyE ,6.6 kb) to

111

2.2 x iO-3 per generation (pET43(b)-amyE, 8.76 kb). Difference in speciffic growth rate (dii) of plasmid free and plasmid bearing cells does not show marked variation with variation in plasmid size and the ratio of growth rates ofplasmid free to plasmid bearing cells (a) was around unity showing the equal competence of plasmid free and plasmid bearing cells. Experimental results showed a correlation between vector size and segregational instability. Based on protein expression, folding and secretion studies, pCS22 vector was found to be a better vedtor compared to other chemical inducible vector and was used for further studies.

Cell immobilization was used as a strategy to improve plasmid stability. Though some improvement in stability of vector was observed but that was at the cost of reduced growth rate. However, complete stability was not attained. Use of par gene for improving plasmid instability showed 100% improvement. The presence ofpar gene in pCS22 vector increased the metabolic load on the cell as analyzed by OUR but it does not affect protein expression.

In order to improve extracellular secretion of recombinant protein, glycine was added to growing culture at the time of induction. Increase in extracellular enzyme activity was observed when the system was treated with 1% glycine aifier induction.

Recombinant bacteria transformed with pCS22 and pCS22par showed approximately four-fold increase in extracellular a-amylase activity. However, no such signifficant effect in secretion was seen using other recombinant systems. It indicates the role of glycine in enhancing the excretion of periplasmic recombinant protein fraction, rather than intracellular recombinant protein fraction.

Keywords: Escherichia coli, Metabolic load, Protein secretion, Periplasmic fraction, Inclusion bodies, Signal sequence, par gene, Cell immobilization

IV

CONTENTS

ACKNOWLEDGEMENT ii

ABSTRACT 111

LIST OF FIGURES ix LIST OF TABLES xii ABBREVIATIONS USED xiv

Page No.

I INTRODUCTION 1-5

2 REVIEW OF LITERATURE 6-38

2.1 Therapeutic proteins 6

2.2 Production of industrially important enzymes using 8 recombinant cells

2.3 Strategies for recombinant heterologous protein hyper- i 2 expression

2.3.1 Choice ofhost 12

2.3.2 Vector characteristics i 4

2.4 Cellular stress response to recombinant vectors 17 2.5 Problems associated with gene expression in E. coli i 9

2.6 Plasmid instability 20

2.6.1 Structural Instability of vector 20

2.6.2 Segregational Instability ofVector 22

2.7 Mechanism ofpiasmid segregation in low copy number 24 plasmids

2.8 Parameters affecting plasmid stability 27 2.9 Models to study the effect of plasmid instability on growth 28

kinetics and protein production

2.10 Strategies to enhance plasmid stability 30

2.10.1 Bioprocess Strategies 36

2.10.2 Cellular/Molecular strategies: Role ofpartitioning locus 38 3

MATERIALS AND METHODS 39-66

3.1

MATERIALS 39

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3.2.

3.2.

Chemicals and reagents 39

Bacterial Strains 41

Plasmids 42

Antibiotics 42

Buffers 43

Reagents 45

Primer sequence for PCR ampliffication 49

METHODS 50

Growth, maintenance and preservation of recombinant E. 50 coli

Medium&growth conditions 50

Isolation ofDNA fflom E.coli cells 50 Genomic DNA isolation fflom B. amyloliquefaciens 51 Restriction digestion of DNA with restriction endonucleases 52

Agarose gel electrophoresis 52

Gel elution 53

Dephosphoylation of Plasmid DNA 53

Ligation 53

Competent cell preparation 54

Transformation of E. coli 55

Primer designing 55

Preparation of primer solutions 56

PCR ampliffication 56

Colony PCR for screening ofrecombinants 57 Screening oftransformants containing the a-amylase gene 57 fragment

Induction ofrecombinant bacteria 58

Fractionation ofrecombinant E.coli for recombinant protein 59

Extracellular fraction 59

Periplasmic protein fraction 59

Cytoplasmic protein fraction 59

Quantitative assay of a-amylase 60

Determination of enzyme activity in different cellular 60 3.1.1

3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.2 3.2.1

3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.2.8 3.2.9 3.2.10 3.2.11

3.2.14 3.2.15 3.2.16

3.2.17 3.2.18 3.2.18.1 3 .2. 18.2 3 .2. 18.3 3.2.19 3.2.20

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66 67-121 67 fflactions

Protein gel electrophoresis

Detection of a-amylase activity by Zyrnogram (in-gel assay) Transmission Electron Microscope (TEM) of recombinant system s

Plasmid instability analysis

Excretion of recombinant protein using glycine Activation of sugarcane bagasse

Immobilization ofrecombinant cells on activated bagasse and adsorption isotherm

Bioprocess Studies Inoculum development Batch fermentation

Volumetric oxygen transfer rate (KLa) and Oxygen uptake rate (OUR)

Continuous culture studies Packed bed reactor studies

Statistical analysis of results

RESULTS AND DISCUSSION

Designing different recombinant expression systems using E, coli

Screening of B. amyloliquefaciens 6 1 0 for u-amylase activity Ampliffication of amyE gene fflom B. amyloliquefaciens Identiffication ofpositive clones by colony PCR

Restriction digestion of a

四

Bioprocess studies with different recombinant system(s) Cell growth and protein expression studies in shake flask Growth studies ofuninduced cells in shake flask

Growth studies of induced cells in shake flask

Recombinant protein expression studies in shake flask Cell growth and protein expression studies in bioreactor Growth studies ofinduced and uninduced recombinant cells 3.2.21

3.2.22 3.2.23

3.2.24 3.2.25 3.2.26 3.2.27

3.2.28 3.2.28.1 3.2.28.2 3.2.28.3

3.2.28.4 3.2.28.5 3.2.29 4 4.1

4.1.i 4.i.2 4.1.3 4.1.4

4.2 4.2.1 4.2.1.1 4.2.1.2 4.2.1.3 4.2.2 4.2.2.

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4.2.2.2 Analysis of recombinant protein in differe凪cellular 95 fractions

4.2.2.3 Oxygen uptake rate for analysis of metabolic burden on 97 recombinant E. coli cells

4.2.2.3.1 Analysis ofvolumetric oxygen uptake rate (KLa) 97

4.2.2.3.2 Analysis of OUR 97

4.2.2.4 Analysis of recombinant bacterial size i 00 4.3 Analysis of segregational instability of recombinant plasmid 101

vectors

4.4 Biochemical strategy of improving plasmid vector stability i 09

4.4.1 Adsorption Isotherm 109

4.4.2 Analysis ofplasmid instability in immobilized recombinant i io E. coli/pCS22 system

4.5 Genetic strategy of improving plasmid stability i i 2 4.5.1 Ampliffication ofpar gene of plasmid pSC i O i i 12 4.5.2 Effect ofpar gene on plasmid maintenance and cellular i i 3

behaviour

4.5.3 Cell growth and protein expression by E. coli/pCS22par- 115 amyE

4.6 Excretion of recombinant protein fflom different recombinant i i 9 systems

CONCLUSION AND SUMMARY 122-124

ANNEXURE-I 125-136

REFERENCES 137-160

ANNEXURE-Il i 6 1 -162 BIODATA

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