High-Level Expression,Purification,and Characterization of the Recombinant Grass Carp Pituitary Adenylate Cyclase-Activating Polypeptide

摘要

Pituitary adenylate cyclase-activating polypeptide-38(PACAP38)is a potent secretagog for growth hormone and gonadotropin in fish species.To obtain recombinant grass carp PACAP38,its open reading frame was subcloned in pET32a(+)vector to express thioredoxin(Trx)-PACAP fusion protein in Escherichia coli BL21(DE3).The resulting expression level of the thioredoxin-PACAP reached 36% of the total proteins,and more than 85% of fusion protein existed as soluble form.Using Ni~(2+)-chelating affinity chromatography,102 mg of Trx-PACAP_(38)with a purity of 97% was obtained from 342 mg of crude proteins from a 1-liter culture of Escherichia coli.The purified Trx-PACAP specifically inhibited T98G human glioblastoma cell proliferation,but the fusion partner had no effect in this regard.Moreover,this inhibition was totally abolished by PACAP-specific antibody.