Biochemical and biophysical insights into the function of the bHLH transcription factor TWIST2 (946.8)

Abstract

TWIST2 is a transcription factor of the basic Helix Loop Helix family, which binds DNA as homo or heterodimer to consensus sequences called E ‐ boxes. Mutations in TWIST2 cause a rare recessive disorder called Setleis Syndrome. To this date, no structural or functional assays have been published using recombinant TWIST2. First, we performed a bioinformatics analysis ranging from multiple sequence alignments to key structural features. These led us to perform a 3D structural prediction of TWIST2 using the Phyre2 server. In order to produce recombinant His tagged TWIST2, the TWIST2 cDNA was cloned into the pQET7 vector and expressed in BL21 pLysS cells. The protein was purified under native conditions by a two‐step purification via Ni‐NTA Affinity followed by Ion Exchange Chromatography. After purification, a band of the expected size of TWIST2 was observed in SDS‐PAGE. Western Blot analysis and Mass Spectrometry validated this finding. TWIST2 functionality was demonstrated by Electrophoretic Mobility Shift Assay (EMSA) with a known E‐box probe. Obtaining recombinant TWIST2 protein will allow future structural and functional assays. These assays will expand the knowledge of this transcription factor and its interactions with DNA to regulate its target genes. Grant Funding Source: Supported by NIH grants (2G12‐RR003051), (8G12‐MD007600) and (R25GM061838)