title: Characterization of a nonglycosylated single chain urinary plasminogen activator secreted from yeast.
abstract: Using site-directed mutagenesis, we have changed the asparagine in human single-chain urinary plasminogen activator (u-PA) at position 302 to an alanine. This alteration removes the only known amino acid residue glycosylated in the protein. The single-chain u-PA containing an alanine residue at position 302 instead of asparagine (scu-PA(N302A] cDNA gene was expressed in the yeast Saccharomyces cerevisiae. Secretion of the protein product into the culture broth was achieved by replacing the human secretion signal codons with those from yeast invertase, adding a yeast promoter from the constitutively expressed glycolytic genes triosephosphate isomerase or phosphoglycerate kinase, and integrating multiple copies of these transcriptional units into the genome of yeast strains carrying the "supersecreting" mutation ssc1. When fermented in a fed-batch mode, these recombinant baker's yeast strains secreted scu-PA(N302A) in a strongly growth-associated manner. Greater than 90% of the u-PA found in the culture broth was in the single-chain form. Scu-PA(N302A) was purified to homogeneity using two chromatography steps. The purified protein had a molecular weight of 47,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lacked any detectable N-linked glycosylation. The in vitro fibrinolytic properties of scu-PA(N302A) were found to be essentially equivalent to those of natural single-chain u-PA derived from the human kidney cell line TCL-598. Since scu-PA(N302A) lacks the immunogenic N-linked carbohydrate pattern of yeast, it may be a useful therapeutic agent which can be produced economically by yeast fermentation.
authors.affiliation: Department of Molecular Biology and Biochemistry, Collaborative Research, Inc., Bedford, Massachusetts 01730.
authors.forename: L M
authors.initials: LM
authors.lastname: Melnick
authors.forename: B G
authors.initials: BG
authors.lastname: Turner
authors.forename: P
authors.initials: P
authors.lastname: Puma
authors.forename: B
authors.initials: B
authors.lastname: Price-Tillotson
authors.forename: K A
authors.initials: KA
authors.lastname: Salvato
authors.forename: D R
authors.initials: DR
authors.lastname: Dumais
authors.forename: D T
authors.initials: DT
authors.lastname: Moir
authors.forename: R J
authors.initials: RJ
authors.lastname: Broeze
authors.forename: G C
authors.initials: GC
authors.lastname: Avgerinos
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D006031:Glycosylation; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D010960:Plasminogen Activators / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D004247:DNA; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator