Pichia Protocols focuses on recent developments of Pichia pastoris as a recombinant protein production system. Highlighted topics include a discussion on the use of fermentors to grow Pichia pastoris, information on the O- and N-linked glycosylation, methods for labeling Pichia pastoris expressed proteins for structural studies, and the introduction of mutations in Pichia pastoris genes by the methods of restriction enzyme-mediated integration (REMI).This volume fully updates and expands upon the first edition of Pichia Protocols, and focuses primarily on information that has come to light since its original publication. Each chapter presents cutting-edge and cornerstone protocols for utilizing P. pastoris as a model recomibinant protein production system.

This book focuses on recent developments of Pichia pastoris as a recombinant protein production system. The volume fully updates and expands upon the first edition, and focuses primarily on information that has come to light since its original publication. Highlighted topics include a discussion on the use of fermentors to grow Pichia pastoris, information on the O- and N-linked glycosylation, methods for labeling Pichia pastoris expressed proteins for structural studies, and the introduction of mutations in Pichia pastoris genes by the methods of restriction enzyme-mediated integration (REMI).

Vectors and Strains for Expression.- DNA-Mediated Transformation.- Rational Design and Optimization of Fed-Batch and Continuous Fermentations.- Saturation of the Secretory Pathway by Overexpression of a Hookworm (Necator americanus) Protein (Na-ASP1).- Purification of the N- and C-Terminal Subdomains of Recombinant Heavy Chain Fragment C of Botulinum Neurotoxin Serotype C.- Rapid Screening of Chromatography Resins for the Purification of Proteins.- Characterization of O-Linked Saccharides on Glycoproteins.- Modification of the N-Glycosylation Pathway to Produce Homogeneous, Human-Like Glycans Using GlycoSwitch Plasmids.- N-Linked Glycan Characterization of Heterologous Proteins.- Heavy Labeling of Recombinant Proteins.- Selenomethionine Labeling of Recombinant Proteins.- Selective Isotopic Labeling of Recombinant Proteins Using Amino Acid Auxotroph Strains.- Classical Genetics.- Identification of Pexophagy Genes by Restriction Enzyme-Mediated Integration.- Characterization of Protein-Protein Interactions.- Localization of Proteins and Organelles Using Fluorescence Microscopy.- Fluorescence Microscopy and Thin-Section Electron Microscopy.