Abstract
FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 × His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.
Highlights
With a rapidly increasing incidence and a low cure rate, lung cancer has been the most common malignancy and leading cause of cancer deaths in the world [1]
In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30
The human non-small-cell lung cancer (NSCLC) cell line A549 and normal human lung fibroblast cell line MRC-5 were obtained from ATCC and were maintained in RPMI 1640 or DMEM supplemented with 10% FCS respectively. pQE-30 plasmid was purchased from Qiagen (Germany)
Summary
With a rapidly increasing incidence and a low cure rate, lung cancer has been the most common malignancy and leading cause of cancer deaths in the world [1]. Genetic alterations and allelic loss of 3p21.3 are among the most frequent and earliest cancer abnormalities detected in the pathogenesis of lung cancers. This phenomenon occurs in almost 100% of small cell lung cancers (SCLCs) and more than 80% of non-small-cell lung cancers (NSCLCs) [3]. FUS1 functions as “gatekeepers” of human cancer and plays a very important role in lung cancer development [4,5]. A majority of human lung cancers have been found loss of expression, haploinsufficiency or deficiency of posttranslational of FUS1 [2,6,7]. The expression and the preparation of recombinant protein FUS1 and its polyclonal antibody should provide effective experimental tools for further identifying its mechanism and biological functions against cancers
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