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.
Yan
Fei
F
College of Life Science, Sichuan University, Chengdu, Sichuan 610041, P. R. China.
Qian
Meilin
M
Yang
Fan
F
Cai
Feng
F
Yuan
Zhu
Z
Lai
Songtao
S
Zhao
Xinyu
X
Gou
Lantu
L
Hu
Zhongguo
Z
Deng
Hongxin
H
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*
D000906:Antibodies; D051017:Apoptosis Regulatory Proteins; C524210:DESI2 protein, Xenopus; D011994:Recombinant Proteins; D029867:Xenopus Proteins