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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(TM) 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.
Inhaltsverzeichnis
Optimising Yeast as a Host for Recombinant Protein Production( review).-Which Yeast Species Shall I Choose? Saccharomyces cerevisiae versus Pichia pastoris (review).-Preparation of Pichia pastoris Expression Plasmids.-Preparation of Saccharomyces cerevisiae Expression Plasmids.-Codon Optimisation for Heterologous Gene Expression in Yeast.-Yeast Transformation to Generate High-yielding Clones.-Screening for High-yielding Pichia pastoris Clones: the Production of G Protein-Coupled Receptors as a Case Study.-Screening for High-yielding Saccharomyces cerevisiae Clones: Using a Green Fluorescent Protein Fusion Strategy in the Production of Membrane Proteins.-The Effect of Antifoam Addition on Protein Production Yields.-Setting up a Bioreactor for Recombinant Protein Production in Yeast.-The Implementation of a Design of Experiments Strategy to Increase Recombinant Protein Yields in Yeast (review).-On-line Analysis and Process Control in Recombinant Protein Production (review).-Monitoring the Biomass Accumulation of Recombinant Yeast Cultures: Off-line Estimations of Dry Cell Mass and Cell Counts.-On-line Monitoring of Biomass Accumulation in Recombinant Yeast Cultures.-Optimising P. pastoris Induction-Optimizing S. cerevisiae Induction Regimes.-Large Scale Production of Membrane Proteins in Pichia pastoris: The Production of G Protein-coupled Receptors as a Case Study.-Large-scale Production of Membrane Proteins in Saccharomyces cerevisiae: Using a Green Fluorescent Protein Fusion Strategy in the Production of Membrane Proteinsl.-Large Scale Production of Secreted Proteins in Pichia pastoris.-Disruption of Yeast Cells to Isolate Recombinant Proteins.
Zusammenfassung
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.
