Fed-Batch Fermentation: A Practical Guide to Scalable Recombinant Protein Production in Escherichia Coli

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 bioreactorThe fed-batch fermentation is designed to be accomplished in a single day with the preparation work being done on the day priorThe fed-batch fermentation described in this book is a robust process and can be easily scaled for CMO production of protein product INDICE: List of figures and tablesFiguresTablesAbout the author1: Introduction to fermentationAbstract1.1 A brief history of early fermentation and the discovery of DNA1.2 The rise of biotechnology I1.3 The rise of biotechnology II2: Generation of a recombinant Escherichia coli expression systemAbstract2.1 Plasmids2.2 Cloning of foreign gene into plasmid2.3 Transcription of gene into messenger RNA (mRNA)2.4 Host cell2.5 Transformation of E. coli2.6 Making competent cells2.7 Expression screening of transformed host cells2.8 Bacterial growth preparation2.9 Streaking and inoculating plates2.10 Generation of a working cell bank (glycerol stock)3: Recombinant fed-batch fermentation using Escherichia coliAbstract3.1 Growth kinetics of E. coli3.2 Reactor kinetics3.3 The bioreactor system3.4 Set-up and performance of a 2 liter fed-batch fermentation3.5 Analysis of fed-batch fermentation3.6 Sample preparation for SDS PAGE4: Escherichia coli produced recombinant protein: Soluble versus insoluble productionAbstract4.1 Introduction4.2 Translation from RNA into protein4.3 The protein4.4 Soluble protein expression in E. coli4.5 The inclusion body4.6 Isolation and solubilization of inclusion bodies4.7 Ni purification of recombinant protein product4.8 Ni purified protein5: The future of Escherichia coli recombinant fermentationAbstractReferencesIndex

• ISBN: 978-0-08-101367-0
• Editorial: Woodhead Publishing
• Encuadernacion: Rústica
• Páginas: 270
• Fecha Publicación: 30/06/2016
• Nº Volúmenes: 1
• Idioma: Inglés