One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.
The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.
Wang
Wei-Ming
WM
Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Lee
A-Young
AY
Chiang
Cheng-Ming
CM
D003001:Cloning, Molecular / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D018808:Transcription Factor AP-1