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Genenase I |
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Substrate Specificity:
Description:
Genenase™ I is a variant of subtilisin BPN´ that has been engineered to have increased specificity by substituting amino acids in its active site (1,2). When designing fusion proteins for cleavage with Genenase I, we recommend the site Pro-Gly-Ala-Ala-His-Tyr. Genenase I will cleave at other histidine residues depending on the surrounding amino acids and the 3-dimensional conformation of the protein. Genenase I cleaves His-Tyr-Glu and His-Tyr-Asp slowly, but will not cleave His-Tyr-Pro or His-Tyr-Ile (2).
Source:
Isolated from a Bacillus subtilis strain carrying the protease gene derived from Bacillus amyloliquefaciens.
Enzyme Properties
Specificity: Genenase I cleaves His-Tyr-Glu and His-Tyr-Asp slowly, but will not cleave His-Tyr-Pro or His-Tyr-Ile (2).
Molecular Weight:
Theoretical: 27.4 kDa and Apparent: 28 kDa
Reaction & Storage Conditions
Unit Definition:
0.5 μg of Genenase I will cleave 50 μg of test substrate to 95% completion in a total reaction volume of 50 μl in 8 hours or less at 23°C in 20 mM Tris-HCl (pH 8.0 @ 25°C) with 200 mM NaCl.
Concentration:
1 mg/ml
Storage Conditions:
20 mM Tris-HCl
200 mM NaCl
2 mM CaCl2
1 mM Dithiothreitol
50% Glycerol
pH 7.2 @ 4°C
Storage Temperature:
-20°C
Notes
General notes:- The rate of Genenase™ I cleavage has been shown to depend on the amino acid following the cleavage site (position P1')(3). Rates of cleavage when P1' is Asp or Glu are slowest, 40-fold slower than when P1' is Arg or Cys. The rate when P1'= Glu or Asp is enhanced 10- and 14-fold, respectively, by including 2 M KCl in the reaction.
References
- Carter, P. and Wells, J.A. (1987) Science, 237, 394-399.
- Carter, P. et al. (1991) Biochemistry, 30, 6142-6148.
- Carter, P. et al. (1989) Proteins: Structure, Function, and Genetics, 6, 240-248.
Companion Products
Enterokinase, light chain
Factor Xa Protease
Furin
Proteinase K
Legal
Licenses/Patents/Disclaimers:
Notice to Buyer/User: The buyer/user has a non-exclusive license to use this Genenase™ I for RESEARCH PURPOSES ONLY. A license to use Genenase™ I for commercial purposes is available from Genencor International, Inc., 180 Kinball Way, South San Francisco, CA 94080.
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