Yeast-derived recombinant human insulin-like growth factor I: Production, purification, and structural characterization

Abstract

Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted from Saccharomyces cerevisiae using a yeast alpha-factor leader to direct secretion. However, approximately 10-20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in beta-turns.

Behavior in the Eukaryotic Secretory Pathway of Insulin-containing Fusion Proteins and Single-chain Insulins Bearing Various B-chain Mutations

“…Like the situation for insulin, IGF-1 bioactivity and normal half-life requires the presence of all three native disulfide bonds (56 -58). Although disulfide mispairing in vivo has not previously been reported for singlechain insulin or proinsulin, it is well known to occur during production of IGF-1 in the secretory pathway (36,37) and upon IGF-1 refolding in vitro (55,59,60). Mispaired disulfide isomers of IGF-1 maintain some biological activity and elements of secondary structure (56,59).…”

Expression of biologically active human insulin‐like growth factor 1 in Arabidopsis thaliana seeds via oleosin fusion technology

“…The prokaryote E. coli is the primary choice for recombinant protein expression because of its facility in handling and cultivation, but direct expression of IGF-1 in E. coli may result in the formation of inclusion body [17], [18] or disulfide-linked aggregates [19]. A yeast expression system has the ability of glycosylation, but the glycosylation is not always correct [14], [19].…”

Improved Secretion of Native Human Insulin-Like Growth Factor 1 from gas1 Mutant Saccharomyces cerevisiae Cells

“…Nevertheless, native rhIGF-1 represents only 10 to 20% of the total rhIGF-1 production; most of the rhIGF-1 is inactive, essentially composed of dimers or multimers. Such forms are due to the formation of incorrect intra-and intermolecular disulfide bonds (4,7,23). In this report we describe an integrated approach to increasing the secretion and/or production in Saccharomyces cerevisiae of monomeric correctly folded rhIGF-1 using an expression system based on the prepro-␣-factor leader sequence.…”