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KMID : 0545120030130040591
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Journal of Microbiology and Biotechnology
2003 Volume.13 No. 4 p.591 ~ p.597
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Secretory Production of Recombinant Urokinase Kringle Domain in Pichia pastoris
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KIM, HYUN-KYUNG
HONG, YONG-KIL/PARK, HYO-EUN/HONG, SUNG HEE
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Abstract
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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 (UKI) as a secreted prntein in high levels is reported. UK1 was expressed in the methylotrophic yeast Pichia pasforis GS115 by fusion of the cDNA spanning from Ser47 to Lys 135 to the secretion signal sequence of a-factor prepro-peptide. In a flask culture. the secreted UKI reached about 1 g/I level after 120 h of methanol induction and was purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the purified UKI revealed that it was cleaved at the Stel3 signal cleavage site. The molecular mass of UKI was determined to he 10,297.01 Da. It was also confirmed that the purified UKI 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 system can be employed to ohtain large amounts of soluble and active UKI.
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