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



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TEV NIa protease
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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

1. Introducing a TEV protease recognition site

  • The seven amino acid consensus sequence is Glu-X-X-Tyr-X-Gln/Ser.
    Cleavage occurs between the conserved Gln and Ser residues. X can be various amino acyl residues but note that not all residues are tolerated. A detailed analysis of altered cleavage sites is described in Dougherty et al. 1989. Virology 171:356-364

  • The cleavage site should be surface exposed.
    Best results will be obtained when TEV protease recognition site is placed between 2 domains. If this is not possible, analysis of secondary structure and surface accessibility will be helpful.

  • Insertion of the cleavage site should not interfere with protein function.
    If a 3D structure or detailed structure/function data are unavailable an alignment of the members of the same protein family combined with secondary structure predictions helps to identify possible sites.

  • If cleavage is not optimal insertion of short linker sequences introducing structural flexibility can improve the efficiency.