Recombinant Protein Production in Yeast: Methods and Protocols PDF
Preview Recombinant Protein Production in Yeast: Methods and Protocols
M M B ™ ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Recombinant Protein Production in Yeast Methods and Protocols Edited by Roslyn M. Bill School of Life & Health Sciences and Aston Research Centre for Healthy Ageing Aston University, Birmingham, UK Editor Roslyn M. Bill School of Life & Health Sciences and Aston Research Centre for Healthy Ageing Aston University Birmingham, UK ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-769-9 e-ISBN 978-1-61779-770-5 DOI 10.1007/978-1-61779-770-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2012933363 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or d issimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com) Dedication For Martin and Alice Preface Advances in our understanding of the biology of the cell rely on our increasing knowledge of protein behaviour and the complex interplay of proteins with other biomolecules. Proteins are essential components of living organisms and have a role in virtually every cellular process: they are enzymes, form cellular scaffolds, and are central to signalling, transport, and regulatory functions. To study these diverse roles, it is necessary to be able to work with suffi cient quantities of suitably stable and functional protein samples. While some proteins can be isolated from native sources for this purpose, many cannot as they are either intrinsically unstable or are present in impractically low quantities. Moreover, the study of mutant forms of a given protein is often central to understanding its structure and activity, and such mutants must be synthesized in vitro. Since the 1970s, recombinant DNA technologies have provided a solution to producing proteins in non-native host cells. However, the attainment of high yields of recombinant proteins, particularly recombinant membrane proteins, is an enduring bottleneck in the post-genomic sciences that is only now being addressed in a truly rational manner. Several host systems have been developed for the production of recombinant proteins, ranging from prokaryotes, such as E scherichia coli , to higher eukaryotes, such as mammalian cell- lines. This book describes strategies and protocols for the use of yeast as a production host. With its extensive literature and sequenced genome, the well-known yeast species, Saccharomyces cerevisiae , provides many opportunities to optimize the functional yields of a target protein of interest. P ichia pastoris , for which there is now a freely available genome sequence, is a popular alternative. Both have been used for the industrial production of pharmaceutical proteins and are described here. In the last few years, signifi cant advances have been made in understanding how a yeast cell responds to the stress of producing a recombinant protein, and how this information can be used to engineer improved host strains. The molecular biology of the expression vector, through the choice of promoter, tag, and codon optimisation of the target gene, is also a key determinant of a high-yielding protein production experiment. The use of statis- tical approaches to examine how parameters such as medium composition, growth variables (e.g., temperature, oxygen availability), and the precise details of the induction regime infl uence recombinant protein yield has also been implemented more widely by researchers in the fi eld. This book examines the process of preparation of expression vectors, transformation to generate high-yielding clones, optimisation of experimental conditions to maximise yields, scale-up to bioreactor formats, and disruption of yeast cells to enable the isolation of the recombinant protein prior to purifi cation. I hope the chapters describing these steps enable you to adopt yeast as a protein production host for your research. Birmingham, UK Roslyn M. Bill vii Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi 1 Optimising Yeast as a Host for Recombinant Protein Production (Review). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Nicklas Bonander and Roslyn M. Bill 2 Which Yeast Species Shall I Choose? Saccharomyces cerevisiae Versus Pichia pastoris (Review) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Richard A.J. Darby, Stephanie P. Cartwright, Marvin V. Dilworth, and Roslyn M. Bill 3 Preparation of Pichia pastoris Expression Plasmids. . . . . . . . . . . . . . . . . . . . . . 25 Christel Logez, Fatima Alkhalfioui, Bernadette Byrne, and Renaud Wagner 4 Preparation of Saccharomyces cerevisiae Expression Plasmids. . . . . . . . . . . . . . . 41 David Drew and Hyun Kim 5 Codon Optimisation for Heterologous Gene Expression in Yeast . . . . . . . . . . 47 Kristina Hedfalk 6 Yeast Transformation to Generate High-Yielding Clones. . . . . . . . . . . . . . . . . 57 Mohammed Jamshad and Richard A.J. Darby 7 Screening for High-Yielding Pichia pastoris Clones: The Production of G Protein-Coupled Receptors as a Case Study. . . . . . . . . . . . . . . . . . . . . . . 65 Shweta Singh, Adrien Gras, Cedric Fiez-Vandal, Magdalena Martinez, Renaud Wagner, and Bernadette Byrne 8 Screening for High-Yielding Saccharomyces cerevisiae Clones: Using a Green Fluorescent Protein Fusion Strategy in the Production of Membrane Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 David Drew and Hyun Kim 9 The Effect of Antifoam Addition on Protein Production Yields. . . . . . . . . . . . 87 Sarah J. Routledge and Roslyn M. Bill 10 Setting Up a Bioreactor for Recombinant Protein Production in Yeast. . . . . . . 99 Sarah J. Routledge and Michelle Clare 11 The Implementation of a Design of Experiments Strategy to Increase Recombinant Protein Yields in Yeast (Review). . . . . . . . . . . . . . . . 115 Nagamani Bora, Zharain Bawa, Roslyn M. Bill, and Martin D.B. Wilks 12 Online Analysis and Process Control in Recombinant Protein Production (Review). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Shane M. Palmer and Edmund R.S. Kunji ix x Contents 13 Monitoring the Biomass Accumulation of Recombinant Yeast Cultures: Offline Estimations of Dry Cell Mass and Cell Counts . . . . . . . . . . . . . . . . . . 157 Shane M. Palmer and Edmund R.S. Kunji 14 Online Monitoring of Biomass Accumulation in Recombinant Yeast Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Shane M. Palmer and Edmund R.S. Kunji 15 Optimising Pichia pastoris Induction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Zharain Bawa and Richard A.J. Darby 16 Optimizing Saccharomyces cerevisiae Induction Regimes . . . . . . . . . . . . . . . . . 191 David Drew and Hyun Kim 17 Large-Scale Production of Membrane Proteins in Pichia pastoris: The Production of G Protein-Coupled Receptors as a Case Study . . . . . . . . . . 197 Shweta Singh, Adrien Gras, Cedric Fiez-Vandal, Magdalena Martinez, Renaud Wagner, and Bernadette Byrne 18 Large-Scale Production of Membrane Proteins in Saccharomyces cerevisiae: Using a Green Fluorescent Protein Fusion Strategy in the Production of Membrane Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 David Drew and Hyun Kim 19 Large-Scale Production of Secreted Proteins in Pichia pastoris. . . . . . . . . . . . . 217 Nagamani Bora 20 Disruption of Yeast Cells to Isolate Recombinant Proteins. . . . . . . . . . . . . . . . 237 Mohammed Jamshad and Richard A.J. Darby Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
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