Yeast secretory expression of insulin precursors

Since the 1980s, recombinant human insulin for the treatment of diabetes me llitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretor y expression system in S. cerevisiae and its subsequent optimisation is des cribed. Expression of proinsulin in S. cerevisiae does not result in effici ent secretion of proinsulin or insulin. However, expression of a cDNA encod ing a proinsulin-like molecule with deletion of threonine(B30) as a fusion protein with the S. cerevisiae alpha-factor prepro-peptide (leader), follow ed either by replacement of the human proinsulin C-peptide with a small C-p eptide (e.g. AAK), or by direct fusion of lysine(B29) to glycine(A1), resul ts in the efficient secretion of folded single-chain proinsulin-like molecu les to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by trypti c transpeptidation in organic-aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, a nd constructed (synthetic) leaders have been developed for efficient secret ory expression of the insulin precursor in the yeasts S. cerevisiae and Pic hia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves t he leader-insulin precursor fusion protein in the late secretory pathway an d the folded insulin precursor is secreted to the culture supernatant. Howe ver, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insu lin precursor fusion protein is secreted to the culture supernatant in a hy perglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease process ing and insulin precursor fermentation yield.