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Endoproteinases

ENP00010-01 - Pro39 Protease, recombinant, His-tag, 500U

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FEATURES

  • Specific cystein protease
  • Used for cleavage of fusion proteins
  • Cleaves substrate both in liquid as well as in a resin-bound state
  • Cleaves amino acid sequence DVLRLGRAGA↓G4,5 at the N-terminus or DVLRLGRAGA↓XIFSS at the C-terminus of the protein, where X is preferably Y or G but can be any amino acid except P4,5

DESCRIPTION

Pro39 is the Semliki Forest Virus (SFV) non-structural protein nsP2 C-terminal protease domain, in a truncated, modified and His-tagged form. Pro39 protease specifically recognizes and cleaves the amino acid sequence DVLRLGRAGAYIFSS or DVLRLGRAGAG. The cleavage is highly specific and active.
The molecular weight of Pro39 is approximately 39 kDa. Since the protease has a His-tag, it can be easily removed from cleavage reactions.
The reaction conditions for cleaving of fusion proteins have to be determined empirically. During cleavage reactions, it is recommended that samples be removed from the reaction mixture at various points in time and analyzed by SDS-PAGE to estimate the yield and extent of digestion, and the purity of the product. Typical parameters that should be varied in pilot experiments include enzyme/substrate ratio, substrate concentration, temperature, reaction time, pH.

CHARACTERISTICS

  • Pro39 cleaves substrates in a wide temperature range +4 ... +36°C (optimal is +30°C), in neutral pH conditions (i.e. pH 7-8) in most commonly used buffer systems, including Tris-HCl, HEPES and phosphate buffers.

  • Pro39 cleaves substrates in the presence of high concentrations of NaCl (up to 4M)4,5 or urea (up to 1M )4,5 .

  • The cleavage of the substrates can be reversibly inhibited by the addition of Zn-ions and reactivated by the addition of EDTA4,5 .

  • Pro39 cleaves substrates containing the recognition site both in liquid as well as in a resin-bound state.

INHIBITORS

  • divalent cations (Ni, Co, Cd, Zn)1,4,5
  • 0.1% SDS4,5
  • 0.5 M guanidine4,5
  • N-ethylmaleimide1
  • Pro39 is resistant to the inhibitors of serine proteases (PMSF), metalloproteases (EDTA), aspartic proteases (pepstatin), and some cystein protease inhibitors (leupeptin and E-64)1 .

REFERENCES

1. Vasiljeva, L. et al, J Biol Chem (2001) 276(33):30786-30793

2. Merits, A. et al, J Gen Virol (2001) 82:765-773

3. Vasiljeva, L. et al, J Biol Chem (2003) 278(43):41636-41645

4. Patent US11/157360

5. Patent PCT/EP2005/006681