Purification and determination of the NH2-terminal amino acid sequence of mouse alpha-amylase secreted from Saccharomyces cerevisiae: correct processing of the secretion signal from pGKL killer 28 kDa precursor protein.
We have previously reported the construction of recombinant mouse salivary alpha-amylase secretion vector in Saccharomyces cerevisiae utilizing novel yeast secretion signal derived from killer 28 kDa precursor protein. Here, we have first purified recombinant mouse alpha-amylase to homogeneity from the culture medium of S. cerevisiae, and determined its NH2-terminal amino acid sequence. The sequencing data indicated that the 28 kDa killer secretion signal-alpha-amylase fusion protein was cleaved accurately at its native processing site, and that both the core-glycosylated and non-glycosylated alpha-amylases possessed the same NH2-terminal amino acid sequences.
Laboratory of Molecular Genetics, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
M
M
Tokunaga
A
A
Kawamura
A
A
Omori
F
F
Hishinuma
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification; D005656:Fungal Proteins / Q000235:genetics; D055646:Killer Factors, Yeast; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009183:Mycotoxins / Q000235:genetics*; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D012463:Saliva / Q000201:enzymology; D000516:alpha-Amylases / Q000235:genetics* / Q000302:isolation & purification
D004792:Enzyme Precursors; D005656:Fungal Proteins; D055646:Killer Factors, Yeast; D009183:Mycotoxins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000516:alpha-Amylases