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

• Yonemura Hiroshi

  Applied Research Department The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Imamura Takayuki

  First Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Soejima Kenji

  First Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Nakahara Yo

  Applied Research Department The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Morikawa Wataru

  Applied Research Department The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Ushio Yoshitaka

  Applied Research Department The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Kamachi Yasuharu

  Applied Research Department The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Nakatake Hiroshi

  First Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Sugawara Keishin

  Applied Research Department The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Nakagaki Tomohiro

  First Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

• Nozaki Chikateru

  First Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN)

書誌事項

タイトル別名

• Preparation of Recombinant α-Thrombin: High-Level Expression of Recombinant Human Prethrombin-2 and Its Activation by Recombinant Ecarin
• Preparation of Recombinant アルファ 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

抄録

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.

収録刊行物

• The Journal of Biochemistry

  The Journal of Biochemistry 135 (5), 577-582, 2004

  社団法人 日本生化学会

キーワード

• α-thrombin
• ecarin
• prethrombin-2
• recombinant

詳細情報

• CRID: 1390001204963803136
• NII論文ID: 130003534698
• NII論文ID: 10016199776
• NII書誌ID: AA00694073
• DOI: 10.1093/jb/mvh070
• COI: 1:CAS:528:DC%2BD2cXlvFKgsr0%3D
• ISSN: 17562651
• ISSN: 0021924X
• NDL書誌ID: 6948895
• PubMed: 15173195
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I6948895
• Web Site: http://academic.oup.com/jb/article-pdf/135/5/577/2548240/mvh070.pdf
• Web Site: https://search.jamas.or.jp/link/ui/2005023051
• 本文言語コード: en
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
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