Production of Recombinant Proteins Edited by Gerd Gellissen Production of Recombinant Proteins. Novel Microbial and Eucaryotic Expression Systems. Edited by Gerd Gellissen Copyright # 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31036-3 Further Titles of Interest G.) Proteins Hansenula polymorpha Biochemistry and Biotechnology Biology and Applications 2001 2002 ISBN 0-471-89906-2 ISBN 3-527-30341-3 G.) Biopharmaceuticals Biochemistry and Biotechnology Biotechnology Second, Completely Revised Edition 2003 Volume 2, Genetic Fundamentals and Genetic ISBN 0-470-84326-8 Engineering 1992 ISBN 3-527-28312-9 Jörg Knäblein and Rainer H.
Stadler, Design, Development and Optimization A.) ISBN 3-527-31184-X Biotechnology Second, Completely Revised Edition M.) Volume 5a, Recombinant Proteins, Monoclonal Antibodies, and Therapeutic Genes DNA-Pharmaceuticals 1998 Formulation and Delivery in Gene Therapy ISBN 3-527-28315-3 and DNA Vaccination 2005 R. Hammelehle ISBN 3-527-31187-4 Pocket Guide to Biotechnology and Genetic Engineering 2003 ISBN 3-527-30895-4 Production of Recombinant Proteins Novel Microbial and Eukaryotic Expression Systems Edited by Gerd Gellissen Edited by & All books published by Wiley-VCH are carefully produced. Nevertheless, authors, Prof.Gerd Gellissen editors, and publisher do not warrant the Ringstrasse 30 information contained in these books, 42489 Wülfrath including this book, to be free of errors. Germany Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at <http://dnb.de> © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into machine language without written permis- sion from the publishers.
Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany Printed on acid-free paper Composition ProSatz Unger,Weinheim Printing Strauss GmbH, Mörlenbach Bookbinding J. Schäffer GmbH i., Grünstadt ISBN 3-527-31036-3 This book is dedicated to my wife Gabi and my sons Benedikt, Georg, and Ulrich.
VII Preface Gene technology has invaded the production of proteins, and especially production processes for pharmaceuticals. At the beginning of this new technology only a limited number of microorganisms was employed for such processes, namely the bacterium Escherichia coli, followed by the baker’s yeast Saccharomyces cerevisiae as a microbial eukaryote. For both organisms a wealth of information was available which stemmed from a long tradition of safe use in science and, in case of the yeast, also from food manufacturing. However, certain limitations and restrictions urged the search for alternatives that were able to meet the requirements and demands for the expression of an ever-growing number of target genes.
As a consequence, a plethora of microbial and cellular expression platforms were developed. Nonetheless, the range of launched products still leans for the most part on production in a restricted set of organisms, with most of the newly identified microbes being applied to research in academia. Despite superior characteristics of some industrially employed platforms, limita- tions and restrictions are still encountered in particular process developments. In a publicly funded program, Rhein Biotech has set out with academic partners in the recent past to identify additional microbes with attractive capabilities that could sup- plement its key system, Hansenula polymorpha.
As such, the Gram-positive Staphylo- coccus carnosus, the thermo- and osmotolerant dimorphic yeast species Arxula adeni- nivorans, the filamentous fungi Aspergillus sojae, and the nonsporulating species Sor- daria macrospora, were developed. This development was supplemented by tools such as the definition of fermentation conditions and a “universal vector” that can be employed to target a range of fungi for the identification of the most suited plat- form in particular process developments. The application of these platforms and tools is included in the business concept of a new German biotech start-up company, MedArtis Pharmaceuticals GmbH, Aachen. The present book is aimed at providing a comprehensive view of these newly iden- tified and defined systems, and comparing them with a range of established and new alternatives.
The book includes the description of two Gram-negative organisms (E. coli and Pseudomonas fluorescens), the Gram-positive Staphylococcus carnosus, four yeast species (Arxula adeninivorans, Hansenula polymorpha, Pichia pastoris and Yarro- wia lipolytica), and the two filamentous fungi Aspergillus sojae and Sordaria macro- spora. The description of these microbial platforms is further supplemented by an overview on expression in mammalian and plant cells. Production of Recombinant Proteins.
