Functional characterization of a purified, recombinant NF-kappaB/IkappaB complex.
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.
Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California, 92037, USA.
C J
CJ
Larson
Y W
YW
Li
F
F
Mercurio
R
R
Müller
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002457:Cell Extracts; D002460:Cell Line; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D009036:Moths / Q000166:cytology / Q000235:genetics / Q000821:virology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D051996:Transcription Factor RelA
D002457:Cell Extracts; D015342:DNA Probes; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human