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biogate21

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专业: 生物大分子结构与功能

[交流] 10个金币求助 High Level Expression of Recombinant Proteins in&n

以下是High Level Expression of Recombinant Proteins in&n的部分内容

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

Regulation of Protein Expression in the pET System
Double induction by IPTG
T7 RNA polymerase (98 kDa)
target gene (only in T7lac vectors)
Highly specific
Compatible with a wide range of expression hosts
requires DE3 lysogen

Compatibility with Toxic Genes

T7 RNA polymerase inhibited by T7 lysozyme

Drawbacks to pET system

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