One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

Abstract

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.

Fig. 1

Outline of the steps involved in the construction of an AP-1 polycistronic expression plasmid and the procedures for purification of recombinant human AP-1 complexes. The transfer vector pET3aTr contains the T7 promoter (p), terminator (t), ribosome-binding site (RBS) and paired restriction enzyme-cutting sites flanking the coding sequence for FLAG-tagged c-Jun (F:c-Jun) or hexahistidine-tagged c-Fos (6His:c-Fos). The NcoI and NdeI sites in pET3aTrF:cJun and pET3aTr-6His:cFos, destroyed following Klenow filled-in reactions, are indicated by parenthesis. In the purification process, guanidine hydrochloride (Gu-HCl) was used to solubilize bacterial inclusion bodies. For details, see Materials and methods.

Fig. 2

Expression and purification of recombinant human AP-1 complexes monitored by Coomassie blue staining and Western blotting. (A) Tracking of F:c-Jun and 6His:c-Fos expression through the purification process. Protein samples were separated by 10% SDS-PAGE and visualized after Coomassie blue staining (top panel) or Western blotting (WB) with anti-FLAG or anti-hexahistidine antibodies (bottom two strips). Lanes 1 and 2 are aliquots of protein samples taken before (T₀) and after 3-hour IPTG induction (T₃). Lane 3, crude bacterial lysate taken after sonication; lane 4, supernatant taken after centrifugation of the crude lysate; lane 5, sample taken after solubilization of inclusion bodies in 6 M guanidine-HCl solution; lane 6, unbound fraction following Ni²⁺-NTA binding; lane 7, purified F:c-Jun/6His:c-Fos from the first elution. Protein size markers (in kDa) are indicated on the left. (B) Expression and purification of c-Jun-containing AP-1 complexes. The position of affinity-tagged c-Jun is indicated by asterisk. (C) Coomassie blue-stained gel of purified AP-1 complexes. Purified protein complexes were separated and visualized as described in (A).

Fig. 3

Coexpression enhances the stability of full-length human JunB expressed in E. coli. Bacterial lysate and sonication supernatant taken from individually expressed F:JunB or coexpressed F:JunB/6His:c-Fos were separated by 10% SDS-PAGE and analyzed by Western blotting with anti-FLAG M2 monoclonal antibody. Lanes 1 and 2, F:JunB expressed from a monocistronic plasmid, pF:JunB-11d, which carries the coding sequence of FLAG-tagged human JunB. Lanes 3 and 4, full-length F:JunB stabilized by coexpressed 6His:c-Fos.

Fig. 4

Purified AP-1 complexes possess sequence-specific DNA-binding activity. (A) Electrophoretic mobility shift assays (EMSA) performed with F:c-Jun/6His:c-Fos. EMSA was conducted as described in Materials and methods using a radiolabeled AP-1 site-containing DNA fragment (117 bp) derived from the HPV-11 upstream regulatory region (URR), in the absence (−) or presence (+) of 10 ng of F:c-Jun/6His:c-Fos with or without an increasing amount of wild-type (WT) or mutant (Mut) cold competitors (10- and 100-fold in excess) or anti-hexahistidine or anti-c-Jun antibodies. (B) DNase I footprinting performed with F:c-Jun/6His:c-Fos and F:c-Jun/6His:FosB. DNase I footprinting was carried out with different amounts (0, 5, 20 and 80 ng) of F:c-Jun/6His:c-Fos or F:c-Jun/6His:FosB as described in Materials and methods using a labeled DNA probe containing a well-characterized AP-1 site (indicated by a box) found in the HPV-11 URR. A hypersensitive site (*) and the protected region are depicted on the right.

Fig. 5

Purified AP-1 complexes exhibit transactivation activity on reconstituted HPV chromatin. In vitro-reconstituted HPV-11 chromatin was incubated with individually expressed and reconstituted c-Jun/6His:c-Fos (lane 2), or coexpressed F:c-Jun/6His:c-Fos (lane 3) or F:c-Jun/6His:FosB (lane 4) and p300 and acetyl-CoA at 30°C for 20 minutes. HeLa nuclear extract (NE) and the pMLΔ53 internal DNA control were then added for preinitiation complex (PIC) assembly at 30°C for 20 minutes. Transcription was initiated by the addition of ribonucleoside triphosphates (NTPs) and continued for an hour. Transcripts synthesized from HPV-11 chromatin and pMLΔ53 DNA were analyzed as described in Materials and methods and visualized following autoradiography.