Intein-mediated rapid purification and characterization of a novel recombinant agonist for VPAC2.
In order to obtain the recombinant VPAC2 agonist efficiently by intein-mediated single column purification, a gene encoding 32-amino acids peptide was designed, synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-ROM was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the rMROM-intein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the rMROM with the homogeneity over 95% was released from the chitin-bound intein tag. The recombinant linear rMROM competitively displaced [125I] PACAP38 on VPAC2 with a half-maximal inhibitory concentration (IC50) of 60 +/- 5 nM, whereas the IC50 of rMROM at human VPAC1 was observed up to 10 microM and no binding was detected at PAC1. rMROM stimulated the cAMP accumulation in Chinese hamster ovary (CHO) cells expressing the human VPAC2 with a half-maximal stimulatory concentration (EC50) of 0.6 nM, which was 500-fold less potent at VPAC1and had no activity on PAC1. An efficient production procedure of a novel recombinant VPAC2-selective agonist was established.
Yu
Rong-jie
RJ
Bio-engineering Institute of Jinan University, Guangzhou 510632, China.
Xie
Qiu-ling
QL
Dai
Yun
Y
Gao
Yuan
Y
Zhou
Tian-hong
TH
Hong
An
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D047668:Inteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000378:metabolism; D051239:Receptors, Vasoactive Intestinal Peptide, Type II / Q000819:agonists*; D013997:Time Factors
C495503:ADCYAP1R1 protein, human; D010455:Peptides; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D051239:Receptors, Vasoactive Intestinal Peptide, Type II