Overexpression, Purification, and Biophysical Characterization of the Heterodimerization Domain of the Core-binding Factor β Subunit

Abstract

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBFα subunit and a non-DNA binding CBFβ subunit that increases the affinity of CBFα for DNA. We have developed a procedure for overexpressing and purifying full-length CBFβ as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBFβ proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBFβ bind to a CBFα·DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBFβ is a mixed α/β protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy. Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBFα subunit and a non-DNA binding CBFβ subunit that increases the affinity of CBFα for DNA. We have developed a procedure for overexpressing and purifying full-length CBFβ as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBFβ proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBFβ bind to a CBFα·DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBFβ is a mixed α/β protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy. The core-binding factor β subunit (CBFβ) 1The abbreviations used are: CBFβ, core-binding factor β subunit; CBFβ(141), the 143-amino acid protein containing residues Gly-Ser followed by the N-terminal 141 amino acids of murine CBFβ; CBFβ(187), the 189-amino acid protein containing residues Gly-Ser followed by amino acids encoding the 187-amino acid isoform of CBFβ; DTT, dithiothreitol; Grx, E. coli glutaredoxin-1; HSQC, heteronuclear single quantum coherence; NH, protein backbone amide NH; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption ionization. is the non-DNA binding subunit of the heterodimeric transcription factor complex called core binding factor (CBF) (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar). CBF binds to target DNA sites with the consensus sequence PyGPyGGT (3Kamachi Y. Ogawa E. Asano M. Ishida S. Murakami Y. Satake M. Ito Y. Shigesada K. J. Virol. 1990; 64: 4808-4819Crossref PubMed Google Scholar, 4Melnikova I.N. Crute B.E. Wang S. Speck N.A. J. Virol. 1993; 67: 2408-2411Crossref PubMed Google Scholar, 5Thornell A. Hallberg B. Grundstrom T. J. Virol. 1991; 65: 42-50Crossref PubMed Google Scholar, 6Meyers S. Downing J.R. Hiebert S.W. Mol. Cell. Biol. 1993; 13: 6336-6345Crossref PubMed Scopus (427) Google Scholar). CBF subunits are encoded by four genes in mammals. CBFA1, CBFA2(AML1), and CBFA3 encode DNA-binding CBFα subunits, and CBFB encodes the common CBFβ subunit (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar,7Bae S.C. Takahashi E. Zhang Y.W. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene (Amst.). 1995; 159: 245-248Crossref PubMed Scopus (152) Google Scholar, 8Bae S.C. Yamaguchi-Iwai Y. Ogawa E. Maruyama M. Inuzuka M. Kagoshima H. Shigesada K. Satake M. Ito Y. Oncogene. 1993; 8: 809-814PubMed Google Scholar, 9Levanon D. Negreanu V. Bernstein Y. Bar-Am I. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Crossref PubMed Scopus (381) Google Scholar, 10Liu P. Tarle S.A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Crossref PubMed Scopus (651) Google Scholar, 11Miyoshi H. Shimizu K. Kozu T. Maseki N Kaneko Y. Ohki M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10431-10434Crossref PubMed Scopus (800) Google Scholar, 12Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Crossref PubMed Scopus (562) Google Scholar). The CBFα subunit can bind DNA in the absence of CBFβin vitro but with 5–10-fold lower affinity than the CBFα/β complex (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar). CBFβ modulates the affinity of the CBFα subunit for DNA without establishing additional contacts on the DNA or changing the magnitude of DNA bending (2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar, 13Golling G. Li L.H. Pepling M. Stebbins M. Gergen J.P. Mol. Cell. Biol. 1996; 16: 932-942Crossref PubMed Google Scholar). The amino acid sequence of CBFβ yields few clues to its tertiary structure or the mechanism by which it modulates the affinity of the CBFα subunit for DNA. The heterodimerization domain in CBFβ has been localized to its N-terminal 135 amino acids, which corresponds to a region of significant homology between CBFβ and its twoDrosophila homologs, Brother (Bro) and Big Brother (Bgb) (13Golling G. Li L.H. Pepling M. Stebbins M. Gergen J.P. Mol. Cell. Biol. 1996; 16: 932-942Crossref PubMed Google Scholar, 14Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). A truncated CBFβ protein containing amino acids 1–141 (CBFβ(141)), which includes the region of homology to Bro and Bgb, stably associates with the CBFα subunit in vitro (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 14Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Further truncation of the C terminus to amino acid 133 results in a protein that weakly associates with CBFα, and C-terminal truncation to amino acid 117 disrupts stable heterodimerization with CBFα (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar,15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). The CBFβ subunit is essential for the in vivo function of at least one of the CBFα subunits that were encoded by theCBFA2 gene (also known as the acute myeloid leukemia 1 orAML1 gene). Homozygous disruption of either theCbfa2 or the Cbfb genes in mice results in identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis (15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 16Niki M. Okada H. Takano H. Kuno J. Tani K. Hibino H. Asano S. Ito Y. Satake M. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5697-5702Crossref PubMed Scopus (164) Google Scholar, 17Okuda T. van Deursen J. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1606) Google Scholar, 18Sasaki K. Yagi H. Bronson R.T. Tominaga K. Matsunashi T. Deguchi K. Tani Y. Kishimoto T. Komori T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12359-12363Crossref PubMed Scopus (328) Google Scholar, 19Wang Q. Stacy T. Binder M. Marı́n-Padilla M. Sharpe A.H. Speck N.