Recombinant Protein Expression In Microbial Systems

With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies. Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes. Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production. With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.

While the choices of microbial and eukaryotic expression systems for production of recombinant proteins are many, most researchers in academic and industrial settings do not have ready access to pertinent biological and technical information since it is normally scattered throughout the scientific literature. This book closes the gap by providing information on the general biology of the host organism, a description of the expression platform, a methodological section -- with strains, genetic elements, vectors and special methods, where applicable -- as well as examples of proteins produced with the respective platform. The systems thus described are well balanced by the inclusion of three prokaryotes (two Gram-negatives and one Gram-positive), four yeasts, two filamentous fungi and two higher eukaryotic cell systems -- mammalian and plant cells. Throughout, the book provides valuable practical and theoretical information on the criteria and schemes for selecting the appropriate expression platform, the possibility and practicality of a universal expression vector, and on comparative industrial-scale fermentation, with the production of a recombinant Hepatitis B vaccine chosen as an industrial example. With a foreword by Herbert P. Schweizer, Colorado State University, USA: "As a whole, this book is a valuable and overdue resource for a varied audience. It is a practical guide for academic and industrial researchers who are confronted with the design of the most suitable expression platform for their favorite protein for technical or pharmaceutical purposes. In addition, the book is also a valuable study resource for professors and students in the fields of applied biology and biotechnology."

The general field of fundamental and applied biotechnology becomes increasingly important for the production of biologicals for human and veterinary use, by using prokaryotic and eukaryotic microorganisms. The papers in the present book are refereed articles compiled from oral and poster presentations from the EFB Meeting on Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology, which was organized in Semmering/A from 5th to 8th October 2000. A special feature of this meeting was the comparison of different classes of host cells, mainly bacteria, yeasts, filamentous fungi, and animal cells, which made obvious that many physiological features of recombinant protein formation, like cell nutrition, stress responses, protein folding and secretion, or genetic stability, follow similar patterns in different expression systems. This comparative aspect is by far the point of most interest because such comparisons are rarely done, and if they are done, their results are most often kept secret by the companies who generated them. Audience: Presently, a comparable book does not exist because the compiling of manuscripts from all fields of biotechnology (prokaryotic as well as eukaryotic, up to animal cell biotechnology) is not done in general. This particularity makes this book very interesting for postgraduate students and professionals in the large field of biotechnology who want to get a more global view on the current state of the expression of recombinant biologicals in different host cell systems, the physiological problems associated with the use of different expression systems, potential approaches to solve such difficulties by metabolic engineering or the use of other host cells, and the cooperation between process development and strain improvement, which is crucial for the optimisation of both the production strain and the process. This book should be in every library of an institution/organization involved in biotechnology.

1. Expression strategy (Michael Dyson) 2. Protein expression in Escherichia coli (Rosalind Kim) 3. Expression engineering of synthetic antibodies using ribosome display (Matthew DeLisa and Lydia M. Contreras Martinez) 4. Refolding proteins from inclusion bodies (Renaud Vincentelli) 5. Selection of protein variants with improved expression using GFP-derived folding and solubility reporters (Geoffrey Waldo and Stéphanie Cabantous) 6. Protein expression in the wheat germ cell-free system (Yaeta Endo and Tatsuya Sawasaki) 7. Saccharomyces cerevisiae ; A microbial eukaryotic expression system (Christine Lang) 8. Expression of proteins in Pichia pastoris (Geoff and Joan Lin-Cereghino and Wilson Leung) 9. Improved baculovirus expression vectors (Linda King, Richard Hitchman and Robert Possee) 10. Transient transfection of insect cells for rapid expression screening and protein production (Robert Novy et al.) 11. Generation of stable CHO cell lines for protein expression (Zhijian Lu et al.) 12. Transient expression in HEK293-EBNA1 cells (Yves Durocher, Roseanne Tom and Louis Bisson) 13. Nisin- and subtilin-controlled gene expression systems for Gram-positive bacteria (Oscar Kuipers and Jan Kok) 14. Protein expression using lentiviral vectors (Bernard Massie, Renald Gilbert and Sophie Broussau) 15. Expression in mammalian cells using BacMam viruses (Yu-Chen Hu and Hsiao-Ping Lee) List of suppliers;Index

More then 20 years have passed now since the first recombinant protein producing microorganisms have been developed. In the meanwhile, numerous proteins have been produced in bacteria, yeasts and filamentous fungi, as weIl as higher eukaryotic cells, and even entire plants and animals. Many recombinant proteins are on the market today, and some of them reached substantial market volumes. On the first sight one would expect the technology - including the physiology of the host strains - to be optimised in detail after a 20 year's period of development. However, several constraints have limited the incentive for optimisation, especially in the pharmaceutical industry like the urge to proceed quickly or the requirement to define the production parameters for registration early in the development phase. The additional expenses for registration of a new production strain often prohibits a change to an optimised strain. A continuous optimisation of the entire production process is not feasible for the same reasons.