Novel Microbial and Eucaryotic Expression Systems. Edited by Gerd Gellissen Copyright # 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31036-3 VIII Preface I would like to thank all academic partners who co-operated in the development of these new platforms. I gratefully acknowledge funding by the Ministry of Economy NRW, Germany (TPW-9910v08).
I would also like to thank D. Ubags, who inspired me to edit this book. I also express my gratitude to all authors for their fine efforts and contributions, and thank Dr. Paul Hardy, Düsseldorf, for carefully reading some of the manu- scripts.
I also acknowledge the continuous support of Dr. Pillmann and her staff at Wiley-VCH. Aachen, October 2004 Gerd Gellissen IX Foreword The availability of ever-increasing numbers of eukaryotic, prokaryotic, and viral gen- omes facilitates the rapid identification, amplification, and cloning of coding se- quences for technical enzymes and pharmaceuticals, including vaccines. To take advan- tage of the treasures of information contained in these sequences, elegant multiplat- form expression systems are needed that fulfill the specific requirements demanded by each potential application; for example, economy in the case of technical enzyme pro- duction, or safety and authenticity in the case of pharmaceutical production.Therefore, while Escherichia coli and other bacteria may be perfectly suited for technical enzyme production or the production of selected pharmaceuticals requiring no special modifi- cation, eukaryotic organisms may be advisable for applications where safety (e., no endotoxin), contamination, or authenticity (e., proper protein modification by glyco- sylation) are of concern.
While the choices of microbial and eukaryotic expression sys- tems for the production of recombinant proteins are many in number, most research- ers in academic and industrial settings do not have ready access to pertinent biological and technical information as it is usually scattered in the scientific literature. This book aims to close this gap by providing, in each chapter, information on the general biology of the host organism, a description of the expression platform, a methodological sec- tion (with strains, genetic elements, vectors and special methods, where applicable), and finally some examples of proteins expressed with the respective platform. The de- scribed systems are well balanced by including three prokaryotes (two Gram-negative and one Gram-positive), four yeasts, two filamentous fungi, and two higher eukaryotic cell systems (mammalian and plant cells). The book is rounded off by providing valu- able practical and theoretical information about criteria and schemes for selection of the appropriate expression platform, about the possibility and practicality of a universal expression vector, and about comparative industrial-scale fermentation.
The produc- tion of a recombinant Hepatitis B vaccine is chosen to illustrate an industrial example. 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.
Fort Collins, Colorado, U. Production of Recombinant Proteins. Novel Microbial and Eucaryotic Expression Systems. Edited by Gerd Gellissen Copyright # 2005 WILEY-VCH Verlag GmbH & Co.
KGaA, Weinheim ISBN: 3-527-31036-3 XI Contents 1 Key and Criteria to the Selection of an Expression Platform 1 Gerd Gellissen, Alexander W. Strasser, and Manfred Suckow 2 Escherichia coli 7 Josef Altenbuchner and Ralf Mattes 2.2 Strains, Genome, and Cultivation 9 2.1 Replication of pMB1-derived Vectors 11 2.4 Regulation of Gene Expression 15 2.5 Transcription and Translation 21 2.4 Transcription Termination and mRNA Stability 26 2.1 Inclusion Body Formation 27 2.1 Chaperones as Facilitators of Folding 28 2.2 Fusion Protein Technology 29 2.3 Secretion into the Periplasm 30 2.4 Disulfide Bond Formation and Folding 31 2.5 Twin Arginine Translocation (TAT) of Folded Proteins 31 2.6 Disulfide Bond Formation in the Cytoplasm 32 2.7 Cell Surface Display and Secretion across the Outer Membrane 33 2.7 Examples of Products and Processes 34 Production of Recombinant Proteins. Novel Microbial and Eucaryotic Expression Systems. Edited by Gerd Gellissen Copyright # 2005 WILEY-VCH Verlag GmbH & Co.
