A novel screening system for yeast strains capable of secreting tissue plasminogen activator.
We have developed a simple screening procedure that allowed us to identify Saccharomyces cerevisiae strains able to secrete human tissue plasminogen activator (tPA) into the culture medium. The screen can be used to isolate more efficient secretor strains and to look for novel tPA analogs. Employing one of these strains to study the effect of glycosylation on secretion, we show that glycosylation in the catalytic domain of tPA plays an important role in folding and/or secretion of the molecule. Removing this glycosylation site resulted in a 3-5-fold reduction in the level of tPA secretion. We anticipate that this system will prove useful in studying yeast secretory pathway as well as structure-function relationships in the tPA molecule.
Gill
G S
GS
Molecular Biology Research, Upjohn Company, Kalamazoo, MI 49007.
Zaworski
P G
PG
Marotti
K R
KR
Rehberg
E F
EF
D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000302:isolation & purification*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator