caa a22 4500 605504989 CHVBK 20210128100619.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6836-2 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6836-2 Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme [Elektronische Daten] [Johara Boldrini-França, Renata Rodrigues, Ludier Santos-Silva, Dayane de Souza, Mário Gomes, Camila Cologna, Edwin de Pauw, Loïc Quinton, Flávio Henrique-Silva, Veridiana de Melo Rodrigues, Eliane Arantes] 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 56mg 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. Springer-Verlag Berlin Heidelberg, 2015 Snake venoms nationallicence Crotalus durissus nationallicence Serine protease nationallicence Hemostasis nationallicence Heterologous expression nationallicence Boldrini-França Johara Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, 14040-903, Ribeirão Preto, SP, Brazil aut Rodrigues Renata Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut Santos-Silva Ludier Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil aut de Souza Dayane Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut Gomes Mário Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut Cologna Camila Department of Chemistry, University of Liège, Liège, Belgium aut de Pauw Edwin Department of Chemistry, University of Liège, Liège, Belgium aut Quinton Loïc Department of Chemistry, University of Liège, Liège, Belgium aut Henrique-Silva Flávio Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil aut de Melo Rodrigues Veridiana Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut Arantes Eliane Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, 14040-903, Ribeirão Preto, SP, Brazil aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 9971-9986 0175-7598 99:23<9971 2015 99 253 https://doi.org/10.1007/s00253-015-6836-2 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6836-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Boldrini-França Johara Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, 14040-903, Ribeirão Preto, SP, Brazil aut NATIONALLICENCE 700 1- Rodrigues Renata Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut NATIONALLICENCE 700 1- Santos-Silva Ludier Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil aut NATIONALLICENCE 700 1- de Souza Dayane Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut NATIONALLICENCE 700 1- Gomes Mário Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut NATIONALLICENCE 700 1- Cologna Camila Department of Chemistry, University of Liège, Liège, Belgium aut NATIONALLICENCE 700 1- de Pauw Edwin Department of Chemistry, University of Liège, Liège, Belgium aut NATIONALLICENCE 700 1- Quinton Loïc Department of Chemistry, University of Liège, Liège, Belgium aut NATIONALLICENCE 700 1- Henrique-Silva Flávio Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil aut NATIONALLICENCE 700 1- de Melo Rodrigues Veridiana Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil aut NATIONALLICENCE 700 1- Arantes Eliane Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, 14040-903, Ribeirão Preto, SP, Brazil aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 9971-9986 0175-7598 99:23<9971 2015 99 253