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pRK1043
(Plasmid #8835)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 8835 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

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
  • 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
  • (Common Sequencing Primers)

Resource Information

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).

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pRK1043 was a gift from David Waugh (Addgene plasmid # 8835 ; http://n2t.net/addgene:8835 ; RRID:Addgene_8835)
  • For your References section:

    Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Kapust RB, Tozser J, Fox JD, Anderson DE, Cherry S, Copeland TD, Waugh DS. Protein Eng. 2001 Dec . 14(12):993-1000. 10.1093/protein/14.12.993 PubMed 11809930