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



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Gene expression in
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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

5. Proteolysis of whole cells

Cell surface proteins containing a TEV protease site can be proteolyzed by adding TEV protease to whole cells.

  • Standard TEV protease assay of whole cells

    Grow cells to OD600=0.3-0.5
    spin, wash in TEV protease buffer
    Resuspend to OD=0.3
    Protease assay:
    180 l cells (OD=0.3)
    Add 2 l Protease buffer 10X
    Add 0.2 l DTT (100 mM stock)
    1-5 l protease (1g/l)
    ___________________
    1-4 h 30oC
    Add 100 mM Iodoacetamide to stop reaction
    TCA precipitate
    Resuspend pellet in 50 l sample buffer
    Boil 10 min
    Run half of the sample on SDS PAGE/Western blot

  • Protease buffer 10X
    0.5 M TrisHCl pH=8.0
    5 mM EDTA

  • TCA precipitation
    Add 190 l TCA (20% stock)
    Vortex, let sit on ice 15 min
    Spin 5 min
    Remove supernatant
    Wash 1x with cold acetone
    Spin 5 min
    Remove supernatant
    Dry pellet under vacuum
    Resuspend pellet in 20l sample buffer

  • To try
    - pcnB strain reduces the copy number of plasmid encoded TEV protease substrates (to avoid overproduction of target protein)
    - Various temperatures (grow cells at 20oC if target protein is somewhat unstable due to TEV protease site insert/ try various temps for protease assay - some sites may be more accessible at higher temps)
    - Cell density (at high cell density some target proteins may not be accessible or cells may inhibit TEV protease)
    - Various TEV protease concentrations