Efficient secretion of Bacillus subtilis levanase by Saccharomyces cerevisiae.
The secretion of Bacillus subtilis (Bs) levanase (Lev) was studied in the yeast Saccharomyces cerevisiae. A set of different yeast expression plasmids, based on the constitutive PGK promoter and harbouring the Bs Lev-encoding gene (sacC), was constructed. In these plasmids, the original Bs signal sequence was either intact, partially deleted or entirely missing. With all constructs, Lev was produced from yeast transformants. However, only when the intact bacterial signal peptide was present was the synthesized enzyme secreted; around 20% was found in the periplasm and 30% in the culture medium. The secreted protein found in the periplasmic space was mainly core-glycosylated and unglycosylated, and had a size of 80-90 and 74 kDa, respectively. In contrast, Lev found in the culture medium was mainly hyper-glycosylated and had a size of 180-200 kDa. Yeast transformants harbouring sacC, but lacking parts of the bacterial signal sequence, only produced cytoplasmic protein which was not glycosylated and had a size of about 74 kDa. The deletion of the entire signal peptide and a further 22 amino acids at the N terminus of mature Lev resulted in a 71-kDa cytoplasmic protein which was not active.
Wanker
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Institut für Biotechnologie, Arbeitsgruppe Genetik, Graz, Austria.
Klingsbichel
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Schwab
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H
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D007444:Inulin / Q000378:metabolism; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007444:Inulin; D010735:Phosphoglycerate Kinase; D006026:Glycoside Hydrolases; C021512:levanase