Download Expresso Solubility and Expression Screening System

Expresso® Solubility and Expression Screening System
Table of Contents
Technical Support............................................................................................................................................ 3
Product Designations .......................................................................................................................................... 3
Introduction......................................................................................................................................................... 5
Special Materials and Equipment Needed ........................................................................................................ 5
Process Workflow ............................................................................................................................................... 6
System Details ..................................................................................................................................................... 7
The pSol Vector Suite ................................................................................................................................... 8
E. cloni® 10G Chemically Competent Cells ................................................................................................ 8
Cloning Strategy........................................................................................................................................... 9
pSol His Control Vector ............................................................................................................................... 9
GH1 Control Insert ...................................................................................................................................... 9
Colony Screening ......................................................................................................................................... 9
Protein Expression ....................................................................................................................................... 9
Protein Purification...................................................................................................................................... 9
SelecTEV™ Protease ................................................................................................................................. 10
Detailed Protocol ............................................................................................................................................... 11
1.
Preparation of Insert DNA ........................................................................................................... 11
2.
Enzyme-free Cloning with the pSol Vectors ................................................................................. 13
3.
Heat Shock Transformation of E. cloni® 10G Chemically Competent Cells ............................... 13
4.
Colony PCR Screening for Recombinants .................................................................................... 15
5.
DNA Purification and Sequencing................................................................................................ 16
6.
Induction of Protein Expression ................................................................................................... 16
7.
Evaluation of Target Protein Expression and Solubility .............................................................. 17
8.
Affinity Purification of 6xHis tagged proteins .............................................................................. 19
9.
Cleavage of Fusion Tag with SelecTEV™ Protease .................................................................... 19
10.
Removing SelecTEV Protease after Cleavage .............................................................................. 19
11.
SelecTEV Cleavage During Dialysis ............................................................................................ 20
Control Information ......................................................................................................................................... 21
References .......................................................................................................................................................... 21
Appendix A: Media Recipes............................................................................................................................. 23
Appendix B: Internal sequencing Primers ..................................................................................................... 23
Appendix C: Colony PCR Screening .............................................................................................................. 24
Appendix D: Vector Map and Sequencing Primers....................................................................................... 25
Appendix E: Cloning Troubleshooting Guide ................................................................................................ 26
Appendix F: Expression/Purification Troubleshooting Guide ..................................................................... 27
Appendix G: Characteristics of Fusion Tags in the pSol Vector Suite ........................................................ 28
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MA158 Rev B