Expression and Characterization of Recombinant Ecarin

Abstract

The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a Km value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the Km for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.

Recombinant snake venom prothrombin activators

“…Our characterization of recombinant ecarin confirmed that r-ecarin could efficiently generate thrombin without any additional co-factors 13 - 15 . We found that although the primary product from digesting prothrombin with ecarin is meizothrombin, the meizothrombin was very rapidly converted to thrombin, and therefore the final product is thrombin.…”

“…In parallel with expressing and characterizing trocarin and oscutarin we also produced and characterized recombinant ecarin from CHO cells as was recently described 13 . Our characterization of recombinant ecarin confirmed that r-ecarin could efficiently generate thrombin without any additional co-factors 13 - 15 .…”

“…During transient transfection procedures FBS was reduced to 1%. Recombinant ecarin was obtained from CHO cells cultured in chemically defined medium as described by Jonebring 13 . Diluted cell culture supernatants containing the respective protein were used in further experiments.…”

Temperature effect on expression of recombinant human prethrombin-2 in Escherichia coli BL21(DE3) ArcticExpress

“…Prethrombin-2, structurally has one glycosylation site (at Asn373) and four disulphide bonds (Cys293-Cys439, Cys348-Cys364, Cys493-Cys507, and Cys521-Cys551) (Soejima et al, 2001). Pretrombin-2 can be converted to thrombin by FXa enzyme activators (Owen et al 1974), or with ecarin, namely metalloprotease in the venom of snake Echis carinatus species (So et al 1992;DiBella et al 1995;Jonebring et al 2012;Yonemura et al 2004;Subroto et al 2016).…”

Cloning, Expression, and Functional Characterization of Autoactivated Human Prethrombin-2 Synthetic Gene by Using Pichia pastoris SMD1168 As a Host

“…To date, there is an effort to express a recombinant h u m a n p r e t h r o m b i n -2 ( r h P T 2 ) h o s t e d b y microorganisms, such Escherichia coli for commercial purposes (Choi et al 1989;So et al 1992;Silaban et al 2014). However, activation of rhPT2 into thrombin via proteolytic cleavage of FXa is replaced by ecarin, the snake venom protease isolated from Echis carinatus (Jonebring et al 2012). The activated rhPT2 needs further purification to remove ecarin, hence decreasing the production efficiency.…”