High-yield expression in E. coli and refolding of the bZIP domain of activating transcription factor 5.
Activating transcription factor 5 (ATF5) recently has been demonstrated to play a critical role in promoting the survival of human glioblastoma cells. Interference with the function of ATF5 in an in vivo rat model caused glioma cell death in primary tumors but did not affect the status of normal cells surrounding the tumor, suggesting ATF5 may prove an ideal target for anti-cancer therapy. In order to examine ATF5 as a pharmaceutical target, the protein must be produced and purified to sufficient quantity to begin analyses. Here, a procedure for expressing and refolding the bZIP domain of ATF5 in sufficient yield and final concentration to permit assay development and structural characterization of this target using solution NMR is reported. Two-dimensional NMR and circular dichroism analyses indicate the protein exists in the partially alpha-helical, monomeric x-form conformation with only a small fraction of ATF5 participating in formation of higher-order structure, presumably coiled-coil homodimerization. Despite the persistence of monomers in solution even at high concentration, an electrophoretic mobility shift assay showed that ATF5 is able to bind to the cAMP response element (CRE) DNA motif. Polyacrylamide gel electrophoresis and mass spectrometry were used to confirm that ATF5 can participate in homodimer formation and that this dimerization is mediated by disulfide bond formation.
Ciaccio
Natalie A
NA
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047, USA.
Moreno
Matthew L
ML
Bauer
Rachel L
RL
Laurence
Jennifer S
JS
D051696:Activating Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D016350:Leucine Zippers; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D020218:Response Elements / Q000235:genetics
D051696:Activating Transcription Factors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase