Fe. Chen et al., Construction, expression, purification and functional analysis of recombinant NF kappa B p50/p65 heterodimer, PROTEIN ENG, 12(5), 1999, pp. 423-428
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.