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Biotechnology and Applied Biochemistry (2009) Immediate Publication, doi:10.1042/BA20090111
Prokaryotic expression, purification and characterization of a novel pro-apoptosis protein hPNAS-4
Hongxin Deng, Qingyuan Jiang, Shufang Liang, Fei Yan, Shengyan Hou, Zhiyong Qian, Jiong Li, Yanjun Wen, Jinliang Yang and Yuquan Wei
Sichuan University, Chengdu, China. zizi2006@yahoo.cn
Recently human PNAS-4 (hPNAS-4) is reported to be a novel pro-apoptotic protein in mammalian cells. However, there is little information about hPNAS-4 characterization and its molecular mechanisms so far. In our present studies, the hPNAS-4 gene was first cloned into the pGEX-6p-1 vector with GST tag to solubly express in E.coli induced with 0.5 mM IPTG at 25℃,and the recombinant hPNAS-4 was purified to near homogeneity with 96% purity by affinity chromatography and anion-exchange chromatography. The purified hPNAS-4 protein was further identified by liquid chromatography-electrospray ionization-mass spectrometry analysis and used to raise polyclonal antibodies which could specifically recognize the hPNAS-4 protein. In addition, hPNAS-4 is localized in cytoplasm and perinuclear. Furthermore the overexpression of hPNAS-4 in human A549 cells resulted in decrease of cell viability via apoptosis. This study represented an important step to investigate the characterization for the new pro-apoptosis protein hPNAS-4, which aids to discover new drug targets for development of effective therapeutic approaches to cancer in future.
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