pRK1043
(Plasmid #8835)

• 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 37°C to 30°C during induction maximizes the yield of soluble MBP-TVMV protease fusion protein. Ampicillin for pRK1043; Chloramphenicol for pRIL
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: tobacco etch virus protease, catalytic domain
• Alt name: TEV protease
• Species: tobacco etch virus
• Insert Size (bp): 700
• Mutation: Autolysis-resistant S219V mutant
• Tags / Fusion Proteins:
  • MBP (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: MBP-F

Resource Information

• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

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

Depositor Comments

pRK1043 overproduces the catalytic domain of tobaco etch virus (TEV) protease in the form of an affinity sandwich, with E. coli maltose-binding protein fused to its N-terminus and a polyarginine tag fused to its C-terminus. TheTEV protease produced by this strain is the autoinactivation-resistant mutant S219V.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 S219V mutant is approximately 2-fold greater than that of the wild-type protease. Shifting the temperature from 37°C to 30°C during induction maximizes the yield of soluble MBP-TEV-Arg fusion protein. Bacterial strain: E.coli BL21(DE3)-RIL (Stratagene).