Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies


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.

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Zhang, D. , Yang, H. , Zhao, X. , Zhu, W. , Feng, Z. , Wan, Y. , Zhao, Z. , Tang, M. , Huang, N. and Wei, Y. (2010) Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies. Journal of Biomedical Science and Engineering, 3, 397-404. doi: 10.4236/jbise.2010.34055.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Murray, T. and Thun, M.J. (2008) Cancer statistics. A Cancer Journal for Clinicians, 58, 71-96.
[2] Kondo, M., Ji, L., Kamibayashi, C., Tomizawa, Y., Randle, D., Sekido, Y., Yokota, J., Kashuba, V., Zabarovsky, E., Kuzmin, I., Lerman, M., Roth, J. and Minna, J.D. (2001) Overexpression of candidate tumor suppressor gene FUS1 isolated from the 3p21.3 homozygous deletion region leads to G1 arrest and growth inhibition of lung cancer cells. Oncogene, 20, 6258-6262.
[3] Lerman, M.I., Glenn, G.M., Daniel, L., Latif, F., Hosoe, S., Brauch, H., Hampsch, K., Delisio, J., Orcutt, M. and Zbar, B. (1990) A new polymorphic probe on chromosome 3p: Lambda LIB28-77 (D3S169E). Nucleic Acids Research, 18, 205.
[4] Lerman, M.I. and Minna, J.D. (2000) The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: Identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Research, 60, 6116-6133.
[5] Zabarovsky, E.R., Lerman, M.I. and Minna, J.D. (2002) Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene, 21, 6915- 6935.
[6] Uno, F., Sasaki, J., Nishizaki, M., Carboni, G., Xu, K., Atkinson, E.N., Kondo, M., Minna, J.D., Roth, J.A. and Ji, L. (2004) Myristoylation of the FUS1 protein is required for tumor suppression in human lung cancer cells. Cancer Research, 64, 2969-2976.
[7] Prudkin, L., Behrens, C., Liu, D.D., Zhou, X., Ozburn, N.C., Bekele, B.N., Minna, J.D., Moran, C., Roth, J.A., Ji, L. and Wistuba, L.L. (2008) Loss and reduction of FUS1 protein expression is a frequent phenomenon in the pathogenesis of lung cancer. Clinical Cancer Research, 14, 41-47.
[8] Ito, I., Ji, L., Tanaka, F., Saito, Y., Gopalan, B., Branch, C.D., Xu, K., Atkinson, E.N., Bekele, B.N., Stephens, L.C., Minna, J.D., Roth, J.A. and Ramesh, R. (2004) Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo. Cancer Gene Therapy, 11, 733-739.
[9] Deng, W.G., Kawashima, H., Wu, G., Jayachandran, G., Xu, K., Minna, J.D., Roth, J.A. and Ji, L. (2007) Synergistic tumor suppression by coexpression of FUS1 and p53 is associated with down-regulation of murine double minute-2 and activation of the apoptotic protease-activating factor 1-dependent apoptotic pathway in human non- small cell lung cancer cells. Cancer Research, 67, 709-717.
[10] Tsukamoto, H., Fukudome, K., Kohara, J., Nakatake, H. and Kimoto, M. (2007) Preparation of recombinant murine tumor necrosis factor-alpha in Escherichia coli: A rapid method to remove tags from fusion proteins by thrombin-cleavage and ion-exchange chromatography. Protein Expression and Purification, 56, 138-144.
[11] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Bio- chemistry, 72, 248-254.
[12] Zhao, X.Y., Li, H.X., Liang, S.F., Yuan, Z., Yan, F., Ruan, X.Z., You, J., Xiong, S.Q., Tang, M.H. and Wei, Y.Q. (2008) Soluble expression of human DRR1 (down-regulated in renal cell carcinoma 1) in Escherichia coli and preparation of its polyclonal antibodies. Biotechnology and Appied Biochemistry, 49, 17-23.
[13] Benzinger, A., Muster, N., Koch, H.B., Yates, J.R. and Hermeking, H. (2005) Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer. Molecular & Cellular Proteomics, 4, 785-795.
[14] Wilkinson, R.J., Elliott, P., Carragher, J.F. and Francis, G. (2004) Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II. Protein Expression and Purification, 35, 334-343.
[15] Ji, L., Nishizaki, M., Gao, B., Burbee, D., Kondo, M., Kamibayashi, C., Xu, K., Yen, N., Atkinson, E.N., Fang, B., Lerman, M.I., Roth, J.A. and Minna, J.D. (2002) Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo. Cancer Research, 62, 2715-2720.

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