TEV NIa protease as a tool for in vitro and in vivo proteolysis



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TEV NIa protease
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© 2001 Webdesign by
Tilman Maier og Lars Jessen
1. Introducing a TEV protease recognition site
2. Expression vectors
3. Purification and storage of TEV protease
4. Proteolysis in vitro
5. Proteolysis of whole cells
6. Proteolysis in vivo
7. References
8. Links

2. TEV protease expression vectors

  • For in vivo proteolysis
    Medium or low copy number vectors such as pBR322, pACYC184 or pHSG575 are recommended. To limit TEV protease levels under non-inducing conditions tight promoters are required. Lac or Tet promoters can be used if lac or tet repressor genes are on the plasmid.
    TEV protease does not lose its activity if it is part of a hybrid protein.

  • For purification of TEV protease
    Follow the suggestions for gene expression in E. coli. The choice of vector system is not so critical. T7 polymerase controlled expression vectors are often used. Growth at 20oC will lead to mainly soluble TEV protease while growth at 37oC will produce inclusion bodies. Inclusion bodies are not a problem because TEV protease can be efficiently refolded. N-terminal His tags facilitate purification and do not interfere with activity or refolding.