Recombinant Protein Production in Yeast: Methods and Protocols

ISBN-13: 9781617797699 / Angielski / Twarda / 2012 / 248 str.

Roslyn M. Bill

Opis

In the last few years, significant 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 optimization of the target gene, is also a key determinant of a high-yielding protein production experiment. Recombinant Protein Production in Yeast: Methods and Protocols examines the process of preparation of expression vectors, transformation to generate high-yielding clones, optimization of experimental conditions to maximize yields, scale-up to bioreactor formats and disruption of yeast cells to enable the isolation of the recombinant protein prior to purification. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.Authoritative and practical, Recombinant Protein Production in Yeast: Methods and Protocols, seeks to aid scientists in adopting yeast as a protein production host.

Spis treści

1 Optimising Yeast as a Host for Recombinant Protein Production( review)

 Nicklas Bonander and Roslyn M Bill

2 Which Yeast Species Shall I Choose? Saccharomyces cerevisiae versus Pichia pastoris (review)

 Richard AJ Darby, Stephanie P Cartwright, Marvin V. Dilworth and Roslyn M Bill

3 Preparation of Pichia pastoris Expression Plasmids

Christel Logez, Fatima Alkhalfioui, Bernadette Byrne, Renaud Wagner

4 Preparation of Saccharomyces cerevisiae Expression Plasmids

David Drew and Hyun Kim

5 Codon Optimisation for Heterologous Gene Expression in Yeast

Kristina Hedfalk

6 Yeast Transformation to Generate High-yielding Clones

Mohammed Jamshad and Richard AJ Darby

7 Screening for High-yielding Pichia pastoris Clones: the Production of G Protein-Coupled Receptors as a Case Study

Shweta Singh, Adrien Gras, Cedric Fiez-Vandal, Magdalena Martinez, Renaud Wagner, Bernadette Byrne

8 Screening for High-yielding Saccharomyces cerevisiae Clones: Using a Green Fluorescent Protein Fusion Strategy in the Production of Membrane Proteins

David Drew and Hyun Kim

9 The Effect of Antifoam Addition on Protein Production Yields

Sarah J Routledge and Roslyn M Bill

10 Setting up a Bioreactor for Recombinant Protein Production in Yeast

Sarah J Routledge and Michelle Clare

11 The Implementation of a Design of Experiments Strategy to Increase Recombinant Protein Yields in Yeast (review)

Nagamani Bora, Zharain Bawa, Roslyn M. Bill and Martin D. B. Wilks

12 On-line Analysis and Process Control in Recombinant Protein Production (review)

Shane M. Palmer and Edmund R.S. Kunji

13 Monitoring the Biomass Accumulation of Recombinant Yeast Cultures: Off-line Estimations of Dry Cell Mass and Cell Counts

Shane Palmer and Edmund R S Kunji

14 On-line Monitoring of Biomass Accumulation in Recombinant Yeast Cultures

Shane Palmer and Edmund R S Kunji

15 Optimising P. pastoris Induction

Zharain Bawa and Richard AJ Darby

16 Optimizing S. cerevisiae Induction Regimes

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

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

David Drew and Hyun Kim

19 Large Scale Production of Secreted Proteins in Pichia pastoris

Nagamani Bora

20 Disruption of Yeast Cells to Isolate Recombinant Proteins

Mohammed Jamshad and Richard AJ Darby