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.
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Harmar, AJ, Arimura, A, Gozes, I, Journot, L, Laburthe, M, Pisegna, JR, et al. International union of pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 1998; 50: 265–70.
Gozes, I, Fridkinb, M, Hill, JM, Brenneman, DE, Pharmaceutical VIP: prospects and problems. Curr Med Chem 1999; 6: 1019–34.
Inagaki, N, Yoshida, H, Mizuta, M, Mizuno, N, Fujii, Y, Gonoi, T, et al. Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells. Proc Natl Acad Sci USA 1994; 91: 2679–83.
Tsutsumi, M, Claus, TH, Liang, Y, Li, Y, Yang, L, Zhu, J, et al. A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal: a potential therapy for type 2 diabetes. Diabetes 2002; 51: 1453–60.
Wei, Y, Mojsov, S, Multiple human receptors for pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide are expressed in a tissue-specific manner. Ann N Y Acad Sci 1996; 805: 624–27.
Yokota, C, Kawai, K, Ohashi, S, Watanabe, Y, Yamashita, K, PACAP stimulates glucose output from the perfused rat liver. Peptides 1995; 16: 55–60.
Yung, SL, Cruz, FD, Hamren, S, Zhu, J, Tsutsumi, M, Bloom, JW, et al. Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide. J Biol Chem 2003; 278: 10273–81.
Chong, S, Mersha, FB, Comb, DG, Scott, ME, Landry, D, Vence, LM, et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 1997; 192: 277–81.
Chong, S, Shao, Y, Paulus, H, Benner, J, Perler, FB, Xu, MQ, Protein splicing involving the Saccharomyces cerevisiae VMA intein: the steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J Biol Chem 1996; 271: 22159–68.
Yu, RJ, Xie, QL, Dai, Y, Gao, Y, Zhou, TH, Hong, A, Intein-mediated rapid purification and characterization of a novel recombinant agonist for VPAC2. Peptides 2006; 27: 1357–66.
Yang, LP, Kong, XP, Yi, XR, Analyzing peptides of low weights molecular with SDS-polyacrylamide gel electrophoresis. Prog Biotechnol 1998; 18: 19–21. Chinese.
Yu, RJ, Hong, A, Dai, Y, Gao, Y, Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide. Acta Biochim Biophys Sin 2004; 36: 759–66.
O'Donnell, M, Garippa, RJ, Rinaldi, N, Selig, WM, Simko, B, Renzetti, L, et al. Ro 25–1553: a novel, long-acting vasoactive intestinal peptide agonist, part I: in vitro and in vivo bronchodilator studies. J Pharmacol Exp Ther 1994; 270: 1282–8.
O'Donnell, M, Garippa, RJ, Rinaldi, N, Selig, WM, Tocker, JE, Tannu, SA, et al. Ro 25–1553: a novel, long-acting vasoactive intestinal peptide agonist, part II: effect on in vitro and in vivo models of pulmonary anaphylaxis. J Pharmacol Exp Ther 1994; 270: 1289–94.
Gourlet, P, Vertongen, P, Vandermeers, A, Vandermeers-Piret, MC, Rathe, J, De Neef, P, et al. The long-acting vasoactive intestinal polypeptide agonist RO 25–1553 is highly selective of the VIP2 receptor subclass. Peptides 1997; 18: 403–8.
Moreno, D, Gourlet, P, De Neef, P, Cnudde, J, Waelbroeck, M, Robberecht, P, Development of selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC2 receptor. Peptides 2000; 21: 1543–49.
Juarranz, MG, Rampelbergh, JV, Gourlet, P, De Neef, P, Cnudde, J, Robberecht, P, et al. Different vasoactive intestinal polypeptide receptor domains are involved in the selective recognition of two VPAC2-selective ligands. Mol Pharmacol 1999; 56: 1280–7.
Langer, I, Gregoire, F, Nachtergael, I, De Neef, P, Vertongen, P, Robberecht, P, Hexanoylation of a VPAC2 receptor-preferring ligand markedly increased its selectivity and potency. Peptides 2004; 25: 275–8.
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This work was supported by grants from the Guangdong Provincial Science Key Foundation of China (No 021202), the Guangdong Provincial Science Foundation of China (No 06300579), the Guangdong Provincial Scientific Strategic Special Project of China (No 2004A10902002), and the National Fifteenth Strategical Plan (No 2002AA2Z3344).
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Yu, Rj., Tam, Nl., Gao, Y. et al. A novel recombinant, VPAC2-selective agonist enhancing insulin release and glucose disposal. Acta Pharmacol Sin 28, 526–533 (2007). https://doi.org/10.1111/j.1745-7254.2007.00529.x
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DOI: https://doi.org/10.1111/j.1745-7254.2007.00529.x
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