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

4. Proteolysis in vitro

  • Reaction conditions

    0.1 mg target protein
    75 µl TEV protease buffer 10X
    1 mM DTT final
    0.005 mg TEV protease
    Add water to 750 µl
    Incubate 3h at 30oC
    Proteolysis can also be done at 4oC even if the optimal temperature is between 30oC and 37oC.

    10X Protease buffer
    0.5 M Tris-HCl pH 8.0
    5 mM EDTA

    Other buffers that should work include PBS, TBS, phosphate and imidazole (try pH range 7- 7.5)

  • Inhibitors
    Iodoacetamide
    N-EM
    0.01% SDS
    also, TEV protease is quite sensitive against denaturants

  • Effect of Detergents
    Detergents tolerated are 0.01% Triton X-100 and 0.01% Dodecylmaltoside.