Annette Frost Pettersson

Secretory expression and characterization of insulin in Pichia pastoris

The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin. The S. cerevisiae mating factor α (α‐factor) prepro‐leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic prepro‐leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin precursor expressed in P. pastoris. In contrast with S. cerevisiae, only insulin precursor and no unprocessed hyperglycosylated α‐factor pro‐leader/insulin precursor fusion protein was secreted from P. pastoris. A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris. A synthetic prepro‐leader, but not an α‐factor prepro‐leader lacking N‐glycosylation sites, facilitated the secretion of the insulin precursor in P. pastoris. P. pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S. cerevisiae. Peptide mapping and MS indicated a structure of the insulin precursor expressed in P. pastoris identical with that of human insulin.

A removable spacer peptide in an α-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 in Saccharomyces cerevisiae and metabolically labeled in order to analyse 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. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).

The role of leaders in intracellular transport and secretion of the insulin precursor in the yeast Saccharomyces cerevisiae.

Pulse-chase analysis of folded and misfolded insulin precursor (IP) expressed in Saccharomyces cerevisiae was performed to establish the requirements for intracellular transport and the influence of the secretory pathway quality control mechanisms on secretion. Metabolic labelling of the IP expressed in S. cerevisiae showed that the effect of a leader was to stabilise the IP in the endoplasmic reticulum (ER), and facilitate intracellular transport of the fusion protein and rapid secretion. The first metabolically labelled IP appeared in the culture supernatant within 2-4 min of chase, and most of the secreted IP appeared within the first 15 min of chase. After enzymatic removal of the leader in a late Golgi apparatus compartment, the IP followed one of two routes: (1) to the plasma membrane and hence to the culture supernatant, or (2) to a Golgi or post-Golgi compartment from which secretion was restricted. Combined secretion and intracellular retention of the IP reflected either saturation of a Golgi or post-Golgi compartment and secretion as a consequence of overexpression, or competition between secretion and intracellular retention. IP which was misfolded, either due to amino acid substitution or because disulphide bond formation had been prevented with dithiothreitol (DTT), was transported from the ER to the Golgi apparatus but then retained in a Golgi or post-Golgi compartment and not exported to the culture supernatant.

Relationship between self-association of insulin and its secretion efficiency in yeast

The folding stability of insulin is positively correlated with the expression yield of the precursor expressed in yeast. Insulin assembles into dimers and hexamers in a concentration-dependent manner and amino acid substitutions that impair the ability of insulin to associate into dimers concomitantly decrease the expression yield (excluding substitutions that enhance folding stability). In contrast, introduction of an amino substitution that enhances the self-association of insulin improved the yeast expression yield. In the monomeric state the majority of the non-polar residues of insulin are exposed to the solvent and assembly into dimers and hexamers shields these from contact with the solvent. It is proposed that self-association enhances the flux of insulin through the secretory pathway by increasing the hydrophilicity, decreasing the surface area as well as decreasing the molar concentration in the secretory pathway.