Expression, purification and activity of pituitary adenylate cyclase-activting polypeptide 38 and its derivative

Objective: To improve the in vivo stability and prolong the half-life of bioactivity of pituitary adenylate cyclase-activting polypeptide 38 (PACAP38). Methods: The amino acid sequence of PACAP38 was modified to obtain a derivative named cPACAP38. The gene fragments encoding PACAP38 and cPACAP38 were designed and synthesized according to the codon bias of E. coil and cloned into vector pET-32a(+). The constructed recombinant plasmids were transformed to E. coli BL21(DE3) for expression under induction of IPTG. The expressed fusion proteins were purified by nickel ion affinity chromatography and digested with enterokinase, by which two target polypeptides were obtained and tested for anorexigenic activities in mice. Mice were treated with the polypeptides for 10 d, then determined for intraperitoneal glucose-resistance as well as serum triglyceride, HDL, LDL and cholesterol contents. Results: Fusion proteins Trx-PACAP38 and Trx-cPACAP38 were expressed in soluble forms, which contained 22% and 25% of total somatic proteins and reached purities of 90% and 98% after purification respectively. Both PACAP38 and cPACAP38 showed anorexigenic activities in mice. However, the anorexigenic activity of cPACAP38 was higher than that of PACAP38. No significant changes were observed in intraperitoneal glucose-resistance as well as serum triglyceride, HDL, LDL and cholesterol contents of mice 10 d after treatment with the fusion proteins. Conclusion: The derivative of PACAP38 with higher in vivo activity was successfully expressed, which laid a foundation of basic study and application of PACAP38.