Purification and characterization of the intrinsically disordered Arabidopsis thaliana protein SOG1

Abstract

SOG1, a transcription factor consisting of a folded NAC (NAM-ATAF-CUC2) domain and an intrinsically disordered C-terminal domain (CTD), co-ordinates the DNA damage response in plants. Here we compare different methods to express and purify recombinant full length Arabidopsis thaliana SOG1. Expression in Sf9 insect cells results in a protein that contains a phosphorylated site that is possibly located on the T423 site in the CTD. This site is reported to be phosphorylated in planta upon aluminium toxicity stress and may affect the transcriptional activity of SOG1 in an yet undetermined way. Expression of SOG1 in E. coli BL21 (DE3) leads to the formation of inclusion bodies, a problem that is resolved by using a cleavable SUMO solubility tag. The resulting protein is not phosphorylated and represents the transcriptional inactive state of SOG1. Both protein preparations show similar CD spectra and melting temperatures. SEC-MALS determined that the proteins, like other NAC transcription factors, form a dimer in solution. Both proteins are also highly non-globular as determined by analytical SEC and are likely stretched out due to their disordered CTD. In electromobility shift assays, both insect and E. coli purified SOG1 proteins bind to a DNA fragment from the promoter region of SMR5, a well established target gene of SOG1, showing the functionality of both purified proteins.

Keywords: Arabidopsis thaliana; DNA damage response; Intrinsic disorder; NAC transcription factor; SOG1; Solubility; protein purification.