Expression and folding of an interleukin‐2‐proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C‐peptide and the N‐terminal fused sequence

Abstract

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsufin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200/xg/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzymelsubstrate rate (I :600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.

Formation of Disulfide Bonds and Homodimers of the Major Cat Allergen Fel d 1 Equivalent to the Natural Allergen by Expression in Escherichia coli

“…Therefore we engineered a head-to-tail construct of the two polypeptide chains of Fel d 1, which we considered would facilitate a uniform and naturallike folding. Interestingly, a high yield of properly refolded protein from the inclusion body was achieved using a similar single polypeptide strategy in the production of recombinant human insulin produced in E. coli (41,42). Similarly, a high level expression E. coli system, which produces proteins without carbohydrates, was also used for the Fel d 1 (2 ϩ 1) hybrid molecule because there is evidence that deglycosylated nFel d 1 is still IgE reactive (20).…”

Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

“…However, the target protein must not be cleaved by this protease, and the protein has to be correctly folded after the enzymatic cleavage or have the correctly folded form before cleavage. Several fused proinsulins that bear a tryptic site (lysine) at the end of the N-fused peptide (instead of the traditional methionine residue), were constructed (Chen et al, 1995;Castellanos-Serra et al, 1996;Nilsson et al, 1996). Using this method, both the N-fused sequence and the C-peptide are removed simultaneously by trypsin, in a single step, without cleavage of the target protein.…”

Methods for protecting silica sorbents used in high-performance liquid chromatography from strongly adsorbed impurities during purification of human recombinant insulin

“…Its manufacturing takes about 25 000 kg per year world wide nowadays [3]. At present, several methods of HRI large-scale production by fermentation of Escherichia coli [4,5] or Saccharomyces cerevisiae [6] are employed. It is clear that the optimal of the HRI production influences the manufacturing profitability, that is why manufacturing optimization becomes very important for process economics.…”