Downstream Processing of Recombinant Hirudin Produced in Saccharomyces cerevisiae

  • Chung, Bong-Hyun (Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Won-Kyung (Korea Research Institute of Bioscience and Biotechnology) ;
  • Rao, K.Jagannadha (Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Chul-Ho (Korea Research Institute of Bioscience and Biotechnology) ;
  • Rhee, Sang-Ki (Korea Research Institute of Bioscience and Biotechnology)
  • 발행 : 1999.04.01

초록

A recombinant form of hirudin, a potent thrombin-specific inhibitor derived from the bloodsucking leech, was expressed as a secretory product in Saccharomyces cerevisiae under the control of GALl0 promoter and the mating factor $\alpha$pre-pro leader sequence. In an attempt to produce recombinant hirudin (r-Hir) of therapeutic purity in large quantities, the fed-batch fermentation was carried out by using this recombinant yeast, and subsequently downstream processing was developed with the preparative-scale column chromatography systems. About 234 mg/l of biologically active r-Hir was produced as a secretory product by the fed-batch fermentation strategy developed for an efficient downstream processing. Using a two-step chromatography process (an anion exchange chromatography followed by the reverse phase HPLC), the r-Hir was purified to>98% with an overall recovery yield of 84%. According to the N-terminal amino acid sequencing, the purified r-Hir was found to have the predicted N-terminal amino acid sequence. The biological activity of the purified r-Hir to inhibit thrombin was also identical to that of the commercial hirudin.

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