Pichia Protocols

ISBN-13: 9781617375637 / Angielski / Miękka / 2010 / 268 str.

James M. Cregg

Opis

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.

Recenzje

From the reviews of the second edition:

"P. pastoris has many superior traits. ... can grow to very high cell densities prior to switching on an inducible promoter (the AOX1 gene) for maximal recombinant protein production. The diversity of such proteins produced by expression strains of P. pastoris is quite amazing and this book describes detailed experimental protocols for a few of them (e.g. hookworm protein, botulinum toxin). ... This is a stand-alone book ... . a book that will prove valuable to yeast molecular geneticists and biotechnologists." (Graeme Walker, Microbiology Today, May, 2008)

Spis treści

Table of Contents Chapter 1 - Introduction [REVIEW CHAPTER] Chapter 2 - Vectors and strains for expression [REVIEW CHAPTER] Chapter 3 - DNA-Mediated Transformation Chapter 4 - Rational design and optimization of fed-batch and continuous fermentations Chapter 5 - Overexpression of a hookworm secretory protein saturates secretory capacity Chapter 6 - Initial processing and purification strategies Chapter 7 - Rapid screening of chromatography resins for the purification of proteins Chapter 8 - Characterization of O-linked saccharides on glycoproteins Chapter 9 - Modification of the N-glycosylation pathway to produce homogeneous human-like glycans using GlycoSwitch plasmids Chapter 10 - N-linked glycan characterization of heterologous proteins Chapter 11 - Heavy labeling of recombinant proteins Chapter 12 - Selenomethionine labeling of recombinant proteins Chapter 13 - Deuterium labeling of proteins Chapter 14 - Classical genetic analysis Chapter 15 - Identification of pexophagy genes by Restriction Enzyme-Mediated Integration (REMI) Chapter 16 - Characterization of protein-protein interactions Chapter 17 - Localization of proteins and organelles using fluorescence microscopy Chapter 18 - Fluorescence microscopy and thin-section electron microscopy

Blurb

The use of Pichia Pastoris as a recombinant protein system has increased greatly in popularity over the years. In Pichia Protocols 2^nd Edition, the author focuses on new information that has come to light in recent years but repeats key basic methods so that researchers are not absolutely dependent on having both editions in order to utilize the system. The chapters in this indispensable laboratory aid cover a wide range of topics associated with Pichia Pastoris. The use of fermentors to grow Pichia Pastoris and good advice on how to go about the initial processing of material from these cultures is discussed. An extensive discussion on how to formally analyze mutations in essential P. pastoris genes is also presented. The final chapters are for cell biologists who are for cell biologists using the Pichia Pastoris system to investigate protein interactions, or to localize proteins in yeast cells. Pichia Protocols 2^nd Edition, is an essential tool for researchers working with the Pichia Pastoris system