High-level expression, purification, and characterization of the recombinant grass carp pituitary adenylate cyclase-activating polypeptide.
Pituitary adenylate cyclase-activating polypeptide-38 (PACAP(38)) is a potent secretagog for growth hormone and gonadotropin in fish species. To obtain recombinant grass carp PACAP(38), 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.
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu.
Zhangzhi
Z
Wang
Mu
M
Yang
Xinyan
X
Wang
Hong
H
Zhou
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011994:Recombinant Proteins