Preparation of Recombinant α-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 α-thrombin. In this system, a high yield of α-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 β-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 μg/ml. Subsequently, the recombinant prethrombin-2 could be activated to α-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant α-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 α-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived α-thrombin. Our system is suitable for the large-scale production of recombinant α-thrombin, which can be used in place of clinically available α-thrombin derived from human or bovine plasma.