Construction, expression, purification and functional analysis of recombinant NF kappa B p50/p65 heterodimer

NF kappa B plays an important role in mediating the gene expression of nume rous cellular processes such as growth, development, the inflammatory respo nse and virus proliferation. The p50/p65 heterodimer is the most abundant f orm of the NF kappa B dimers and plays a more elaborate role in gene regula tion. 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 expressi on vector which contains two ribosomal binding sites to facilitate the coex pression of the polypeptides in the p50/p65 NF kappa B heterodimer. The sec ond method uses a mixed protein refolding strategy. Both methods yield crys tallizable protein. Electrophoretic mobility shift assays confirm that the DNA binding affinity is independent of the method used to purify; the prote in. These methods will facilitate the numerous studies on various NF kappa B/Rel family members.