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Rapid Selection of Multiple Gene Integrant for the Production of Recombinant Hirudin in Hansenula polymorpha

Kim Hwa Young    (Korea Research Institute of Bioscience and Biotechnology   ); Sohn Jung Hoon    (Korea Research Institute of Bioscience and Biotechnology   ); Kim Chul Ho    (Korea Research Institute of Bioscience and Biotechnology   ); Rao K. Jagannadha    (Korea Research Institute of Bioscience and Biotechnology   ); Choi Eui Sung    (Korea Research Institute of Bioscience and Biotechnology   ); Kim Myung Kuk    (Central Research Institute, Dong Kook Pharmaceutical Co. Ltd.   ); Rhee Sang Ki    (Korea Research Institute of Bioscience and Biotechnology  );
  • 초록

    For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeast Hansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using the MOX promoter of H. polymorpha and the mating factor $\alpha$ secretion signal of S. cerevisiae. Multiple integrants of a transforming vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression in H. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.


  • 주제어

    hirudin .   Hansenula polymorpha .   G418 resistance .   multicopy integration .   MOX promoter.  

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