|
| E. coli strain BL21(DE3)-RIL/pRK792 |
| Description: | This derivative of E. coli BL21(DE3) overproduces the catalytic domain of tobbaco etch virus (TEV) protease in the form of an MBP fusion protein that cleaves itself in vivo to yield a TEV protease catalytic domain with an N-terminal His-tag and a C-terminal polyarginine tag. The TEV protease produced by this strain is the autoinactivation-resistant mutant S219P. |
| Plasmids: | The TEV protease expression
vector pRK792. The tRNA accessory plasmid pRIL (Stratagene) |
| Replicon: | ColE1/pBR322
(pRK792) p15A (pRIL) |
| Antibotic Resistance: | Ampicillin
(pRK792) Chloramphenicol (pRIL) |
| Promotor: | tac (pRK792) |
| Inducer: | IPTG (1 mM) |
| Notes: | The pRIL plasmid (Stratagene) improves the yield of TEV protease by increasing the supply of argU tRNA in the cell (for AGG and AGA codons). The catalytic activity of the S219P mutant is approximately 2-fold lower than that of the wild-type protease. Shifting the temperature from 37 °C to 30 °C during induction maximizes the yield of soluble TEV protease. |
| Literature Citations: | Kapust, R. B., Tözsér, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Prot. Eng. 14: 993-1000. |
| Nucleotide Sequence: |  |
| Amino Acid Sequence: |
|
|