Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Secretory Production of Recombinant Urokinase Kringle Domain in Pichia pastoris

  • Kim, Hyun-Kyung (Cancer Research Institute, Catholic Research Institute of Medical Sciences) ;
  • Hong, Yong-Kil (Department of Neurosurgery, The Catholic University of Korea) ;
  • Park, Hyo-Eun (Cancer Research Institute, Catholic Research Institute of Medical Sciences) ;
  • Hong, Sung-Hee (Laboratory of Experimental Therapeutics, Korea Institute of Radiological and Medical Sciences) ;
  • Joe, Young-Ae (Cancer Research Institute, Catholic Research Institute of Medical Sciences)
  • Published : 2003.08.01
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Abstract

Human urokinase kringle domain, sharing homology with angiostatin kringles, has been shown to be an inhibitor of angiogenesis, which can be used for the treatment of cancer, rheumatoid arthritis, psoriasis, and retinopathy. Here, the expression of the kringle domain of urokinase (UK1) as a secreted protein in high levels is reported. UK1 was expressed in the methylotrophic yeast Pichia pastoris GS115 by fusion of the cDNA spanning from Ser47 to Lys135 to the secretion signal sequence of ${\alpha}-factor$ prepro-peptide. In a flask culture, the secreted UK1 reached about 1 g/l level after 120h of methanol induction and was purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the purified UK1 revealed that it was cleaved at the Ste13 signal cleavage site. The molecular mass of UK1 was determined to be 10,297.01 Da. It was also confirmed that the purified UK1 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, or epidermal growth factor, in a dose-dependent manner. These results suggest that a P. pastoris sytem can be employed to obtain large amounts of soluble and active UK1.

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