Recombination and functional studies of a dual-action peptide for diabetes

Abstract

Objective: To study a recombined chimeric peptide consisting of lysozyme N-terminal sequence and exendin-4 (shortly LYZ(N)–EX4) as a dual-action peptide for diabetes.Methods: LYZ(N)–EX4 was recombined into plasmid pET-32a(+) and expressed in Escherichia coli. The fusion protein was separated by affinity chromatography and hydrolyzed by enterokinase to prepare LYZ(N)–EX4. The chimeric peptide was digested by thrombin and the digests were analyzed by HPLC. The secondary peptides were identified by mass spectrometry. Biological activities of the thrombin digests were determined in vitro, using NIT-1 cells for insulin promoting action and using human white blood cells (WBC) for anti-AGEs action.Results: The fusion protein was highly expressed in E. coli and LYZ(N)–EX4 was obtained via hydrolysis of the fusion protein. The thrombin digests of LYZ(N)–EX4 were separated by HPLC into two peaks, which were identified as LYZ(N) and EX4 by mass spectrametry. Functional studies found that the digests were able to antagonize the effects of AGEs on expression of RAGE mRNA in WBC, promote cell activity, stimulate PDX-1 mRNA expression and increase insulin secretion by NIT-1 cells, suggesting the actions of LYZ(N) and EX4 on the cells.Conclusions: LYZ(N)–EX4 was sensitive to thrombin digestion, and the secondary peptides LYZ(N) and EX4 could function as anti-AGEs and insulin-promoting peptides, respectively.