Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme
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| 024 | 7 | 0 | |a 10.1007/s00253-015-6836-2 |2 doi |
| 035 | |a (NATIONALLICENCE)springer-10.1007/s00253-015-6836-2 | ||
| 245 | 0 | 0 | |a Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme |h [Elektronische Daten] |c [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] |
| 520 | 3 | |a 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. | |
| 540 | |a Springer-Verlag Berlin Heidelberg, 2015 | ||
| 690 | 7 | |a Snake venoms |2 nationallicence | |
| 690 | 7 | |a Crotalus durissus |2 nationallicence | |
| 690 | 7 | |a Serine protease |2 nationallicence | |
| 690 | 7 | |a Hemostasis |2 nationallicence | |
| 690 | 7 | |a Heterologous expression |2 nationallicence | |
| 700 | 1 | |a Boldrini-França |D Johara |u 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 |4 aut | |
| 700 | 1 | |a Rodrigues |D Renata |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | |
| 700 | 1 | |a Santos-Silva |D Ludier |u Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil |4 aut | |
| 700 | 1 | |a de Souza |D Dayane |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | |
| 700 | 1 | |a Gomes |D Mário |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | |
| 700 | 1 | |a Cologna |D Camila |u Department of Chemistry, University of Liège, Liège, Belgium |4 aut | |
| 700 | 1 | |a de Pauw |D Edwin |u Department of Chemistry, University of Liège, Liège, Belgium |4 aut | |
| 700 | 1 | |a Quinton |D Loïc |u Department of Chemistry, University of Liège, Liège, Belgium |4 aut | |
| 700 | 1 | |a Henrique-Silva |D Flávio |u Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil |4 aut | |
| 700 | 1 | |a de Melo Rodrigues |D Veridiana |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | |
| 700 | 1 | |a Arantes |D Eliane |u 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 |4 aut | |
| 773 | 0 | |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/23(2015-12-01), 9971-9986 |x 0175-7598 |q 99:23<9971 |1 2015 |2 99 |o 253 | |
| 856 | 4 | 0 | |u https://doi.org/10.1007/s00253-015-6836-2 |q text/html |z Onlinezugriff via DOI |
| 898 | |a BK010053 |b XK010053 |c XK010000 | ||
| 900 | 7 | |a Metadata rights reserved |b Springer special CC-BY-NC licence |2 nationallicence | |
| 908 | |D 1 |a research-article |2 jats | ||
| 949 | |B NATIONALLICENCE |F NATIONALLICENCE |b NL-springer | ||
| 950 | |B NATIONALLICENCE |P 856 |E 40 |u https://doi.org/10.1007/s00253-015-6836-2 |q text/html |z Onlinezugriff via DOI | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Boldrini-França |D Johara |u 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 |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Rodrigues |D Renata |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Santos-Silva |D Ludier |u Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a de Souza |D Dayane |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Gomes |D Mário |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Cologna |D Camila |u Department of Chemistry, University of Liège, Liège, Belgium |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a de Pauw |D Edwin |u Department of Chemistry, University of Liège, Liège, Belgium |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Quinton |D Loïc |u Department of Chemistry, University of Liège, Liège, Belgium |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Henrique-Silva |D Flávio |u Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a de Melo Rodrigues |D Veridiana |u Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Arantes |D Eliane |u 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 |4 aut | ||
| 950 | |B NATIONALLICENCE |P 773 |E 0- |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/23(2015-12-01), 9971-9986 |x 0175-7598 |q 99:23<9971 |1 2015 |2 99 |o 253 | ||