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