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

FEBS Letters

Hardy, Eugenio¹  Besada, Vladimir¹  Silva, Alejandro¹  Herrera, Luis¹  Gonzalez, Marta¹  Fernandez, Cesar¹  Castellanos-Serra, Lila R.¹  Santos, Alicia¹  Ubieta, Raimundo¹  Falcon, Viviana¹  Perez, Gudelia¹  Vispo, Nelson S.¹ 

[1] Center for Genetic Engineering and Biotechnology, P.O. Box 6162, La Habana, Cuba

关键词: Recombinant insulin;    Proinsulin folding;    Enzymatic processing;   

DOI  :  10.1016/0014-5793(95)01437-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown