Cloning, expression and purification of NF-kappaB p50 transcription factor from human using E.coli host cells
Main Article Content
Abstract
NF-κB is a transcription factor, which plays a central role in regulating the immune system. It controls up to 500 genes involved in immune responses and also coordinates both innate and adaptive immunity. NF-κB participates in cellular responses to diverse stimuli such as stress, cytokines, free radicals, ultraviolet radiation, and bacterial or viral antigens. Incorrect regulation of NF-κB has been linked to a number of human diseases such as cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development. Among human NF- κB transcription factors, NF- κB p50 is one of the most important transcription factor which is found in all type of cells. In this study, we cloned the gene encoding for human NF-κB1 p50 and inserted the gene in to pET-M expression vector (modified form of pET-32a expression vector). Vector pET-M containing NF-κB1 p50 gene was transformed and expressed in BL21(DE3) E.coli cells. Recombinant NF-κB1 p50 protein was purified using nikel bead column with the purity close to 99%. As a result the obtained NF-κB1 p50 with high purity can be used for testing and screening of potential drug for NF-κB related diseases.
References
[2] T.D. Gilmore, Introduction to NF-kappaB: players, pathways, perspectives, Oncogene, 25 (2006) 6680-6684.
[3] S.J.a.R.D. W., Molecular cloning: A laboratory manual 3rd ed NY., (2001).
[4] B. Barre, N.D. Perkins, A cell cycle regulatory network controlling NF-kappaB subunit activity and function, The EMBO journal, 26 (2007) 4841-4855.
[5] N.D. Perkins, The importance of the p50 NF-kappaB subunit, Cell cycle, 14 (2015) 2877-2878.
[6] C.W. Muller, F.A. Rey, S.C. Harrison, Comparison of two different DNA-binding modes of the NF-kappa B p50 homodimer, Nature structural biology, 3 (1996) 224-227.
[7] X. Tong, L. Yin, R. Washington, D.W. Rosenberg, C. Giardina, The p50-p50 NF-kappaB complex as a stimulus-specific repressor of gene activation, Molecular and cellular biochemistry, 265 (2004) 171-183.
[8] V.S. Nguyen, C. Jobichen, K.W. Tan, Y.W. Tan, S.L. Chan, K. Ramesh, Y. Yuan, Y. Hong, J. Seetharaman, K.Y. Leung, J. Sivaraman, Y.K. Mok, Structure of AcrH-AopB Chaperone-Translocator Complex Reveals a Role for Membrane Hairpins in Type III Secretion System Translocon Assembly, Structure, 23 (2015) 2022-2031.
[9] D.J. Chen, Y.M. Xu, J.Y. Du, D.Y. Huang, A.T. Lau, Cadmium induces cytotoxicity in human bronchial epithelial cells through upregulation of eIF5A1 and NF-kappaB, Biochemical and biophysical research communications, 445 (2014) 95-99.
[10] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic acids research, 22 (1994) 4673-4680.
[11] S. Liptay, R.M. Schmid, N.D. Perkins, P. Meltzer, M.R. Altherr, J.D. McPherson, J.J. Wasmuth, G.J. Nabel, Related subunits of NF-kappa B map to two distinct loci associated with translocations in leukemia, NFΚB and NFΚB2, Genomics, 13 (1992) 287-292.