High-Level Expression, Purification, and Characterization of the Recombinant Grass Carp Pituitary Adenylate Cyclase-Activating Polypeptide
2008
WANG, Zhangzhi | Yang, Mu | WANG, Xinyan | Zhou, Hong
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 Ni²⁺-chelating affinity chromatography, 102 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-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.
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