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
