Production Of Recombinant Proteins - Novel Microbial And Eukaryotic Expression Systems

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."

Gerd Gellissen is Senior Scientist at Rhein Biotech GmbH,Düsseldorf and Associate Professor of Microbiology at the Heinrich-Heine University of Düsseldorf.
He has published approximately 130 research papers and co-authored 7 patents. Professor Gellissen is editor of the Wiley-VCH book "Hansenula polymorpha" (2002).

1 Key and Criteria to the Selection of an Expression Platform (Gerd Gellissen, Alexander W.M. Strasser, and Manfred Suckow).

2 Escherichia coli (Josef Altenbuchner and Ralf Mattes).

2.1 Introduction.

2.2 Strains, Genome, and Cultivation.

2.3 Expression Vectors.

2.4 Regulation of Gene Expression.

2.5 Transcription and Translation.

2.6 Protein Production.

2.7 Examples of Products and Processes.

2.8 Conclusions and Future Perspectives.

Appendix.

References.

3 Pseudomonas fluorescens (Lawrence C. Chew,Tom M. Ramseier, Diane M. Retallack, Jane C. Schneider, Charles H. Squires, and Henry W. Talbot).

3.1 Introduction.

3.2 Biology of Pseudomonas fluorescens.

3.3 History and Taxonomy of Pseudomonas fluorescens Strain Biovar I MB101.

3.4 Cultivation.

3.5 Genomics and Functional Genomics of P. fluorescens Strain MB101.

3.6 Core Expression Platform for Heterologous Proteins.

3.7 Production of Heterologous Proteins in P. fluorescens.

3.8 Conclusions.

Appendix.

References.

4 Staphylococcus carnosus and other Gram-positive Bacteria (Roland Freudl).

4.1 Introduction.

4.2 Major Protein Export Routes in Gram-positive Bacteria.

4.3 Extracytosolic Protein Folding.

4.4 The Cell Wall as a Barrier for the Secretion of Heterologous Proteins.

4.5 Degradation of Exported Proteins by Cell-associated and Secreted Proteases.

4.6 Staphylococcus carnosus.

Appendix.

References.

5 Arxula adeninivorans(Erik Böer, Gerd Gellissen, and Gotthard Kunze).

5.1 History of A. adeninivorans Research.

5.2 Physiology and Temperature-dependent Dimorphism.

5.3 Genetics and Molecular Biology.

5.4 Arxula adeninivorans as a Gene Donor.

5.5 The A. adeninivorans-based Platform.

5.6 Conclusions and Perspectives.

Acknowledgments.

Appendix.

References.

6 Hansenula polymorpha (Hyun Ah Kang and Gerd Gellissen).

6.1 History, Phylogenetic Position, Basic Genetics and Biochemistry of H. polymorpha.

6.2 Characteristics of the H. polymorpha Genome.

6.3 N-linked glycosylation in H. polymorpha.

6.4 The H. polymorpha-based Expression Platform.

6.5 Product and Process Examples.

6.6 Future Directions and Conclusion.

Appendix.

References.

7 Pichia pastoris (Christine Ilgen, Joan Lin-Cereghino, and James M. Cregg).

7.1 Introduction.

7.2 Construction of Expression Strains.

7.3 Post-translational Modification of Secreted Proteins.

7.4 Conclusions.

Acknowledgments.

Appendix.

References.

8 Yarrowia lipolytica (Catherine Madzak, Jean-Marc Nicaud, and Claude Gaillardin).

8.1 History, Phylogenetic Position, Basic Genetics, and Biochemistry.

8.2 Characteristics of the Y. lipolytica Genome.

8.3 Description of the Expression Platform.

8.4 Examples.

8.5 Transformation Methods.

8.6 Conclusions and Future Trends.

Acknowledgments.

Appendix.

References.

9 Aspergillus sojae (Margreet Heerikhuisen, Cees van den Hondel, and Peter Punt).

9.1 Introduction.

9.2 Taxonomy.

9.3 The Expression Platform.

9.4 Aspergillus sojae as a Cell Factory for Foreign Proteins.

9.5 Strain Development.

9.6 Outlook.

Acknowledgments.

