title: Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme.
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.
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, Ribeirão Preto, SP, 14040-903, Brazil.
authors.forename: Johara
authors.initials: J
authors.lastname: Boldrini-França
authors.affiliation: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil.|INCT, Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Belo Horizonte, MG, Brazil.
authors.forename: Renata
authors.initials: R
authors.lastname: Santos Rodrigues
authors.affiliation: Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil.
authors.forename: Ludier Kesser
authors.initials: LK
authors.lastname: Santos-Silva
authors.affiliation: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil.|INCT, Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Belo Horizonte, MG, Brazil.
authors.forename: Dayane Lorena Naves
authors.initials: DL
authors.lastname: de Souza
authors.affiliation: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil.|INCT, Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Belo Horizonte, MG, Brazil.
authors.forename: Mário Sérgio Rocha
authors.initials: MS
authors.lastname: Gomes
authors.affiliation: Department of Chemistry, University of Liège, Liège, Belgium.
authors.forename: Camila Takeno
authors.initials: CT
authors.lastname: Cologna
authors.affiliation: Department of Chemistry, University of Liège, Liège, Belgium.
authors.forename: Edwin
authors.initials: E
authors.lastname: de Pauw
authors.affiliation: Department of Chemistry, University of Liège, Liège, Belgium.
authors.forename: Loïc
authors.initials: L
authors.lastname: Quinton
authors.affiliation: Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil.
authors.forename: Flávio
authors.initials: F
authors.lastname: Henrique-Silva
authors.affiliation: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brazil.|INCT, Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Belo Horizonte, MG, Brazil.
authors.forename: Veridiana
authors.initials: V
authors.lastname: de Melo Rodrigues
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, Ribeirão Preto, SP, 14040-903, Brazil. ecabraga@fcfrp.usp.br.
authors.forename: Eliane Candiani
authors.initials: EC
authors.lastname: Arantes
mesh_terms: D000818:Animals; D001777:Blood Coagulation; D002417:Cattle; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology*; D017839:Crotalus; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature; D015032:Zinc / Q000378:metabolism
chemical_list: D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D005344:Fibrinopeptide A; D003300:Copper; D005340:Fibrinogen; D057057:Serine Proteases; D015032:Zinc
keywords: Crotalus durissus; Hemostasis; Heterologous expression; Serine protease; Snake venoms