In-vitro processing of yeast alpha-factor leader fusion proteins using a soluble yscF (Kex2) variant.
The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the alpha-factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscFs). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for alpha-factor leader-hIGF1 and alpha-factor leader-hirudin fusion proteins.
Ciba-Geigy Ltd., Biotechnology Department, Basel, Switzerland.
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D003124:Colorimetry; D005285:Fermentation; D005656:Fungal Proteins / Q000378:metabolism*; D006629:Hirudins / Q000378:metabolism; D015151:Immunoblotting; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D000072235:Mating Factor; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D012995:Solubility; D013381:Subtilisins; D014170:Transformation, Genetic
D005656:Fungal Proteins; D006629:Hirudins; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D007334:Insulin-Like Growth Factor I; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae