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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 |
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 µl cells (OD=0.3)
Add 2 µl Protease buffer 10X
Add 0.2 µl DTT (100 mM stock)
1-5 µl protease (1µg/µl)
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1-4 h 30oC
Add 100 mM Iodoacetamide to stop reaction
TCA precipitate
Resuspend pellet in
50 µl 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 µl 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 20µl 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
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TEV NIa protease as a tool for in vitro and in vivo proteolysis
