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Informationen zum Autor Garner G. Moulton has a Masters of Science in Biology from the Program of Cell and Molecular Biology at San Diego State University. He has been working in biotech and non-profit research for over 25 years, and has focussed specifically on protein purification and fermentation process development over the last 17 years. During the past 4 years he has been employed by the Infectious Disease Research Institute (IDRI) in Seattle, WA, USA, where he is a principal research associate in charge of the Process Development Department. He has developed many robust fermentation processes that can be adapted to many different recombinant products without having to re-optimize conditions of growth and expression. Numerous projects have been successfully transferred to a contract manufacturer for scale-up production and use in clinical trials. Klappentext 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. Zusammenfassung 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. Inhaltsverzeichnis List of figures and tables Figures Tables About the author 1: Introduction to fermentation Abstract 1.1 A brief history of early fermentation and the discovery of DNA 1.2 The rise of biotechnology I 1.3 The rise of biotechnology II 2: Generation of a recombinant Escherichia coli expression system Abstract 2.1 Plasmids 2.2 Cloning of foreign gene into plasmid 2.3 Transcription of gene into messenger RNA (mRNA) 2.4 Host cell 2.5 Transformation of E. coli 2.6 Making competent cells 2.7 Expression screening of transformed host cells 2.8 Bacterial growth preparation 2.9 Streaking and inoculating plates 2.10 Generation of a working cell bank (glycerol stock) 3: Recombinant fed-batch fermentation using Escherichia coli Abstract 3.1 Growth kinetics of E. coli 3.2 Reactor kinetics 3.3 The bioreactor system 3.4 Set-up and performance of a 2 liter fed-batch fermentation 3.5 Analysis of fed-batch fermentation 3.6 Sample preparation for SDS PAGE 4: Escherichia coli produced recombinant protein: Soluble versus insoluble production Abstract 4.1 Introduction 4.2 Translation from RNA into protein 4.3 The protein 4.4 Soluble protein expression in E. coli 4.5 The inclusion body 4.6 Isolation and solubilization of inclusion bodies 4.7 Ni purification of recombinant protein product 4.8 Ni purified protein 5: The future of Escherichia coli recombinant fermentation Abstract References Index...
Sommario
- List of figures and tables
- About the author
- 1: Introduction to fermentation
- Abstract
- 1.1 A brief history of early fermentation and the discovery of DNA
- 1.2 The rise of biotechnology I
- 1.3 The rise of biotechnology II
- 2: Generation of a recombinant Escherichia coli expression system
- Abstract
- 2.1 Plasmids
- 2.2 Cloning of foreign gene into plasmid
- 2.3 Transcription of gene into messenger RNA (mRNA)
- 2.4 Host cell
- 2.5 Transformation of E. coli
- 2.6 Making competent cells
- 2.7 Expression screening of transformed host cells
- 2.8 Bacterial growth preparation
- 2.9 Streaking and inoculating plates
- 2.10 Generation of a working cell bank (glycerol stock)
- 3: Recombinant fed-batch fermentation using Escherichia coli
- Abstract
- 3.1 Growth kinetics of E. coli
- 3.2 Reactor kinetics
- 3.3 The bioreactor system
- 3.4 Set-up and performance of a 2 liter fed-batch fermentation
- 3.5 Analysis of fed-batch fermentation
- 3.6 Sample preparation for SDS PAGE
- 4: Escherichia coli produced recombinant protein: Soluble versus insoluble production
- Abstract
- 4.1 Introduction
- 4.2 Translation from RNA into protein
- 4.3 The protein
- 4.4 Soluble protein expression in E. coli
- 4.5 The inclusion body
- 4.6 Isolation and solubilization of inclusion bodies
- 4.7 Ni purification of recombinant protein product
- 4.8 Ni purified protein
- 5: The future of Escherichia coli recombinant fermentation
- References
- Index
