title: A novel pro-apoptosis protein PNAS-4 from Xenopus laevis: cloning, expression, purification, and polyclonal antibody production.
abstract: 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.
authors.affiliation: College of Life Science, Sichuan University, Chengdu, Sichuan 610041, P. R. China.
authors.forename: Fei
authors.initials: F
authors.lastname: Yan
authors.forename: Meilin
authors.initials: M
authors.lastname: Qian
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Feng
authors.initials: F
authors.lastname: Cai
authors.forename: Zhu
authors.initials: Z
authors.lastname: Yuan
authors.forename: Songtao
authors.initials: S
authors.lastname: Lai
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhao
authors.forename: Lantu
authors.initials: L
authors.lastname: Gou
authors.forename: Zhongguo
authors.initials: Z
authors.lastname: Hu
authors.forename: Hongxin
authors.initials: H
authors.lastname: Deng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D029867:Xenopus Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics*
chemical_list: D000906:Antibodies; D051017:Apoptosis Regulatory Proteins; C524210:DESI2 protein, Xenopus; D011994:Recombinant Proteins; D029867:Xenopus Proteins