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 proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/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 enzyme/substrate 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.