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KMID : 0545119990090020179
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Journal of Microbiology and Biotechnology
1999 Volume.9 No. 2 p.179 ~ p.183
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Downstream Processing of Recombinant Hirudin Produced in Saccharomyces cerevisiae
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CHUNG, BONG HYUN
KIM, WON KYUNG/RAO, K. JAGANADHA/KIM, CHUL-HO/RHEE, SANG-KI
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Abstract
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A recombinant form of hirudin, a potent thrombin-specific inhibitor derived from the bloodsucking leech, was expressed as a secretory product in Saccharomyees cerevisiae under the control of CAL10 promoter and the mating factor α 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 ㎎/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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