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Pichia protocols / edited by James M. Cregg. — 2nd ed. — Totowa, N.J. : Humana Press, c2007.—(58.17/M592/v.389)

Contents

    Contents
    
    Preface v
    Contributors ix
    1 Introduction: Distinction Between Pichia pastoris and Other Expression Systems 1
    2 Vectors and Strains for Expression 11
    3 DNA-Mediated Transformation 27
    4 Rational Design and Optimization of Fed-Batch and Continuous Fermentations 43
    5 Saturation of the Secretory Pathway by Overexpression of a Hookworm (Necator americanus) Protein (Na-ASP1) 65
    6 Purification of the N- and C-Terminal Subdomains of Recombinant Heavy Chain Fragment C of Botulinum Neurotoxin Serotype C 77
    7 Rapid Screening of Chromatography Resins for the Purification of Proteins 99
    8 Characterization of O-Linked Saccharides on Glycoproteins 107
    9 Modification of the N-Glycosylation Pathway to Produce Homogeneous Human-Like Glycans Using GlycoSwitch Plasmids 119
    10 N-Linked Glycan Characterization of Heterologous Proteins 139
    11 Heavy Labeling of Recombinant Proteins 151
    12 Selenomethionine Labeling of Recombinant Proteins 165
    13 Selective Isotopic Labeling of Recombinant Proteins Using Amino Acid Auxotroph Strains 175
    14 Classical Genetics 189
    15 Identification of Pexophagy Genes by Restriction Enzyme~Mediated Integration 203
    16 Characterization of Protein-Protein Interactions: Application to the Understanding of Peroxisome Biogenesis 219
    17 Localization of Proteins and Organelles Using Fluorescence Microscopy 239
    18 Fluorescence Microscopy and Thin-Section Electron Microscopy 251