Expression and secretion of staphylococcal nuclease in yeast: effects of amino-terminal sequences.
Staphylococcus aureus nuclease A hybrid genes, encoding proteins OmpA-nuclease, lipo-nuclease and Pin-nuclease, were cloned downstream of the yeast GAL10 inducible promoter. OmpA-nuclease and lipo-nuclease contain the mature staphylococcal nuclease sequence preceded by the Escherichia coli OmpA and lipoprotein signal sequences, respectively, whereas Pin-nuclease lacks a defined signal sequence at its amino terminus. We found that: (a) the nuclease gene products synthesized in yeast are active, but they do not affect cell growth; (b) OmpA-nuclease and lipo-nuclease are partially processed and constitute approximately 1.0-1.5% of the yeast cell protein; (c) OmpA and lipoprotein signal sequences function similarly in secretion, allowing 35-40% of the processed nuclease to be translocated into the yeast periplasm; and (d) Pin-nuclease, which lacks hydrophobic sequences at its amino-terminus, is accumulated at a level tenfold lower than the hybrid proteins that do contain signal sequences. Nevertheless, 50% of the enzyme activity of Pin-nuclease in yeast is localized in the periplasmic space.
Pines
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O
Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
London
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A
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics*
D008836:Micrococcal Nuclease