High Level Expression of Recombinant Proteins in E. coli

相关专题

 

High Level Expression?

Protein Biochemistry
Westerns and pull-down assays
Enzyme Assays
Sub-mg quantities

Protein Structure
Structural Biologists
NMR and X-ray crystallography
10’s of mg quantities

Part I. Expression Systems

Expression System Components
Target Protein
mRNA
cDNA
genomic DNA
Expression Vector (plasmids)
pET
pQE
pGEX
Expression Strain (E. coli )
BL21(DE3)
Rosetta(DE3) plus pRARE
Tuner(DE3) lacZY deletion mutant
ADA494(DE3) trxB mutant
Origami(DE3) trxB and gor mutations

Expression Plasmid Elements
Origin of Replication (ColE1)
Selectable marker (ampicillin, kanamycin or chloramphenicol resistance)
Multiple cloning site
Transcription promoter (T7, T5 or Tac)
Regulated expression of target protein

Regulation of Protein Expression in the lac Operon

pET Expression System
Most comprehensive commercial expression system
Uses non-native T7 RNA polymerase
~33 different vectors available