This volume provides readers with a useful and comprehensive resource of laboratory protocols for working with Pichia pastoris . The chapters in this book are organized into seven parts: introduction, bioinformatics, cell and systems biology, gene expression cassettes and vectors, Pichia pastoris expression strains, upstream bioprocessing, and downstream processing and product analysis. This book covers basic and newer laboratory methods, starting from the gene and genetic construct to mature (yet reasonable) amounts of a recombinant product being produced, purified, and characterized. 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 tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and thorough, Pichia pastoris: Methods and Protocols is a valuable resource for both new and experienced researchers who want to learn more about this important and developing field.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt

A Brief History of the Pichia pastoris Expression System.- Pichia pastoris Genomic Resources: Accessibility, Recommendations, and Pitfalls.- Isolation of Pichia pastoris Genomic DNA for Long-Read DNA Sequencing and PCR Applications.- Pichia pastoris RNA-Sequencing and Data Analysis.- Localization of Proteins in the Secretory Pathway of Pichia pastoris using Fluorescence Microscopy.- Auxin-Induced Degradation of Target Proteins in Pichia pastoris.- Establishing Reference Genes of Pichia pastoris Quantitative Real-Time Polymerase Chain Reaction Analyses.- ID-MS Based Quantitative Analysis of Metabolites in Pichia pastoris: A Step-By-Step Protocol.- High Throughput Stationary 13 C-Based Metabolic Flux Analysis of Pichia pastoris Strains.- Determination of Biomass Elemental Composition of Pichia pastoris.- Pichia pastoris Expression Plasmid Assembly using Type IIS Restriction Enzymes.- Autonomously Replicating Sequence-Bearing Plasmids Utilized in Pichia pastoris.- Overlap Extension Construction and Direct Cloning of Loop-Out Prone DNA Expression Cassettes in Pichia pastoris: 75k--Secalin from Secale cereale (rye) as a Case Study.- In Vivo Plasmid Assembly in NHEJ-Deficient Pichia pastoris Strains.- Protein Targeting Signals and Strategies for Pichia pastoris.- Cloning of MF Secretion Variants for Protein Secretion in Pichia pastoris.- Lytic Polysccharide Monooxygenases as Reporters for Signal Peptide Evaluation in Pichia Pastoris.- Exploting Self-Processing 2A Peptides for Multi-Gene Expression in Pichia pastoris.- Retrofitting of AOX1 Promoter-Based Pichia pastoris Expression Strains for Methanol-Independent Protein Production.- Selective Cultivation of Methylotrophic Pichia pastoris Laboratory and Natural Strains.- Transformation of Pichia pastoris by Electroportation.- High Throughput Transformation of Chemical Competent Pichia pastoris Cells using Lithium Acetate/Chloride and Carrier DNA.- High-Efficiency Transformation of Integrative Libraries under Zeocin Selection in Pichia pastoris.- High-Throughput Generation of Pichia pastoris Knock-Out Strains by using CRISPR/Cas9.- Engineering Pichia pastoris Strains using CRISPR/Cas9 Technologies: The Basic Protocol.- Adaptive Laboratory of Pichia pastoris by Serial Batch Cultivation.- Classical Genetics of Pichia pastoris.- Pichia pastoris Random Mutagenesis and Screening.- Cryo-Preservation of Pichia pastoris Strains for Long-Term Storage.- Adapting Pichia pastoris Cultivation Media to REACH Regulations.- Fed-Batch Operational Strategies for Efficient and Robust Pichia pastoris Cultivations Achieving High Cell Densities.- A Guide to Fed-Batch Cultivation of Pichia pastoris on Laboratory Scale.- Pichia pastoris Continuous Cultivations for Strain Characterization and Continuous Biomanufacturing.- Best Practices for Protein Expression in Pichia pastoris Combining Design of Experiments with an Automated Platform for Parallelized Cultivation in Small-Scale Stirred Tank Bioreactors.- Kinetic Modeling of Pichia pastoris Bioreactor Systems under Different Operating Conditions: Batch, Fed-Batch, and Chemostat.- On-Line Monitoring and Control of Methanol Concentration in Pichia pastoris Fermentations.- Off-Gas Balancing for Bioprocess Monitoring Pichia pastoris Bioprocesses.- A Single-Use Technology Platform: How to Set Up Bioprocesses with Pichia pastoris.- A Pichia pastoris Cell-Free Protein Synthesis Platform.- Clarification of Pichia pastoris Broth by TFF with Ceramic Membranes.- Pichia pastoris Cell Disruption by High-Pressure Homogenization (HPH).- Concentration and Purification of Proteins Produced by Pichia pastoris by TFF with Ceramic Membranes.- Protein Purification Protocols for Recombinant Enzymes Produced in Pichia pastoris.- Characterization of Low Molecular Weight Variants (Fragmentation) in Pichia pastoris by CE-SDS.