FEBS Letters | |
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 | |
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
【 预 览 】
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