Isolation and characterization of human pro-urokinase and its mutants accumulated within the yeast secretory pathway.
Human pro-urokinase (pro-UK) and two pro-UK deletion mutants, one lacking the epidermal growth factor(EGF)-like domain, and the other lacking both the EGF-like domain and the kringle domain, were produced in Saccharomyces cerevisiae. This was done using the yeast GAL7 promoter and the prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). Although biologically active and heavily glycosylated pro-UKs were secreted into the culture medium, the amounts were extremely small. On the other hand, large amounts of pro-UKs of a single-chain form were accumulated inside the cells, exceeding 3-4% of total cellular proteins. The intracellular pro-UKs were N-glycosylated, probably with a single core carbohydrate unit, and amino acid sequencing of their N termini revealed that the secretion signal of MPR was correctly processed. Biologically active pro-UKs were recovered in high yields by means of solubilization with 4.5 M guanidine.HCl and subsequent dialysis for refolding. The refolded yeast pro-UK was indistinguishable from human kidney-derived pro-UK in terms of specific enzymatic activity and its secondary structure, as determined by circular dichroism spectroscopy.
Hiramatsu
R
R
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Horinouchi
S
S
Beppu
T
T
D000595:Amino Acid Sequence; D002874:Chromosome Mapping; D004591:Electrophoresis, Polyacrylamide Gel; D005343:Fibrinolytic Agents / Q000302:isolation & purification; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D005343:Fibrinolytic Agents; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator