GLYCOSYLATION OF A PROTEIN PRECURSORIN YEAST: SECRETION AND PROTEOLYTIC PROCESSING EFFICIENCY

Abstract

The 65aa peptide hirudin (rHV2Lys“), which is synthesized in the salivary glands of the leech Hirudo medicinalis, has been secreted from a transformed Saccharomyces cerevisiae strain expressing a MFal::rHV2Lys” chimeric gene. The ‘prepro’ part of the protein precursor is derived from the MFal gene providing an homologous secretion signal in yeast. A large amount of hirudin-related material detected in the culture supernatant using Western analysis is heterogeneousandof higher apparent molecular weight than mature hirudin. Endoglycosidase F (Endo F) treatment reduces the heterogeneity and generates a predominant form of about 15 kDa which would correspond to pro-rHV2Lys”. In the fusion protein only the MFa1 derived ‘pro’ peptide can be N-glycosylated. After amplification of the yeast KEX2 gene, which encodes the processing protease, the amount of precursor material was diminished and the level of biologically active rHV2Lys” was augmented more than five-fold. Mutations have been introduced in the MFal ‘pro’ peptide, which eliminate one or more of the glycosylationsites. All of these modifications result in significantly reduced secretion of active hirudin. Amplification of KEX2 leads to an increase in production rates for most of the precursors containing variant ‘pro’ peptides as comparedto the wild type form.