Article (Scientific journals)
Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme
Boldrini-Franca, Johara; Santos Rodrigues, Renata; Santos-Silva, Ludier Kesser et al.
2015In Applied Microbiology and Biotechnology, 99, p. 9971-9986
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Keywords :
venomics; toxins; serine protease
Abstract :
[en] 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 kcat/Km 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.
Disciplines :
Chemistry
Author, co-author :
Boldrini-Franca, Johara;  Universidade de São Paulo - Brazil
Santos Rodrigues, Renata;  Universidade Federal de Uberlândia - Brazil
Santos-Silva, Ludier Kesser;  Universidade Federal de São Carlos - Brazil
Naves de Souza, Dayane;  Universidade Federal de Uberlândia - Brazil
Rocha Gomes, Mario Sergio;  Universidade Federal de Uberlândia - Brazil
Cologna Takeno, Camila;  Universidade de Sao Paulo - Brazil
De Pauw, Edwin  ;  Université de Liège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Quinton, Loïc  ;  Université de Liège > Département de chimie (sciences) > Chimie biologique
Henrique-Silva, Flavio;  Universidade Federal de São Carlos - Brazil
de Melo Rodrigues, Veridiana;  Universidade Federal de Uberlândia - Brazil
Candiani Arantes, Eliane;  Universidade de São Paulo - Brazil
Language :
English
Title :
Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme
Publication date :
2015
Journal title :
Applied Microbiology and Biotechnology
ISSN :
0175-7598
eISSN :
1432-0614
Publisher :
Springer Verlag, Berlin, Germany
Volume :
99
Pages :
9971-9986
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 29 June 2016

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