An Increasing Number Of Recombinant Therapeutic Proteins Are Currently Being Developed, Tested In Clinical Trials And Marketed For Used. Most Of The Recombinant Therapeutic Proteins Are Being Successfully Produced Into Escherichia Coli And Pichia Pastoris Expression System. These Two Expression Systems Are Very Much Efficient And Cost Effective. This Book Takes A Close Look Of These Two Expression Systems And Fermentation Conditions, Purification Strategies Of Different Recombinant Proteins. This Book Also Discusses The Market Size And Cost Analysis For The Production Of Different Therapeutic Proteins And Some General Experimental Protocols For Production. Contents Part I: Recombinant Protein Expression Into Escherichia Coli And Fermentation Conditions; Chapter 1: Introduction; Chapter 2: Construction Of Efficient Expression Vector (Plasmid); Chapter 3: Factors Affecting Transcription, Promoters, Upstream Elements, Transcriptional Terminators, Transcriptional Antitermin, Tightly Regulated Expression Systems; Chapter 4: Mrna Stability; Chapter 5: Factors Affecting Translation, Mrna Translational Initiator, Translational Enhancers, Translational Termination; Chapter 6: Expression Of Target Protein And The Compartments Of Expression, Cytoplasmic Expression, Periplasmic Expression, Extracellular Secretion; Chapter 7: Fusion Proteins; Chapter 8: Post-Translational Protein Folding; Chapter 8: Codon Usage; Chapter 10: Protein Degradation; Chapter 11: Fermentation Conditions For High-Density Cell Cultivation (Hdcc), Growth Medium, Efficient Production Of Recombinant Protein In Hdcc, Nutrient Feeding Strategy In Hdcc; Chapter 12: One Examples Of Protein Production Using E. Coli Expression System; Chapter 13: Conclusion. Part Ii: Recombinant Protein Expression Into Yeast, Pichia Pastoris And Fermentation Conditions; Chapter 1: Introduction; Chapter 2: Why P. Pastoris? Chapter 3: Construction Of Expression Strains, Expression Vectors, Alternative Promoters, Host Strains, Methanol Utilisation Phenotype, Protease-Reduced Host Strains, Integration Of Expression Vectors Into The P. Pastoris Genome, Generating Multicopy Strains; Chapter 4: Post-Translational Modifications Of Secreted Proteins, Secretion Signal Selection, N-Linked Glycosylation; Chapter 5: Production Of Recombinant Proteins In Fermenter Cultures Of The Yeast, Pichia Pastoris, Conceptual Basis For The P. Pastoris Expression System, High-Level Expression In Fermenter Cultures, Protein-Specific Adjustments To Improve Yield, Glycosylation Of Recombinant Proteins, Secretion Signals; Chapter 6: One Examples Of Protein Producing Using P. Pastoris Expression System, Chapter 7: Conclusion. Part Iii: Purification Strategies For Recombinant Proteins; Chapter 1: Purification Of Proteins; Chapter 2: Conventional Chromatography, Ion Exchange Chromatography, Reversed Phase Chromatography, Gel Permeation Chromatography, Affinity Chromatography, Affinity Tags, Cleavage, Conclusion. Part Iv: Market Size And Cost Analysis For The Production Of Therapeutic Proteins; Chapter 1: Market Size Of Therapeutic Proteins; Chapter 2: Outline Structure Of A Productin Unit And Cost Analysis For The Production Of Three Therapeutic Proteins. Part V: General Experimental Protocols; Chapter 1: Different Experimental Protocols, Preparation Of Genome Dna For E. Coli, A Differnt Method For Preparation Of Genomic Dna From Bacteria, Preparation Of Proteins From Periplasm (Osmotic Shock Method), Preparation Of Proteins From Outer Membrane, Transformation Of Plasmid Dna Into E. Coli (Calcium Chloride/Heat Shock Method), Transformation Of Plasmid Dna Into E. Coli (Electroporation), Sds-Page For Large Proteins, Sds-Page For Small Peptide, Pcr Amplification Of Dna, Protein Quantification: Brandford Method, Trans-Bloting For Protein, Restriction Enzyme Digestion Of Dna, Phenol/Chloroform Extraction Of Dna, Ethanol Precipitation Of Dna, Agarose Gel Electrophoresis, Transformation Of E. Coli By Electroporation (Alternative Method), Wizard Tm Pcr Preps Dna Purification System For Rapid, Purification Of Dna Fragments, Alternate Method For Purifying Dna From Agarose Gels, Southern Blotting, Rt Pcr Protocol, Using Superscript Reverse Transcriptase, Preparation Of Sequencing Gels, Isolation Of Rna From Mammalian Cells Using Rnazoltm (Teltest), Preparation For Yeast Transformation, Yeast Transformation, Digesting Prsq-Ura3 With Bamhi, Genomic Dna Preparation Of Yeast, Ligation (Circularisation) Of Genomic Dna Fragments, E. Coli Transformation (Alternate Method), Dna Miniprep From E. Coli (Alternate Method), Basic Plasmid Dna Isolation Protocol, Identification And Determination Of Amount Rec-Hum Proteins Via An Immunoenzymatic Test (Elisa), Determination Of Host Dna Contaminant Into R Hu Protein Through Dot Blot Method, Protocols For Down-Stream Processing.

Gene Expression Systems: Using Nature for the Art of Expression offers detailed information on a wide variety of gene expression systems from an array of organisms. It describes several different types of expression systems including transient, stable, viral, and transgenic systems. Each chapter is written by a leader in the field. The book includes timelines and examples for each expression system, and provides an overview of the future of recombinant protein expression. Provides detailed information on expression systems Covers a variety of promoters and host organisms enabling researchers to tailor protocols to their specific needs Includes timelines and examples Compares pros and cons of each method

Advances in protein expression technologies have mushroomed in recent years. In this book current and emerging expression technologies are reviewed. Reviews of the molecular genetics of expression systems in various organisms are presented. Topics covered include: Expression of extremophilic proteins; expression in E. coli, Bacillus spp., Saccharomyces cerevisiae, and methylotrophic yeasts; insect cell expression and the baculovirus system; and Chinese Hamster Ovary (CHO) cell lines for large-scale protein production. Also covered are two emerging expression systems, Methylobacterium extorquens AM1 and Caulobacter crescentus.