High-yield expression in E. coli and refolding of the bZIP domain of activating transcription factor 5.

Protein Expression and Purification
Natalie A CiaccioJennifer S Laurence

Abstract

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 electroph...Continue Reading

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Citations

May 14, 2009·Molecular Pharmaceutics·Natalie A Ciaccio, Jennifer S Laurence
May 9, 2014·Bioconjugate Chemistry·Brittney J MillsJennifer S Laurence
Mar 29, 2014·The Journal of Biological Chemistry·D Fernando EstradaEmily E Scott
Jun 17, 2020·Current Research in Structural Biology·Zhou YinGabby Rudenko

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