A novel recombinant, VPAC2-selective agonist enhancing insulin release and glucose disposal.
OBJECTIVE
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 beta-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.


CONCLUSIONS
An efficient production procedure of a recombinant VPAC2-selective agonist with corresponding effects on glucose disposal was established.
Yu
Rong-jie
RJ
Bio-engineering Institute of Ji-nan University, Guangzhou 510632, China.
Tam
Ngai-lik
NL
Gao
Yuan
Y
Zeng
Zhi-hong
ZH
Zhou
Tian-hong
TH
Hong
An
A
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis / Q000494:pharmacology*; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide / Q000187:drug effects; D051239:Receptors, Vasoactive Intestinal Peptide, Type II / Q000819:agonists*; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014660:Vasoactive Intestinal Peptide / Q000096:biosynthesis / Q000494:pharmacology
D007004:Hypoglycemic Agents; D007328:Insulin; D010446:Peptide Fragments; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D011994:Recombinant Proteins; C409214:vasoactive intestinal peptide (1-7)-GRF (8-27), Lys(15)-Arg(16)-Leu(27)-; D014660:Vasoactive Intestinal Peptide; D005947:Glucose