Secretion of human blood coagulation factor XIIIa by the yeast Yarrowia lipolytica.
The industrial yeast, Yarrowia lipolytica, secretes high yields of an alkaline extracellular protease (AEP), which is synthesized as a preproprotein encoded by the XPR2 gene. We investigated the possibility of using this system for the secretion of human coagulation factor XIII subunit a (FXIIIa). This protein is naturally secreted in the plasma by an unknown, signal peptide-independent mechanism and has so far been found to be nonsecretable in yeast. We have designed six hybrid genes encoding fusion proteins between increasing portions of the AEP preprodomain and the precursor or mature forms of FXIIIa. All constructs directed translocation of the FXIIIa precursor into the endoplasmic reticulum. Transport of the translocated and core-glycosylated hybrid precursor to the Golgi apparatus appeared to be strongly rate limiting, and most of the precursors appeared to be partially proteolysed. One of these constructs directed the extracellular secretion of a low amount of hyperglycosylated FXIIIa. These results indicate that fusion to the yeast AEP signal peptide and dipeptide stretch allows FXIIIa to be translocated, albeit inefficiently, through the endoplasmic reticulum and to follow a classical secretory transit.
Laboratoire de Génétique INRA-CNRS, Institut National Agronomique Paris-Grignon, Thiverval Grignon, France.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D008969:Molecular Sequence Data; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics*; D012697:Serine Endopeptidases / Q000235:genetics*; D011503:Transglutaminases / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011503:Transglutaminases; D012697:Serine Endopeptidases; C067468:Yarrowia lipolytica alkaline extracellular protease