[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
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