TY - JOUR
T1 - One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system
AU - Wang, Wei Ming
AU - Lee, A. Young
AU - Chiang, Cheng Ming
N1 - Funding Information:
We thank Paul Dobner for providing pCMV-Fra1 and pCMV-Fra2 plasmids, James Goodrich for pET-Jun and pET-6His-c-Fos, Curt Pfarr for pcDNA3.1-hJunD, Song Tan for pET3aTr and pST39, and Yu-Chung Yang for pMT3-HA:JunB. We are also grateful to Shwu-Yuan Wu for many helpful discussions in the development of the purification protocol. This work is supported in part by Grants CA103867 and CA124760 from the National Institutes of Health and is Report CSCN #033 from University of Texas Southwestern Medical Center Simmons Comprehensive Cancer Center.
PY - 2008/5
Y1 - 2008/5
N2 - 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 Jun × 4 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.
AB - 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 Jun × 4 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.
KW - AP-1
KW - Affinity purification
KW - FLAG tag
KW - Hexahistidine tag
KW - Inclusion bodies
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U2 - 10.1016/j.pep.2008.01.016
DO - 10.1016/j.pep.2008.01.016
M3 - Article
C2 - 18329890
AN - SCOPUS:40949111823
SN - 1046-5928
VL - 59
SP - 144
EP - 152
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -