Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein.
Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.
D000135:Acid Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014415:Tunicamycin / Q000494:pharmacology; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014415:Tunicamycin; D000135:Acid Phosphatase; D000516:alpha-Amylases; D011761:Pyrrolidonecarboxylic Acid