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

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

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

Hiroshi Yonemura,Takayuki Imamura,Kenji Soejima,Yo Nakahara,Wataru Morikawa,Yoshitaka Ushio,Yasuharu Kamachi,Hiroshi Nakatake,Keishin Sugawara,Tomohiro Nakagaki,Chikateru Nozaki,

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.

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin. Publishing Authors By Initials

H Yonemura,T Imamura,K Soejima,Y Nakahara,W Morikawa,Y Ushio,Y Kamachi,H Nakatake,K Sugawara,T Nakagaki,C Nozaki,

For similar investigative techniques: genetic techniques: gene transfer techniques: transfection research abstracts see: investigative techniques: genetic techniques: gene transfer techniques: transfection research

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

PUBLICATION TYPE: Journal Article

Journal: Journal of biochemistry

VOLUME: 135

Page Numbers: 577-82

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: May

YEAR: 2004

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

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LANGUAGE: eng

NlmUniqueID: 376600

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

KEYWORDS: Transfection

MESH TERMS: metabolism

Chemical & Substance for Abstract: Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin. Information

Substance Name: ecarin

Registry Number: EC 3.4.99.27

Grant and Affiliation Information for Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.

AFFILIATION: Applied Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), Kawabe, Kyokushi, Kikuchi, Kumamoto 869-1298.

Country: Japan

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MEDLINETA: J Biochem

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