Recombinant Grass Carp Of Pacap <sub> 38 </ Sub> Was Highly Expressed, Separation, Purification And Biological Activity Of Research

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP₃₈),a member of the glucagon/secretin peptide family, exerts pleiotropic physiological functions in both central nervous system and peripheral organs in vertebrates. PACAP is a potent secretagogue for growth hormone and gonadotropin in fish species. Recently, the role of PACAP₃₈ as a novel growth hormone-releasing factor has received increasing attention in fish model.In present study, to obtain the economical and active grass carp PACAP₃₈ in large scare preparation, its open reading frame (ORF) was synthesized by three olignucleo -tides which were designed according to the codon preference of Escherichia coli using two-step PCR strategy. This ORF was subcloned in pThioHis A and pET32a (+) vector, and then Top10-pThioHis A-PACAP₃₈, BL21(DE3)-pThioHis A-PACAP₃₈ and BL21(DE3)-pET32a(+)-PACAP₃₈ expression systems were constructed, respectively. Results showed that BL21(DE3)-pET32a(+)-PACAP₃₈ expression system was the best one to express recombinant PACAP₃₈. In this system, the resulting expression level of the thioredoxin-PACAP₃₈ (Trx-PACAP₃₈) reached 36% of the total proteins in large scare preparation, and more than 85% of fusion protein existed as the soluble form. Using Ni2+-chelating affinity chromatography, 99 mg of Trx-PACAP₃₈ with a purity of 97% was obtained from 342 mg of crude proteins from a 1-liter culture of Escherichia coli. The purified Trx-PACAP₃₈ was biologically active to specifically inhibit T98G human glioblastoma cell proliferation while the fusion partner had no effect in this regard, and this inhibition was totally abolished by PACAP₃₈ specific antibody.This efficient method for expression and purification of recombinant grass carp PACAP₃₈ may provide a general approach to produce other small peptides. Meanwhile, the purified recombinant grass carp PACAP₃₈ will not only be useful for fish culture, but also allow further studies on the physiological role of PACAP in fish growth and reproduction.