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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.
Highlights
The core-binding factor  subunit (CBF)1 is the non-DNA binding subunit of the heterodimeric transcription factor complex called core binding factor (CBF) (1, 2)
The CBF subunit is essential for the in vivo function of at least one of the CBF␣ subunits that were encoded by the CBFA2 gene
Based on the high level of overexpression obtained for E. coli glutaredoxin (25), we reasoned that a fusion vector with CBF fused to the C-terminal end of glutaredoxin should give very high levels of expression
Summary
The core-binding factor  subunit (CBF)1 is the non-DNA binding subunit of the heterodimeric transcription factor complex called core binding factor (CBF) (1, 2). We have developed a procedure for overexpressing and purifying full-length CBF as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system.
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