Recombinant NFkB p65 protein was expressed from a full-length cDNA clone in E. coli and has a 14 amino acid truncation at the C-term. This clone had five point mutations compared to the p65 sequence listed under accession no. AAA36408: L159V, P180S, F309S, A439V and V462M. The protein was purified by affinity chromatography, followed by gel filtration. NFkB p65 is a subunit of the NFkB transcription factor complex that plays a significant role in the regulation of genes that control various biological processes, including inflammation, differentiation, tumorigenesis, and cell growth and survival. NFkB is comprised of homo- or heterodimers of different subunits of the structurally related Rel family of transcription factors that includes p50 (NF-kB1), p52 (NF-kB2), p65 (RelA), RelB and c-Rel. NFkB p65, RelB and c-Rel contain a transactivation domain (TD) in their C-termini, which is required for the transport of active NFkB complexes into the nucleus. In contrast, subunits p50 and p52 do not contain transactivation domains; they are unable to transactivate on their own and must form heterodimers with p65, RelB or c-Rel. The p50/p65 heterodimers and the p50 homodimers are the most common dimers found in the NFkB signaling pathway. Inactive NFkB dimers are sequestered in the cytoplasm of cells by the IkB family of inhibitory proteins. Activation of NFkB by external inducers such as lipopolysaccharide, TNF or IL-1, results in the phosphorylation and degradation of the IkB proteins. This releases NFkB dimers, which subsequently translocate to the nucleus where they activate appropriate target genes.
