PRODUCTION OF HUMAN GASTRIC LIPASE IN THE FISSION YEAST SCHIZOSACCHAROMYCES-POMBE

A cDNA encoding human gastric lipase (hGL) has been expressed on multi copy plasmids in the fission yeast Schizosaccharomyces pombe (Sp). Act ive lipase is secreted from transformants containing the hGL cDNA unde r the control of either the Sp adh1 promoter (Padh1) or the plant caul iflower 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 mo lecular mass to natural hGL is secreted by Sp, although the major secr eted product is of a higher molecular mass than either native hGL or r ecombinant hGL produced in the budding yeast Saccharomyces cerevisiae (Sc). Several distinct hGL are present within cells at all growth phas es. Treatment of these proteins with endoglycosidase H gives rise to a single species equivalent in size to deglycosylated natural hGL, indi cating that most of these are glycosylation intermediates. An hGL of s imilar molecular mass accumulates intracellularly in Sp when a modifie d version of cDNA is used which lacks the sequence encoding the natura l secretory signal peptide. Production of hGL markedly slows the growt h rate of Sp. The average copy number per cell of the plasmid expressi ng the hGL cDNA from the recombinant Padh1 is 2-3, as compared with 11 -12 for the control plasmid.