Appendix.

References.

10 Sordaria macrospora (Ulrich Kück and Stefanie Pöggeler).

10.1 Introduction.

10.2 General Biology.

10.3 Morphological Characterization, Molecular Phylogeny and Life Cycle of Sordaria macrospora.

10.4 Generation of Sterile Mutants as Host Strains.

10.5 S. macrospora as a Safe Host for Heterologous Gene Expression.

10.6 Molecular Genetic Techniques Developed for S. macrospora.

10.7 Isolation and Characterization of Strong Promoter Sequences from S. macrospora.

10.8 Construction of Vectors for Efficient Gene Expression in S. macrospora.

10.9 Successful Expression of the Recombinant EGFP Reporter Gene in S. macrospora.

10.10 Conclusions.

Acknowledgments.

Appendix.

References.

11 Mammalian Cells(Volker Sandig, Thomas Rose, Karsten Winkler, and Rene Brecht).

11.1 Why Use Mammalian Cells for Heterologous Gene Expression?

11.2 Mammalian Cell Lines for Protein Production.

11.3 Mammalian Expression Systems.

11.4 Mammalian Cell-based Fermentation Processes.

11.5 Conclusions.

Acknowledgments.

Appendix.

References.

12 Plant Cells (Rainer Fischer, Richard M Twyman, Jürgen Drossard, Stephan Hellwig and Stefan Schillberg).

12.1 General Biology of Plant Cells.

12.2 Description of the Expression Platform.

12.3 Examples of Recombinant Proteins Produced in Plant Cell Suspension Cultures.

12.4 Methodology Using Model Tobacco Cell Suspension Lines to Produce Recombinant Antibodies.

12.5 Conclusions.

Appendix.

References.

13 Wide-Range Integrative Expression Vectors for Fungi, based on Ribosomal DNA Elements (Jens Klabunde, Gotthard Kunze, Gerd Gellissen, and Cornelis P. Hollenberg).

13.1 Why is a Wide-range Expression Vector Needed?

13.2 Which Elements are Essential for a Wide-range Expression Vector?

13.3 Structure of the Ribosomal DNA and its Utility as an Integration Target.

13.4 Transformation Based on rDNA Integration.

13.5 rDNA Integration as a Tool for Targeting Multiple Expression Cassettes.

13.6 Conclusions and Perspectives.

Acknowledgments.

References.

14 Comparative Fermentation (Stephan Hellwig, Christoph Stöckmann, Gerd Gellissen, and Jochen Büchs).

14.1 Introduction.

14.2 Escherichia coli.

14.3 Staphylococcus carnosus.

14.4 Arxula adeninivorans.

14.5 Hansenula polymorpha.

14.6 Sordaria macrospora.

Appendix.

A14.1 Escherichia coli Media.

A14.2 Staphylococcus carnosus Media.

A14.3 Yeast Media.

A14.4 Sordaria macrospora Media.

References.

15 Recombinant Hepatitis B Vaccines: Disease Characterization and Vaccine Production (Pascale Brocke, Stephan Schaefer, Karl Melber,Volker Jenzelewski, Frank Müller, Ulrike Dahlems, Oliver Bartelsen, Kyung-Nam Park, Zbigniew A. Janowicz, and Gerd Gellissen).

15.1 Introduction.

15.2 Virus and Disease Characteristics.

15.3 Recombinant Vaccine Production.

15.4 HepavaxGene.

15.5 Hepatitis B Vaccines: Past, Present, and Future.

15.6 Conclusions.

References.

16 Biopharmaceuticals and the Industrial Environment (Georg Melmer).

16.1 Introduction.

16.2 Early Success Stories.

16.3 The Bumpy Road Appeared.

16.4 The Breakthrough in Many Areas.

16.5 Which are the Current and Future Markets?

16.6 The Clinical Development of Biopharmaceuticals.

16.7 Drug Delivery and Modification of Proteins.

16.8 Expression Systems for Commercial Drug Manufacture.

16.9 Will Demand Rise?

16.10 Conclusions and Perspectives.

References.

Subject Index.