Preparation of Recombinant  -Thrombin: High-Level Expression of Recombinant Human Prethrombin-2 and Its Activation by Recombinant Ecarin

Abstract

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.

Crystal Structure of Thrombin in Complex with S-Variegin: Insights of a Novel Mechanism of Inhibition and Design of Tunable Thrombin Inhibitors

“…Lyophilized recombinant human thrombin [18], [19] and s-variegin were dissolved and mixed in buffer containing 50 mM HEPES (pH 7.4) and 375 mM NaCl to final concentrations of 20 mg/ml and 3 mg/ml (1∶1.5 molar ratio), respectively. Crystallization was achieved using the hanging drop vapor diffusion method.…”

Successful treatment of collagen-induced arthritis in non-human primates by chimeric anti-osteopontin antibody

“…V H and V L of 2K1 cDNA fragments were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) and were then inserted into pCAG-γ1 with the human γ1 constant region gene and pCAG-κ vector with human kappa chain constant region, respectively [11,12]. Chinese hamster ovary (CHO) cells were transfected with both pCAG-κ1 and pCAG-κ carrying 2K1 V H and V L cDNA by electroporation.…”

Expression and Characterization of Recombinant Ecarin

“…Ecarin is a component of the venom from the saw-scaled viper Echis carinatus that can convert prethrombin-2 to thrombin [12] or prothrombin to meizothrombin, which subsequently will be converted to thrombin by auto-catalytic activity [8]. In contrast to FXa-like snake venom prothrombin activators, the ecarin protease activity does not depend on additional co-factors or calcium ions neither on the presence of a GLA domain [9].…”