OVEREXPRESSION, PURIFICATION, AND BIOPHYSICAL CHARACTERIZATION OF THEHETERODIMERIZATION DOMAIN OF THE CORE-BINDING FACTOR-BETA SUBUNIT

Core-binding factors (CBF) are heteromeric transcription factors essen tial for several developmental processes, including hematopoiesis. CBF s contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF be ta subunit that increases the affinity of CBF alpha for DNA. We have d eveloped a procedure for overexpressing and purifying full-length CBF beta as web as a truncated form containing the N-terminal 141 amino ac ids using a novel glutaredoxin fusion expression system. Substantial q uantities of the CBF beta proteins can be produced in this manner allo wing for their biophysical characterization. me show that the full-len gth and truncated forms of CBF beta bind to a CBF alpha.DNA complex wi th very similar affinities, Sedimentation equilibrium measurements sho w these proteins to be monomeric. Circular dichroism spectroscopy demo nstrates that CBF beta is a mixed alpha/beta protein and NMR spectrosc opy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.