Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion.
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.
Wang
P
P
Institute of Molecular Medicine, Nanjing, 210093, People's Republic of China.
Zhang
J
J
Sun
Z
Z
Chen
Y
Y
Liu
J N
JN
D001665:Binding Sites; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010843:Pichia / Q000235:genetics*; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D005340:Fibrinogen; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D002266:Carboxymethylcellulose Sodium; C067551:saruplase