Mutations of the alpha-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast.
The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture medium. To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MEL1 promoter and MEL1 sp coding sequence (MEL1sp). The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MEL1sp. S. cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1. Using site-directed mutagenesis, several mutations were introduced into the MEL1sp. Two mutations were identified which dramatically increased the secretion of both Echis and PAI-1 to levels similar to those achieved when using the yeast MF alpha 1 pre-pro secretory leader. In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase in the secretion levels of those two proteins.
Hofmann
K J
KJ
Department of Cellular and Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.
Schultz
L D
LD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D005800:Genes, Fungal; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides; D010957:Plasmids; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D012440:Saccharomyces / Q000235:genetics*; D014170:Transformation, Genetic; D014757:Viper Venoms / Q000096:biosynthesis; D000519:alpha-Galactosidase / Q000235:genetics*
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D021382:Protein Sorting Signals; D014757:Viper Venoms; C057986:echistatin; D000519:alpha-Galactosidase