pRK792
(Plasmid #8830)

• Depositing Lab: David Waugh
• Publication: Kapust et al Protein Eng. 2001 Dec . 14(12):993-1000.

Backbone

• Vector backbone: pMal-C2
• Backbone manufacturer: New England Biolabs
• Backbone size w/o insert (bp): 6700
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol and Ampicillin, 25 & 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): BL21(DE3)-RIL
• Growth instructions: Shifting the temperature from 37C to 30C during induction maximizes the yield of soluble TEV protease. Ampicillin for pRK792; Chloramphenicol for pRIL
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: TEV protease, S219P mutant
• Alt name: tobacco etch virus protease
• Insert Size (bp): 750
• Mutation: S219P mutation
• Tags / Fusion Proteins:
  • MBP (cleaved) (N terminal on backbone)
  • His (N terminal on insert)
  • polyarginine (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SacI (not destroyed)
• 3′ cloning site: BamHI (not destroyed)
• 5′ sequencing primer: N/A
• 3′ sequencing primer: M13-F20

Resource Information

• A portion of this plasmid was derived from a plasmid made by: ATCC
• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

pRK792 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.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 less than that of the wild-type protease. Bacterial strain: E.coli BL21(DE3)-RIL (Stratagene).