KGaA, Weinheim ISBN: 3-527-31036-3 XII Contents 2.8 Conclusions and Future Perspectives 35 Appendix 36 References 37 3 Pseudomonas fluorescens 45 Lawrence C. Squires, and Henry W.2 Biology of Pseudomonas fluorescens 47 3.3 History and Taxonomy of Pseudomonas fluorescens Strain Biovar I MB101 47 3.5 Genomics and Functional Genomics of P. fluorescens Strain MB101 49 3.6 Core Expression Platform for Heterologous Proteins 52 3.1 Antibiotic-free Plasmids using pyrF and proC 52 3.2 Gene Deletion Strategy and Re-usable Markers 53 3.3 Periplasmic Secretion and Use of Transposomes 54 3.4 Alternative Expression Systems: Anthranilate and Benzoate-inducible Promoters 54 3.7 Production of Heterologous Proteins in P.8 Conclusions 60 Appendix 61 References 62 4 Staphylococcus carnosus and other Gram-positive Bacteria 67 Roland Freudl 4.2 Major Protein Export Routes in Gram-positive Bacteria 68 4.1 The General Secretion (Sec) Pathway 69 4.2 The Twin-Arginine Translocation (Tat) Pathway 71 4.3 Extracytosolic Protein Folding 73 4.4 The Cell Wall as a Barrier for the Secretion of Heterologous Proteins 75 4.5 Degradation of Exported Proteins by Cell-associated and Secreted Proteases 75 4.2 Microbiological and Molecular Biological Tools 77 4. carnosus as Host Organism for the Analysis of Staphylococcal-related Pathogenicity Aspects 77 4.4 Secretory Production of Heterologous Proteins by S.
carnosus 78 Contents XIII 4.1 The Staphylococcus hyicus Lipase: Secretory Signals and Heterologous Expression in S.2 Use of the Pre-pro-part of the S. hyicus Lipase for the Secretion of Heterologous Proteins in S.3 Process Development for the Secretory Production of a Human Calcitonin Precursor Fusion Protein by S.5 Surface Display on S. carnosus 82 Appendix 83 References 84 5 Arxula adeninivorans 89 Erik Böer, Gerd Gellissen, and Gotthard Kunze 5.2 Physiology and Temperature-dependent Dimorphism 91 5.3 Genetics and Molecular Biology 96 5.4 Arxula adeninivorans as a Gene Donor 97 5. adeninivorans-based Platform 99 5.2 Heterologous Gene Expression 99 5.6 Conclusions and Perspectives 105 Acknowledgments 105 Appendix 105 References 108 6 Hansenula polymorpha 111 Hyun Ah Kang and Gerd Gellissen 6.1 History, Phylogenetic Position, Basic Genetics and Biochemistry of H.2 Characteristics of the H.3 N-linked glycosylation in H.
polymorpha-based Expression Platform 120 6.3 Plasmids and Available Elements 124 6.5 Product and Process Examples 127 6.6 Future Directions and Conclusion 129 6.1 Limitations of the H. polymorpha-based Expression Platform 129 6.2 Impact of Functional Genomics on Development of the H. polymorpha RB11-based Expression Platform 130 Appendix 132 References 136 7 Pichia pastoris 143 Christine Ilgen, Joan Lin-Cereghino, and James M.1 Introduction 143 XIV Contents 7.2 Construction of Expression Strains 144 7.1 Expression Vector Components 145 7.1 Methanol Utilization Phenotype 148 7.2 Protease-deficient Host Strains 149 7.5 Construction of Expression Strains 149 7.7 Growth in Fermentor Cultures 151 7.3 Post-translational Modification of Secreted Proteins 152 7.4 “Humanization” of N-linked Carbohydrate 156 7.4 Conclusions 157 Acknowledgments 157 Appendix 158 References 160 8 Yarrowia lipolytica 163 Catherine Madzak, Jean-Marc Nicaud, and Claude Gaillardin 8.1 History, Phylogenetic Position, Basic Genetics, and Biochemistry 163 8.2 Historical Perspective on the Development of Studies 164 8.3 Secretion of Proteins 165 8.4 Production of Heterologous Proteins and Glycosylation 166 8.2 Characteristics of the Y.3 Description of the Expression Platform 168 8.2 Selection Markers and Expression Signals 170 8.2 The Expression Cassette 172 8.2 Secretion Targeting Signals 175 8.3 Shuttle Vectors for Heterologous Protein Expression 176 8.1 Examples of Mono-copy Integrative Vectors 177 8.2 Homologous Multiple Integrations 178 8.
