Efficient intra- and extracellular production of human beta-1,4-galactosyltransferase in Saccharomyces cerevisiae is mediated by yeast secretion leaders.
We have compared the function of homologous and heterologous secretion leaders to mediate production of human beta-1,4-galactosyltransferase (Gal-Tf) in the yeast Saccharomyces cerevisiae. Although all genes encoding leader/Gal-Tf fusions were transcribed by strong yeast promoters, only low production levels were obtained for full-length Gal-Tf containing the human membrane-anchor region. In contrast, a gene fusion encoding the membrane-anchor region of the yeast alpha-1,2-mannosyltransferase (Mnt1) fused to soluble Gal-Tf yielded high mRNA and intracellular protein levels. Gal-Tf could also be produced extracellularly using a fusion of the pre-pro region of the yeast Mf alpha 1 precursor (MF alpha 1) to soluble Gal-Tf; a fusion containing only the pre-region of Mf alpha 1 was synthesized intracellularly, but did not lead to Gal-Tf activity in the culture medium. All yeast-produced Gal-Tf proteins were enzymatically active. These results demonstrate that yeast secretion leaders are advantageous to achieve efficient production of active Gal-Tf in S. cerevisiae.
Schwientek
T
T
Institut für Mikrobiologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Ernst
J F
JF
D001692:Biological Transport; D015152:Blotting, Northern; D002451:Cell Compartmentation; D005822:Genetic Vectors; D006801:Humans; D008364:Mannosyltransferases / Q000096:biosynthesis / Q000235:genetics; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C068701:KRE2 protein, S cerevisiae; D008364:Mannosyltransferases; D009237:N-Acetyllactosamine Synthase