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 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.