Expression and purification of recombinant human c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro.
c-Fos and c-Jun are members of the AP-1 family of transcriptional activators that regulate the expression of genes during cell proliferation. To facilitate in vitro studies of mechanisms of transcriptional activation by c-Jun and c-Fos we developed a method for obtaining recombinant c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Full-length human c-Fos and c-Jun were expressed in Escherichia coli. The expression of c-Fos was dependent on a helper plasmid that encodes rare (Arg)tRNAs. Both over-expressed c-Fos and c-Jun were recovered from inclusion bodies. A c-Fos/c-Jun complex was generated by co-renaturation and purified via a His-tag on the full-length human c-Fos. The resulting c-Fos/c-Jun bound DNA with high affinity and specificity, and activated transcription in a reconstituted human RNA polymerase II transcription system. The availability of active recombinant human c-Fos/c-Jun will allow future biochemical studies of these important transcriptional activators.
Ferguson
H A
HA
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Campus Box 215, Boulder, CO 80309-0215, USA.
Goodrich
J A
JA
D018983:DNA Footprinting; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000302:isolation & purification / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1