Expression of active human uterine tissue plasminogen activator in yeast.
Tissue-type plasminogen activator (tPA) cDNA derived from human uterine mRNA was inserted into different yeast expression vectors. All such expression plasmids carried a yeast acid phosphatase (PHO5) promoter, a 2-micron plasmid replication origin, transcription termination signals, and a selectable TRP1 gene. Plasmid pYBDT-10 contained the entire tPA coding region ("pre-pro-tPA"), pYBDT-10-PRO contained a sequence encoding the putative pro-tPA precusor, and pYBDT-6 contained only a mature tPA cDNA fused precisely in frame to the sequence encoding the entire signal peptide of acid phosphatase. All constructions directed the synthesis of single-chain tPA proteins that were readily precipitated with a specific antibody directed against human uterine tPA. Electrophoretic mobilities were approximately the same as those of the Bowes melanoma single-chain tPA and a 68-kD protein marker. Treatment of immunoprecipitates with endoglycosidase H resulted in increased electrophoretic mobilities, suggesting that these yeast products are glycosylated. Despite the use of either human or yeast signal sequences, however, tPA produced in yeast was not secreted into the culture medium, but rather was found only in cells following disruption with glass beads. Although this cellular tPA exhibited fibrinolytic activity, most of the activity was associated with large cellular debris.
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*; D014170:Transformation, Genetic; D014599:Uterus / Q000378:metabolism*
D004247:DNA; D010959:Tissue Plasminogen Activator