pRK1035
(Plasmid #8832)

• Depositing Lab: David Waugh
• Publication: Nallamsetty et al Protein Expr Purif. 2004 Nov . 38(1):108-15.

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): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): BL21(DE3)
• Growth instructions: Shifting the temperature from 37C to 30C during induction maximizes the yield of soluble TVMV protease.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: TVMV protease
• Alt name: tobacco vein mottling virus protease
• Species: tobacco vein mottling virus
• Insert Size (bp): 750
• Mutation: synthetic gene with codons optimized for E. coli
• Tags / Fusion Proteins:
  • MBP (cleaved) (N terminal on insert)
  • His (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: MBP-F

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

pRK1035 overproduces the catalytic domain of tobbaco vein mottling virus (TVMV) protease in the form of an MBP fusion protein that cleaves itself in vivo to yield a TVMV protease catalytic domain with an N-terminal His-tag. The specificity of TVMV protease is distinct from that of TEV protease. The canonical recognition site for TVMV protease is ETVRFQS, with cleavage occurring between Q and S. Shifting the temperature from 37C to 30C during induction maximizes the yield of soluble TVMV protease. The TVMV protease open reading frame on pRK1035 was constructed from synthetic oligonucleotides and has been codon-optimized for expression in E. coli.