Overexpression, purification, and biophysical characterization of the heterodimerization domain of the core-binding factor beta subunit.

The Journal of Biological Chemistry
X HuangJohn H Bushweller

Abstract

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.

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Citations

May 7, 2003·Blood Cells, Molecules & Diseases·Lina ZhangJohn H Bushweller
Jan 8, 2011·Protein Science : a Publication of the Protein Society·Panagiotis L KastritisJoël Janin
Mar 31, 2005·Proteomics·Riccardo SgarraGuidalberto Manfioletti
Aug 10, 2001·Chemistry : a European Journal·T SöhnelP Schwerdtfeger
Dec 6, 2018·Journal of Cellular Physiology·Deli HongGary S Stein
Dec 6, 2000·Seminars in Cell & Developmental Biology·N AdyaP P Liu

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