内容简介:  

Table of Contents
Materials vii
Purchaser Notification x
Product Qualification  xii
Introduction 1
Overview 1
Experimental Outline  3
Recombination and Integration in Pichia 7
Methods 11
Pichia Strains  11
E coli Strains  13
Selecting a Pichia Expression Vector  14
pHIL-D2  16
pPIC35 17
pHIL-S1 18
pPIC9 19
Signal Sequence Processing  20
Cloning into the Pichia Expression Vectors 21
Transformation into E coli  26
Preparation of Transforming DNA 27
Growth of Pichia for Spheroplasting  30
Preparation of Spheroplasts 32
Transformation of Pichia  34
Screening for Mut+ and MutS Transformants  36
PCR Analysis of Pichia Integrants 40
Expression of Recombinant Pichia Strains  42
Analysis by SDS-Polyacrylamide Gel Electrophoresis  45
Optimization of Pichia Protein Expression 47
Scale-up of Expression 49
Protein Purification and Glycosylation 51
Recipes 53
E coli Media Recipes  53
Pichia Media Recipes  54
Appendix  59
Electroporation of Pichia  59
PEG 1000 Transformation Method for Pichia  60
Lithium Chloride Transformation Method  61
Total DNA Isolation from Pichia 62
Detection of Multiple Integration Events  63
Procedure for Total RNA Isolation from Pichia  64
β-Galactosidase Assay  65
Technical Service  67
References 69

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