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

We report the expression in E. coli of a proinsulin fusion protein car rying a modified interleukin-2 N-terminal peptide linked to the N-term inus of proinsulin by a lysine residue, The key aspects investigated w ere: (a) the expression of the fused IL2-PI gene, (b) the folding effi ciency of the insulin precursor when still carrying the N-fused peptid e and (c) the selectivity of the enzymatic cleavage reaction with tryp sin in order to remove simultaneously the C-peptide and the N-terminal extension, It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 mu g/ml to yield about 80% of correctly folde d proinsulin and then it was converted into insulin by prolonged react ion (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrat e rate (1:600), 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.