Glycosylation of Prourokinase Produced by Pichia pastoris Impairs Enzymatic Activity but Not Secretion

Authors: Wang P.^{1, 2}; Zhang J.¹; Sun Z.^{1, 2}; Chen Y.¹; Liu J-N.^{1, 2}

Source: Protein Expression and Purification, Volume 20, Number 2, November 2000 , pp. 179-185(7)

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Abstract:

Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture. The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography. More than 99% of pro-UK in the culture medium was found in single-chain form. This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion. The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells. However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis. Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK. By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent. It was concluded that glycosylation of pro-UK by yeast P. pastoris interferes with the catalytic site but not secretion of this protein. Copyright 2000 Academic Press.

Language: English

Document Type: Research article

Affiliations: 1: Nanjing University, Nanjing, 210093, People's Republic of China 2: Vascular Research Laboratory, Harvard Medical School, Boston, Massachusetts, 02215

Publication date: 2000-11-01