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J. Biochem, 2004, Vol. 135, No. 5 577-582
© 2004 The Japanese Biochemical Society

BIOTECHNOLOGY

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

Hiroshi Yonemura*,1, Takayuki Imamura*,2, Kenji Soejima2, Yo Nakahara1, Wataru Morikawa1, Yoshitaka Ushio1, Yasuharu Kamachi1, Hiroshi Nakatake2, Keishin Sugawara1, Tomohiro Nakagaki2 and Chikateru Nozaki§,2

1 Applied Research Department and 2 First Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), Kawabe, Kyokushi, Kikuchi, Kumamoto 869-1298

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 ß-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 {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.

* These authors contributed equally to this work.

§ To whom correspondence should be addressed. Tel: +81-968-37-3100, Fax: +81-968-37-3616, E-mail: nozaki{at}kaketsuken.or.jp


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