Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin

• Published in: Journal of biochemistry (Tokyo) 2004-05, Vol.135 (5), p.577-582
• Main Authors: Yonemura, Hiroshi, Imamura, Takayuki, Soejima, Kenji, Nakahara, Yo, Morikawa, Wataru, Ushio, Yoshitaka, Kamachi, Yasuharu, Nakatake, Hiroshi, Sugawara, Keishin, Nakagaki, Tomohiro, Nozaki, Chikateru
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Animals; Biotechnology - methods; Blotting, Western; Cattle; Cell Line; Chickens; CHO Cells; Chromatography, Affinity; Cricetinae; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endopeptidases - chemistry; Enzyme Activation; Enzyme Precursors - isolation & purification; Enzyme Precursors - metabolism; Genetic Vectors; Humans; Kinetics; Methotrexate - pharmacology; Mice; Multiple Myeloma - metabolism; Mutation; Plasmids - metabolism; Platelet Aggregation; Promoter Regions, Genetic; Prothrombin - isolation & purification; Prothrombin - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Sepharose - chemistry; Tetrahydrofolate Dehydrogenase - genetics; Thrombin - metabolism; Time Factors; Transfection
• ISSN: 0021-924X
• DOI: 10.1093/jb/mvh070

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.