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A Novel Pro-apoptosis Protein PNAS-4 from Xenopus laevis: Cloning, Expression, Purification, and Polyclonal Antibody Production

Fei Yan1,2#, Meilin Qian1#, Fan Yang2, Feng Cai1, Zhu Yuan1,2, Songtao Lai2, Xinyu Zhao1,2, Lantu Gou2, Zhongguo Hu1,2, and Hongxin Deng2*

1College of Life Science, Sichuan University, Chengdu, Sichuan 610041, P. R. China

2State Key Laboratory of the Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Guo Xue Xiang, No. 37, Chengdu, Sichuan 610041, P. R. China; fax: (8628) 8516-4060; E-mail: denghongx@yahoo.com.cn

* To whom correspondence should be addressed.

# Fei Yan and Meilin Qian contributed equally to this work.

Received December 20, 2006; Revision received February 13, 2007
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.
KEY WORDS: xPNAS-4, apoptosis, Escherichia coli, protein purification, antibody production

DOI: 10.1134/S0006297907060107