Production of Recombinant Human Antithrombin by Pichia pastoris(Microbial Physiology and Biotechnology)

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概要

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• This paper deals with the production of recombinant human antithrombin (rAT) by the methylotrophic yeast Pichia pastoris. In preliminary methanol-limited fed-batch fermentation, the rAT concentration reached 324 mg/l at 192 h of cultivation, but the specific heparin cofactor (HC) activity of rAT in the culture supernatant was 10% of that of plasma-derived antithrombin (pAT). To improve the specific HC activity of rAT, effort was first focused on the optimization of culture pH and media composition, resulting in protection of rAT against pH-dependent instability and proteolytic degradation. However, even in the optimized methanol-limited fed-batch fermentation, the specific HC activity of rAT in the culture supernatant was still 20% that of pAT. To investigate the unknown mechanisms involved in the decreased specific HC activity of rAT, the culture supernatant of mock-transfected cells was prepared by methanol-limited fed-batch fermentation. When pAT was added to this supernatant, a rapid decrease in HC activity was observed; the residual HC activity was 26% after 24 h of incubation at 25℃. The loss of pAT activity was prevented by addition of a formaldehyde scavenger, aminourea, to the supernatant. In addition, alcohol oxidase activity was observed in the supernatant, resulting in the accumulation of formaldehyde in the culture broth. These results suggest that the formaldehyde produced by methanol oxidation in the culture broth of P. pastoris might decrease the HC activity of rAT during fermentation. Replacing the methanol with glycerol as the carbon source improved the specific HC activity of rAT from 20% to above 40% of that of pAT. In the glycerol-limited fed-batch fermentation, rAT is expressed at 100 mg/l under the control of the truncated mutated AOX2 promoter.

• 社団法人日本生物工学会の論文
• 2005-03-25

著者

• Ohya Tomoshi

  Protein Research Laboratory, Mitsubishi Tanabe Pharma Corporation

• Kobayashi Kaoru

  Protein Research Laboratory, Mitsubishi Tanabe Pharma Corporation

• Ohya Tomoshi

  Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation

• Ohya Tomoshi

  大阪大学 工学研究科生命先端工学専攻

• Ohya Tomoshi

  Protein Reserach Laboratories Pharmaceuticals Research Division Mitsubishi Pharma Corporation

• Kuwae Shinobu

  Protein Research Laboratory, Pharmaceutical Research Unit, Mitsubishi Pharma Corporation

• OHYAMA MASAO

  Protein Research Laboratory, Pharmaceutical Research Division, Mitsubishi Pharma Corporation

• OHI HIDEYUKI

  Protein Research Laboratory, Pharmaceutical Research Division, Mitsubishi Pharma Corporation

• Ohda Toyoo

  Bioscience Research Drug Discovery Laboratories Pharmaceutical Research Division Yoshitomi Pharmaceu

• Kobayashi Kaoru

  Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation

• Ohyama Masao

  Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation

• Ohi Hideyuki

  Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation

• Kuwae Shinobu

  Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation

• Kuwae Shinobu

  Research Center For Cell And Tissue Culture Faculty Of Agriculture Kyoto University

• Kobayashi Kaoru

  Protein Research Laboratory Mitsubishi Tanabe Pharma Corporation

• Ohya Tomoshi

  Protein Research Laboratory Mitsubishi Tanabe Pharma Corporation

関連論文

• Overexpression of GADD34 Enhances Production of Recombinant Human Antithrombin III in Chinese Hamster Ovary Cells(CELL AND TISSUE ENGINEERING)
• Development of a Fed-Batch Culture Process for Enhanced Production of Recombinant Human Antithrombin by Chinese Hamster Ovary Cells(CELL AND TISSUE ENGINEERING)
• Production of Recombinant Human Antithrombin by Pichia pastoris(Microbial Physiology and Biotechnology)
• High-Level Production of Prourokinase-Annexin V Chimeras in the Methylotrophic Yeast Pichia pastoris (BIOCHEMICAL ENGINEERING)
• High Level Secretion of Recombinant Human Serum Albumin by Fed-Batch Fermentation of the Methylotrophic Yeast, Pichia pastoris, Based on Optimal Methanol Feeding Strategy
• Addition of Oleic Acid Increases Expression of Recombinant Human Serum Albumin by the AOX2 Promoter in Pichia pastoris
• High-Level Expression of Recombinant Human Serum Albumin from the Methylotrophic Yeast Pichia pastoris with Minimal Protease Production and Activation
• THE EFFECTS OF CODING REGION ON ITS GENE EXPRESSION IN THE Chlamydomonas CHLOROPLAST
• Effect of Coding Regions on Chloroplast Gene Expression in Chlamydomonas reinhardtii
• Derivation of a Glycerol-negative Mutant of a Methylotrophic Yeast, Candida boidinii, and Accumulation of Glycerol in Methanol Medium(Microbiology & Fermentation Industry)
• Overexpression of GADD34 Enhances Production of Recombinant Human Antithrombin III in Chinese Hamster Ovary Cells
• Production of Glycerol from Methanol by a Mutant Strain of Candida boidinii No. 2201

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