Prokaryotic expression, purification and characterization of a novel pro-apoptosis protein hPNAS-4.
Recently, hPNAS-4 [human PNAS-4; also known as C1orf121 (Genbank accession number NP-057160)] was reported to be a novel pro-apoptotic protein in mammalian cells. However, at present there is little information about hPNAS-4 and its molecular mechanisms. In our present studies, the hPNAS-4 gene was first cloned into the pGEX-6p-1 vector with a GST (glutathione transferase) tag to express in soluble form in Escherichia coli induced with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) at 25 degrees C, 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-ESI (electrospray ionization)-MS analysis and used to raise polyclonal antibodies that could specifically recognize the hPNAS-4 protein. In addition, hPNAS-4 is localized to the cytoplasm and the perinuclear compartment. Furthermore, the overexpression of hPNAS-4 in human A549 lung cancer 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 should aid the discovery of new drug targets for the development of effective therapeutic approaches to cancer in the future.
Deng
Hongxin
H
State Key Laboratory of Biotherapy, West China Hospital and College of Life Science, Sichuan University, Chengdu 610041, People's Republic of China.
Jiang
Qingyuan
Q
Liang
Shufang
S
Yan
Fei
F
Hou
Shengyan
S
Qian
Zhiyong
Z
Li
Jiong
J
Wen
Yanjun
Y
Yang
Jinliang
J
Wei
Yuquan
Y
D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D019759:Carbon-Nitrogen Lyases; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D000906:Antibodies; D051017:Apoptosis Regulatory Proteins; D011993:Recombinant Fusion Proteins; D019759:Carbon-Nitrogen Lyases; C000604991:DESI2 protein, human