Secretion of mouse alpha-amylase from fission yeast Schizosaccharomyces pombe: presence of chymostatin-sensitive protease activity in the culture medium.
We have constructed two secretion vectors for Schizosaccharomyces pombe using an SV40 promoter and the secretion signals of the pGKL killer toxin complex derived from Kluyveromyces lactis. Although indigenous secretory glycoproteins tend to accumulate in the periplasmic space of S. pombe, we have succeeded in the secretion of mouse alpha-amylase into the culture medium. The efficiency of secretion, processing pattern, stability and culture conditions for mouse alpha-amylase were studied in S. pombe. The 128 kDa killer secretion signal was more effective in directing secretion of mouse alpha-amylase than the 28 kDa killer secretion signal. We detected a chymostatin-sensitive protease activity in the culture medium of S. pombe, which digests mouse alpha-amylase secreted into the culture medium. The addition of 5 micrograms/ml chymostatin was shown to protect mouse alpha-amylases from this degradation.
Tokunaga
M
M
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Kawamura
A
A
Yonekyu
S
S
Kishida
M
M
Hishinuma
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D010450:Endopeptidases / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*; D000516:alpha-Amylases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*
D009842:Oligopeptides; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C002101:chymostatin; D000516:alpha-Amylases; D010450:Endopeptidases; D002918:Chymotrypsin