Protein Engineering, Vol. 12, No. 5, 423-428,
May 1999
© 1999 Oxford University Press
Construction, expression, purification and functional analysis of recombinant NF
B p50/p65 heterodimer
Department of Biology and 1 Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92037, USA
NF
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 NFB dimers and plays a more elaborate role in gene regulation. Biochemical research on p50/p65 NFB 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 NFB 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 NFB 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 NFB/Rel family members.
Keywords: NFB/protein analysis/protein crystallography/protein purification/recombinant expression
2 To whom correspondence should be addressed
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