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Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme.
- Source :
-
Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2015 Dec; Vol. 99 (23), pp. 9971-86. Date of Electronic Publication: 2015 Jul 31. - Publication Year :
- 2015
-
Abstract
- Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.
- Subjects :
- Animals
Blood Coagulation
Cattle
Cloning, Molecular
Copper metabolism
Enzyme Inhibitors metabolism
Enzyme Stability
Fibrinogen metabolism
Fibrinopeptide A metabolism
Gene Expression
Hydrogen-Ion Concentration
Hydrolysis
Kinetics
Pichia genetics
Pichia metabolism
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins isolation & purification
Recombinant Proteins metabolism
Serine Proteases chemistry
Serine Proteases genetics
Temperature
Zinc metabolism
Crotalid Venoms enzymology
Crotalus
Serine Proteases isolation & purification
Serine Proteases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1432-0614
- Volume :
- 99
- Issue :
- 23
- Database :
- MEDLINE
- Journal :
- Applied microbiology and biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 26227411
- Full Text :
- https://doi.org/10.1007/s00253-015-6836-2