Expression and reconstitution of NF-kappaB from insect cells using a baculovirus vector.
NF-kappaB is a pleiotropic transcriptional activator originally identified by its ability to regulate immunoglogulin kappa light chain expression. Purification of this DNA-binding complex demonstrated that NF-kappaB is a heterodimer composed of two subunits, NFKB1 and RelA. Previous studies have shown that truncated versions of these proteins could be expressed and purified from bacterial cells. In the present study, we utilize a baculovirus expression vector system (BEVS) to overexpress each subunit independently to produce homodimers or together to reconstitute functional NF-kappaB. These proteins can be enriched to >70% homogeneity on a kappaB-agarose DNA- affinity column. The purified proteins are active in DNA binding as measured by electrophoretic mobility shift assays. Finally, transcriptional activation of these recombinant proteins can be measured by their ability to activate a kappaB-CAT reporter plasmid in transiently transfected/infected SF-9 cells. Thus, BEVS provides a method for production of full-length, transcriptionally active NF-kappaB proteins.
Coleman
T A
TA
Human Genome Sciences, Inc., Rockville, Maryland 20850, USA. Tim_Coleman@hgsi.com
Huddleston
K A
KA
Ruben
S M
SM
Rosen
C A
CA
Gentz
R
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019281:Dimerization; D017930:Genes, Reporter; D005822:Genetic Vectors; D016328:NF-kappa B / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000166:cytology; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D016328:NF-kappa B; D011994:Recombinant Proteins; D051996:Transcription Factor RelA