Overexpression, purification, and biophysical characterization of the heterodimerization domain of the core-binding factor beta subunit.
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.
Huang
X
X
Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, USA.
Crute
B E
BE
Sun
C
C
Tang
Y Y
YY
Kelley
J J
JJ
Lewis
A F
AF
Hartman
K L
KL
Laue
T M
TM
Speck
N A
NA
Bushweller
J H
JH
D002942:Circular Dichroism; D050657:Core Binding Factor alpha Subunits; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D019281:Dimerization; D004926:Escherichia coli; D005822:Genetic Vectors; D054477:Glutaredoxins; D010088:Oxidoreductases; D010957:Plasmids; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
D050657:Core Binding Factor alpha Subunits; D004268:DNA-Binding Proteins; D054477:Glutaredoxins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D010088:Oxidoreductases