Functional characterization of a purified, recombinant NF-kappaB/IkappaB complex. |
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Authors: | C J Larson Y W Li F Mercurio R Müller |
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Institution: | Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California, 92037, USA. |
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Abstract: | The NF-kappaB signal transduction pathway involves the interaction of several NF-kappaB and IkappaB family members that are activated by a diverse range of extracellular signals and that stimulate many different cellular responses. The biochemical regulation of this cascade can be studied by establishing a cell-free system using purified proteins. As a first step toward establishing an in vitro model incorporating multiple combinations of NF-kappaB and IkappaB proteins, we produced purified human p65 (RelA) and human IkappaBalpha proteins and tested their functionality. Full-length RelA and IkappaBalpha proteins were overproduced by coinfection of TN5-JE cells with two recombinant baculoviruses. RelA and IkappaBalpha formed a stable complex that could be purified to >95% homogeneity. Protein-protein interactions, protein-DNA binding, and protein phosphorylation were similar to the native proteins. |
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