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

• Published in: FEBS letters Vol. 378; no. 2; pp. 171 - 176
• Main Authors: Castellanos-Serra, Lila R., Hardy, Eugenio, Ubieta, Raimundo, Vispo, Nelson S., Fernandez, Cesar, Besada, Vladimir, Falcon, Viviana, Gonzalez, Marta, Santos, Alicia, Perez, Gudelia, Silva, Alejandro, Herrera, Luis
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 08.01.1996
• Subjects: Amino Acid Sequence; C-Peptide - metabolism; Carboxypeptidase B; Carboxypeptidases - metabolism; Enzymatic processing; Escherichia coli - genetics; Gene Expression; Insulin - metabolism; Interleukin-2 - chemistry; Interleukin-2 - genetics; Lysine - chemistry; Molecular Sequence Data; Peptide Fragments - metabolism; Proinsulin - chemistry; Proinsulin - genetics; Proinsulin folding; Protein Folding; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant insulin; Trypsin - metabolism; Recombinant insulin; Enzymatic processing; Proinsulin folding
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(95)01437-3

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 μg/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.