title: Expression, identification and biological effects of the novel recombination protein, PACAP38-NtA, with high bioactivity.
abstract: Pituitary adenylate cyclase‑activating polypeptide (PACAP) is a type of neuropeptide with multiple biological functions. However, it has a short half‑life period in the body, ~3 to 5 min, restricting its further development as a drug that can promote the recovery of nerve injury. In vitro and in vivo experiments have shown that PACAP can repair the epithelial cell on the surface of the injured cornea, as PACAP can act on the trigeminal nerve cell to secrete other active neurotransmitters, which can promote corneal epithelial cell proliferation and differentiation. In the present study, PACAP is connected to the N‑terminal agrin domain (NtA) with a genetic engineering method, which allows the function of repairing the injured nerve. Notably, the recombinant polypeptide can interact with laminin, improving the biological effect of PACAP in repairing the injured nerve. In the study, the recombinant protein was constructed by combining PACAP38 and NtA by genetic engineering, and it is expressed in the pronucleus escherichia coli. The recombinant protein, PACAP38‑NtA, is obtained with a two‑step purification method, including anion‑exchange chromatography and Ni‑affinity chromatography, with the purity reaching >90%. The in vitro experiment has shown that this recombinant protein not only has the neurotrophy and neural restoration function of PACAP, but also has the function of an anchoring protein as laminin interacts with NtA. According to the in vitro anti‑apoptosis, PC12 axon growth and ELISA experiments, this protein has the biological activity of a recombinant protein. PACAP38‑NtA also has an anchoring function as NtA and laminin interact with good biological activity.
authors.affiliation: Biomedicine Institute, College of Life Science and Technology, Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou, Guangdong 510632, P.R. China.
authors.forename: Lusheng
authors.initials: L
authors.lastname: Wu
authors.affiliation: Biomedicine Institute, College of Life Science and Technology, Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou, Guangdong 510632, P.R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Biomedicine Institute, College of Life Science and Technology, Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou, Guangdong 510632, P.R. China.
authors.forename: Xiaojia
authors.initials: X
authors.lastname: Chen
authors.affiliation: Biomedicine Institute, College of Life Science and Technology, Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou, Guangdong 510632, P.R. China.
authors.forename: An
authors.initials: A
authors.lastname: Hong
mesh_terms: D018171:Agrin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000378:metabolism*; D006801:Humans; D007797:Laminin / Q000378:metabolism; D016716:PC12 Cells; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: C495401:ADCYAP1 protein, human; D018171:Agrin; D007797:Laminin; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011993:Recombinant Fusion Proteins