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Center for Structural Biology

pH6TevMut

For expression of His-tagged TEV protease in E. coli.

General Information:

  • Ref: Protein Sci. 16:2360-7 (2007)
  • This construct is NOT the same as pS219V or pETTev
  • T7lac promoter
  • low copy
  • Kan resistance
  • dna sequence
  • Encodes S219V, L56V, S135G triple point mutant of the TEV protease catalytic domain
  • S219V mutant has been shown to be more stable and more efficient as a catalyst than the wild-type protease
  • L56V/S135G mutations improve solubility
  • TEV protease recognition sequence: ENLYFQ↓G
  • MW = 28.5kDa
  • Amino Acid Sequence:

MGSSHHHHHH GGGESLFKGP RDYNPISSTI CHLTNESDGH TTSLYGIGFG PFIITNKHLF RRNNGTLVVQ SLHGVFKVKN TTTLQQHLID GRDMIIIRMP KDFPPFPQKL KFREPQREER ICLVTTNFQT KSMSSMVSDT SCTFPSGDGI FWKHWIQTKD GQCGSPLVST RDGFIVGIHS ASNFTNTNNY FTSVPKNFME LLTNQEAQQW VSGWRLNADS VLWGGHKVFM VKPEEPFQPV KEATQLMNEL VYSQ

Protocol: TEV protease expression and purification

 

Standard cleavage conditions:

  • 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA and 1mM DTT
  • TEV protease tolerates a variety of buffers including phosphate, MES, and acetate.
  • It is better not to agitate the solution when cleaving with TEV as shaking can cause precipitation.

Reagents that inhibit TEV protease:

  • Zinc (≥ 5mM)
  • Possibly high salt concentrations. (We found no difference in activity in 0.5M, 0.3M, 0.15M, or 0M salt. David Waugh reports that it is 50% less active in 0.5M NaCl vs. no salt. See David Waugh’s TEV Protease FAQ.)
  • Low pH. (We found no difference in protease activity between pH 5-7.5, but have not tested lower or higher pH’s. Waugh states that the protease is active between pH 4-9. Others have found no difference in activity between pH 6-9.)
  • Possibly detergents. There’s some debate about this. (See Mohanty et al. Protein Expr. Purif. 2003 Jan;27(1):109-14 and Lundback et al. Anal. Biochem. 2008 Nov 1;382(1):69-71.)
  • Iodoacetamide and other reagents that react with cysteine. (TEV is a cysteine protease.)
  • Low temperature. (Waugh has found that TEV protease is three-fold less active at 4°C than at 20°C. Maximal activity was found at 34°C.)

Reagents that do not inhibit TEV protease:

  • Glycerol or sorbitol (up to 40%)
  • EDTA (1mM)
  • PMSF, AEBSF (1mM)
  • TLCK (1mM)
  • Bestatin (1mg/mL)
  • Pepstatin A (1mM)
  • E-64 (3mg/mL)
  • Roche Complete protease inhibitor cocktail