Construction, expression, purification and functional analysis of recombinant NFκB p50/p65 heterodimer

NF kappa B plays an important role in mediating the gene expression of numerous cellular processes such as growth, development, the inflammatory response and virus proliferation. The p50/p65 heterodimer is the most abundant form of the NF kappa B dimers and plays a more elaborate role in gene regulation. Biochemical research on p50/p65 NF kappa B has not benefited however from the availability of easily purified recombinant protein, We report two methods for the large scale expression and purification of recombinant NF kappa B p50/p65 heterodimer, The first utilizes a bacterial double expression vector which contains two ribosomal binding sites to facilitate the coexpression of the polypeptides in the p50/p65 NF kappa B heterodimer. The second method uses a mixed protein refolding strategy. Both methods yield crystallizable protein. Electrophoretic mobility shift assays confirm that the DNA binding affinity is independent of the method used to purify; the protein. These methods will facilitate the numerous studies on various NF kappa B/Rel family members.