Fed-batch Fermentation is primarily a practical guide for recombinant protein production in E. coli using a Fed-batch Fermentation process. Ideal users of this guide are teaching labs and R&D labs that need a quick and reproducible process for recombinant protein production. It may also be used as a template for the production of recombinant protein product for use in clinical trials. The guide highlights a method whereby a medium cell density - final Ods = 30-40 (A600) - Fed-batch Fermentation process can be accomplished within a single day with minimal supervision. This process can also be done on a small (2L) scale that is scalable to 30L or more. All reagents (media, carbon source, plasmid vector and host cell) used are widely available and are relatively inexpensive. This method has been used to produce three different protein products following cGMP guidelines for Phase I clinical studies.•This process can be used as a teaching tool for the inexperienced fermentation student or researcher in the fields of bioprocessing and bioreactors. It is an important segue from E. coli shake flask cultures to bioreactor. •The fed-batch fermentation is designed to be accomplished in a single day with the preparation work being done on the day prior. •The fed-batch fermentation described in this book is a robust process and can be easily scaled for CMO production of protein product

List of figures and tables vii

About the author xi

1 Introduction to fermentation 1

1.1 A brief history of early fermentation and the discovery of DNA 2

1.2 The rise of biotechnology I 6

1.3 The rise of biotechnology II 14

2 Generation of a recombinant Escherichia coli expression system 31

2.1 Plasmids 32

2.2 Cloning of foreign gene into plasmid 37

2.3 Transcription of gene into messenger. RNA (mRNA) 38

2.4 Host cell 42

2.5 Transformation of E. coli 43

2.6 Making competent cells 48

2.7 Expression screening of transformed host cells 50

2.8 Bacterial growth preparation 53

2.9 Streaking and inoculating plates 57

2.10 Generation of a working cell bank (glycerol stock) 61

3 Recombinant fed-batch fermentation using Escherichia coli 63

3.1 Growth kinetics of E. coli 64

3.2 Reactor kinetics 67

3.3 The bioreactor system 84

3.4 Set-up and performance of a 2 liter fed-batch fermentation 93

3.5 Analysis of fed-batch fermentation 104

3.6 Sample preparation of SDS PAGE 106

4 Escherichia coli produced recombinant protein: Soluble versus insoluble production 109

4.1 Introduction 110

4.2 Translation from RNA into protein 111

4.3 The protein 119

4.4 Soluble protein expression in E. coli 127

4.5 The inclusion body 129

4.6 Isolation and solubilization of inclusion bodies 135

4.7 Ni purification of recombinant protein product 136

4.8 Ni purified protein 138

5 The future of Escherichia coli recombinant fermentation 141

References 151

Index 165

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