Production of human gastric lipase in the fission yeast Schizosaccharomyces pombe.
A cDNA encoding human gastric lipase (hGL) has been expressed on multicopy plasmids in the fission yeast Schizosaccharomyces pombe (Sp). Active lipase is secreted from transformants containing the hGL cDNA under the control of either the Sp adh1 promoter (Padh1) or the plant cauliflower mosaic virus (CaMV) 35S promoter. Cell-wall-associated lipase activities are greatest in the early logarithmic growth phase and with Padh1. Western blot analysis indicates that a protein of identical molecular mass to natural hGL is secreted by Sp, although the major secreted product is of a higher molecular mass than either native hGL or recombinant hGL produced in the budding yeast Saccharomyces cerevisiae (Sc). Several distinct hGL are present within cells at all growth phases. Treatment of these proteins with endoglycosidase H gives rise to a single species equivalent in size to deglycosylated natural hGL, indicating that most of these are glycosylation intermediates. An hGL of similar molecular mass accumulates intracellularly in Sp when a modified version of cDNA is used which lacks the sequence encoding the natural secretory signal peptide. Production of hGL markedly slows the growth rate of Sp. The average copy number per cell of the plasmid expressing the hGL cDNA from the recombinant Padh1 is 2-3, as compared with 11-12 for the control plasmid.
Smerdon
G R
GR
Department of Biological Sciences, University of Exeter, UK.
Aves
S J
SJ
Walton
E F
EF
D000426:Alcohol Dehydrogenase / Q000235:genetics; D017796:Caulimovirus / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D018628:Gene Dosage; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006596:Hexosaminidases; D006801:Humans; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development; D013270:Stomach / Q000201:enzymology*; D014170:Transformation, Genetic; D014280:Triglycerides / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D000426:Alcohol Dehydrogenase; D008049:Lipase; D006596:Hexosaminidases; C005830:tributyrin