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A novel pro-apoptosis protein PNAS-4 from Xenopus laevis: cloning, expression, purification, and polyclonal antibody production

Human PNAS-4 was identified as a novel pro-apoptotic protein in mammalian cells. Here we report the cloning, expression, purification, and antibody production of a PNAS-4 homolog (named xPNAS-4) from Xenopus laevis, an extensively used model organism in exploring gene functions during embryonic deve...

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Bibliographic Details
Published in:Biochemistry (Moscow) 2007-06, Vol.72 (6), p.664-671
Main Authors: Yan, Fei, Qian, Meilin, Yang, Fan, Cai, Feng, Yuan, Zhu, Lai, Songtao, Zhao, Xinyu, Gou, Lantu, Hu, Zhongguo, Deng, Hongxin
Format: Article
Language:English
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Summary:Human PNAS-4 was identified as a novel pro-apoptotic protein in mammalian cells. Here we report the cloning, expression, purification, and antibody production of a PNAS-4 homolog (named xPNAS-4) from Xenopus laevis, an extensively used model organism in exploring gene functions during embryonic development. Recombinant histidine-tagged xPNAS-4 protein was expressed in Escherichia coli as insoluble inclusion bodies. The inclusion bodies were subsequently dissolved in 8 M urea and purified to near homogeneity by Ni2+ affinity chromatography. The resulting denatured protein was refolded by stepwise dilution of urea concentration via dialysis. This procedure yielded about 4 mg refolded protein per liter of E. coli culture with a purity of 95%. The purified protein was identified by liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) and used to raise anti-xPNAS-4 polyclonal antibodies that were suitable for detecting the expression of PNAS-4 in X. laevis embryos by Western blotting. The availability of recombinant protein and specific polyclonal antibodies will provide a valuable tool in studying apoptotic mechanisms of this protein. To our knowledge, this is the first report to demonstrate the presence of PNAS-4 in X. laevis.
ISSN:0006-2979
1608-3040
0320-9725
DOI:10.1134/S0006297907060107