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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
Author(s) -
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
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01437-3
Subject(s) - proinsulin , peptide , fusion protein , biochemistry , cyanogen bromide , chemistry , trypsin , enzyme , protein folding , carboxypeptidase , endopeptidase , c peptide , peptide sequence , insulin , recombinant dna , biology , gene , endocrinology
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.