A novel recombinant, VPAC2-selective agonist enhancing insulin release and glucose disposal
Abstract
Aim: To obtain the recombinant, VPAC2 -selective ( VPAC2: type 3 receptor of pituitary adenylate cyclase activating polypeptide which shared by vasoactive intestinal peptide) agonist with effects on glucose disposal by intein-mediated, single column purification.
Methods: A gene encoding 32-amino acid peptide named rMBAY was designed and synthesized and cloned into Escherichia coli expression vector, pKYB (NEB, USA). The recombinant vector was transferred into E coli ER2566 strain and the target protein was overexpressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the fusion protein was purified
by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by β-mercaptoethanol and the target peptide, rMBAY, was released from the chitin-bound intein tag.
Results: Approximately 53 mg rMBAY with the purity over 95% was obtained by single column purification from 1 L induced culture fermented in 5 L fermenter. The results of the competitive binding assay and cAMP accumulation assay indicated that the recombinant rMBAY had special binding selectivity and potency for VPAC2. The recombinant peptide, rMBAY, enhanced insulin release and decreased the plasma glucose level after intraperitoneal injection (50 ng/kg) with a high concentration of glucose (1.8 mmol/kg) in the NIH mice.
Conclusion: An efficient production procedure of a recombinant VPAC2-selective agonist with corresponding effects on glucose disposal was established.
Keywords:
Methods: A gene encoding 32-amino acid peptide named rMBAY was designed and synthesized and cloned into Escherichia coli expression vector, pKYB (NEB, USA). The recombinant vector was transferred into E coli ER2566 strain and the target protein was overexpressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the fusion protein was purified
by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by β-mercaptoethanol and the target peptide, rMBAY, was released from the chitin-bound intein tag.
Results: Approximately 53 mg rMBAY with the purity over 95% was obtained by single column purification from 1 L induced culture fermented in 5 L fermenter. The results of the competitive binding assay and cAMP accumulation assay indicated that the recombinant rMBAY had special binding selectivity and potency for VPAC2. The recombinant peptide, rMBAY, enhanced insulin release and decreased the plasma glucose level after intraperitoneal injection (50 ng/kg) with a high concentration of glucose (1.8 mmol/kg) in the NIH mice.
Conclusion: An efficient production procedure of a recombinant VPAC2-selective agonist with corresponding effects on glucose disposal was established.