A REMOVABLE SPACER PEPTIDE IN AN ALPHA-FACTOR-LEADER INSULIN PRECURSOR FUSION PROTEIN IMPROVES PROCESSING AND CONCOMITANT YIELD OF THE INSULIN PRECURSOR IN SACCHAROMYCES-CEREVISIAE

An alpha-factor leader/insulin precursor fusion protein was produced i n Saccharomyces cerevisiae and metabolically labeled in order to analy se the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introducti on of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creatin g a N-terminal extension of the insulin precursor, greatly increased t he Kex2p catalytic efficiency and the fermentation yield of insulin pr ecursor. The N-terminal extension features a Lys to allow subsequent p roteolytic removal by trypsin or the Achromobacter lyticus Lys-specifi c protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu- Ala dipeptides from the extension was inhibited by adding a Glu N-term inally to the extension, Unexpectedly, this modified N-terminal extens ion (EEAEAEAK) was partially cleaved after the Lys during fermentation . This monobasic proteolytic activity was demonstrated to be associate d with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).