to Pichia pastoris -- Classical Genetic Manipulation -- Transformation -- Small Vectors for Expression Based on Dominant Drug Resistance with Direct Multicopy Selection -- The Generation of Multicopy Recombinant Strains -- Isolation of Nucleic Acids -- Generation of Protease-Deficient Strains and Their Use in Heterologous Protein Expression -- Glycosylation Profiling of Heterologous Proteins -- High Cell-Density Fermentation -- Use of Pichia pastoris as a Model Eukaryotic System -- Secretion of Recombinant Human Insulin-Like Growth Factor I (IGF-I) -- Secretion of scFv Antibody Fragments -- Expression of Tetanus Toxin Fragment C -- Expression of EGF and HIV Envelope Glycoprotein -- Expression of an Integral Membrane Protein, the 5HT5A Receptor -- Localization of the myc-Tagged 5HT5A Receptor by Immunogold Staining of Ultrathin Sections.

The Pichia pastoris expression system has rapidly become one of the key protein expression systems used in commercial, academic, and government laboratories today. In Pichia Protocols, David Higgins and James Cregg compile for the first time an all-inclusive collection of powerful experimental procedures to manipulate Pichia pastoris for gene expression purposes. The methods-developed by world-class researchers in their own laboratories-contain detailed descriptions of available expression strains, as well as vectors, and step-by-step instructions for the construction of expression strains and for their use in the production of foreign proteins from shake-flask or fermentor cultures. General procedures for the purification of foreign proteins are also provided, along with protocols for analyzing foreign protein products for glycosylation and subcellular location. Pichia Protocols captures the collective expertise of the international P. pastoris scientific community in close detail. It conveys a breadth of experience that will permit any researcher in molecular biology to quickly construct an expression strain designed to produce a desired foreign protein, and to use this state-of-the-art molecular and cellular biological system successfully in every area of biological research today.