Gene Synthesis, Expression In Yeast And Purification Of Hirudin

Hirudin is a small protein secreted from the salivary gland of medicinal Jeech. Natural hirudin includes many variants which are composed of 65or 65 amino acids with 3 disulfide linkage. The sixty-third amino acid is a sulfated tyrosine. Hirudin is a nonglycosylated polypeptide and shows good stability.Hirudin is an highly efficient and specific thrombin inhibitor. It is clinically used to prevent the formation of thrombus. Since rare natural hirudin is available, recombinant-DNA techniques have been used to prepare hirudin.In this research the hirudin gene was synthesized and put into yeast system for expression. The efficiency of gene expression was valued and some factors affecting it were probed. According to the reported amino acid sequence of hirudin variants and the coden usage pattern in Saccharomyces cerevisiae, the HV1 gene was synthesized in 12 oligo-nucleotide fragments. After purification, the fragments were ligated into a complete HV1 gene and amplified by PCR. Then it was inserted into a cloning vector pBS-SK(+) and sequenced. In order to get the protein secreted, the coding sequence of the signal peptide of yeast α-factor was added to the front of the gene. The correctly modified HVl gene was inserted into yeast expression vector pYC-DE-2 and identified. The recombinant plasmid was transformed into the yeast strain S. cerevisiae BJ1990 to carry out the primary expression experiment. In culture supernant of screened positive clone, the hirudin activity was detected to be 30 ATU/ml. The expression level was higher than that of HV2 in yeast and HVl in prokaryotic system. A 4-step procedure was carried out to purify the hirudin: (1)precipitation with (NH₄)₂SO₄; (2) gel filtration with a column of sephadex G50; (3) an ion-exchange chromatography on Q sepharose HP column; (4) HPLC cation-exchange chromatography . Purity of the recombinant hirudin obtained from this procedure is higher than 99% and the sequence of N terminal amino acids coincided with the expected . The production of pure hirudin is 2mg/L The specific activity of antithrombin is 8000ATU/mg.It was proved by this research that the synthesized hirudin gene had been correctly expressed in yeast and successfully secreted from...