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

3. Purification and storage of TEV protease

  • Purification of soluble 6His TEV protease

    Grow cells in double rich medium*
    at 20oC to OD=0.8
    Induce (e.g. 0.01-0.1 mM IPTG)
    Let grow overnight
    Harvest, wash cells e.g. in 50 mM NaH2PO4, 100 mM NaCl, pH 8.0
    Lyse cells e.g. by passing twice through a French Press (1500 psi)
    Purify TEV protease by Ni-chromatography using standard protocol supplied by e.g. Qiagen
    After elution with imidazole dialyze immediately against 50 mM Tris HCl pH=7.5 plus 1 mM EDTA, 5 mM DTT
    Do not concentrate TEV protease too much (0.5 - 1 mg/ml)
    Spin 10,000 rpm for 20 min
    Take supernatant

    Add glycerol for storage at -80oC. Freezing in the absence of glycerol will kill the activity.

    * If TEV protease expression is IPTG-dependent do not use BactoTryptone in your medium since it contains lactose. Change BactoTryptone by NZ amine A (available from SIGMA)

  • Purification of insoluble 6His TEV protease

    Grow cells in double rich medium*
    37oC to OD=0.8

    Fully induce (e.g. 1 mM IPTG)
    Let grow overnight
    Harvest, wash cells e.g. in 50 mM NaH2PO4, 100 mM NaCl, pH 8.0
    Lyse cells e.g. by passing twice through a French Press (1500 psi)
    Harvest inclusion bodies by centrifugation (27,000xg, 20', 4oC)
    Solubilize inclusion bodies by resuspending the pellet in 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris/HCl, pH 8.0
    Remove insoluble aggregates by centrifugation (27,000xg, 10', room temperature
    Purify TEV protease by Ni-chromatography using standard protocol
    After elution with imidazole dialyze against 8 M urea in 50 mM Tris HCl pH=7.5 plus 1 mM EDTA, 5 mM DTT
    Refold TEV protease by dilution

  • TEV protease storage buffer

    50 mM Tris HCl pH=7.5
    1 mM EDTA
    5 mM DTT
    50% glycerol
    0.1% Triton X100
    Store at -80oC