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3444-3449Crossref PubMed Scopus (1029) Google Scholar). In humans, chromosomal rearrangements that disrupt the CBFA2 and CBFB genes are associated with a variety of leukemias, including de novo acute myeloid leukemias t(8;21)(q22;q22), inv(16)(p13;q22), t(16;16), and del(16)(q22), acute lymphocytic leukemias t(12;21)(p13;q22), and therapy-related leukemias and myelodysplasias t(3;21)(q26;q22) (10Liu P. Tarle S.A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Crossref PubMed Scopus (651) Google Scholar, 11Miyoshi H. 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The of CBFβ and its are similar to of and the mechanism by which CBFβ the CBFα·DNA complex is The CBFβ subunit is essential of the CBF complex and is in a significant of leukemias, it and target for biophysical and In this a novel for the CBFβ subunit in and a with which can of CBFβ We that a truncated form of CBFβ, CBFβ(141), to contain heterodimerization domain (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google binds to CBFα with affinity very similar to that of the full-length The heterodimerization domain in a structure that which it in the of the full-length We that the full-length and truncated proteins are and contain a of and The encoding the fusion protein in the DNA sequence to coli the A. 1991; PubMed Scopus Google using the and The the for at its The a followed by encoding a for the DNA encoding the (2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google using the and A for at the of the and a followed by a to the The were with the and between the and sites of The the for the of DNA and and or for protein The encoding the glutaredoxin protein to by in that encodes with a containing the of which a containing (2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar). A for this encoding a sites and were and the between the and the of The in were by are to the E. coli were with the and a single used to of containing at at a of the used to 1 of to A of and by the to and at this for were by in of and by The were at expression of full-length CBFβ, E. coli and were in the identical manner as for expression of or CBFβ(187), E. coli with the in as the were at and were to the a The for and the were by four a at The by at for The a of in 1 1 at 1 and the with a of in the containing the fusion protein were by which the of the glutaredoxin as by A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The were and by on a to The a of in 1 and with the at The were by and by to The fusion protein by with of of fusion protein with of to and at for The by of The protein a lower a of in 1 1 DTT, and with a of in the at The CBFβ were by and to by 1 of a of is protein used for NMR the a of in 1 DTT, and at The protein by were at The fusion protein in the manner as for the fusion on and the fusion protein by with of of fusion protein in a containing 1 1 and at by the for at The by of with a at The protein a lower by a of in 1 1 DTT, and with a to in the at The were by and to by The protein a of in 1 1 DTT, and with the to the full-length protein the of the gel using with as by the gel on a using and as by the The for the and proteins were on the of the and of the proteins as by L. G. T. Sci. 1995; PubMed Scopus Google Scholar). A of for by at the at the for were to the of by N-terminal of the residues at the at A sequence of that at the the sequence of the amino acids with The of and for a CBFα·DNA complex were by The of acid the murine protein the DNA-binding in and as B.E. Lewis A.F. Bushweller J.H. Speck N.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). binding were that the of DNA of the domain for the DNA and the of domain of CBFβ for the complex A of DNA and the with of CBFβ were used in binding and the were by and for were the as (15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). The are as the of the of CBFβ, and is as the of CBFβ at complex as the of and complex is the at the in the absence of PubMed Scopus Google equilibrium were at in a at of and or and using were to in 1 were using J. Full Text PDF PubMed Scopus Google Scholar). and were as Sedimentation for of in Scholar). CBFβ(141), the for the at by the to additional of in the the H. PubMed Scopus Google Scholar, Chem. PubMed Scopus Google Scholar). CBFβ(187), the for its at by and the additional in the Circular dichroism were at on a using amide were using the known protein The protein were to of were used for measurements in the The were by of a of the A of were at 1 to for protein and at a of with a The for were using the of and 1990; PubMed Scopus Google Scholar, P. PubMed Scopus Google using by proteins at a were the and the were for of structure and were of which than and measurements were on a NMR with and were at with of the proteins in were using the sequence P. T. J. Chem. Scopus Google Scholar). The of complex and for these and of 1 the of N.A. G. T. J.D. 1994; PubMed Scopus Google for of were A of used between to of V. A. J. Scholar). 1 were with of the and for and and for were with to the of the and for and and for the and for 1 were to the and of and as P. T. J. Chem. Scopus Google Scholar). The were using using the P. V. G. K. J. Scopus Google Scholar). The of the in the were by the using the in the M. P. K. J. 1995; PubMed Scopus Google Scholar). The in the were to the of the The 1 were by the to the 1 in which is the at and is the at 1 and and were by least of the to the in 1 using the S.A. to the of The of the to 1 by of the with of at as M. J. Chem. 1991; Scopus Google Scholar). of the for and were a of A. PubMed Scopus Google for backbone with 1 by the The biophysical and to to CBFβ quantities of to CBFβ in a of including fusion with these and the quantities of CBFβ for biophysical and have a novel fusion on E. on the of coli glutaredoxin A. 1991; PubMed Scopus Google that a fusion with CBFβ to the C-terminal of glutaredoxin very of A the have in which the sequence for E. has been to that of a containing the sequence for The sequence the of the fusion protein with additional amino acids are at the N-terminal to the used for A similar fusion has been with the protein S. 1993; PubMed Scopus Google this for it for proteins with and for NMR spectroscopy. fusion a on the of a A. 1991; PubMed Scopus Google these it to the in the for expression of by a encoding a between the for and the 1 the fusion of expression of protein that is a of with a of the and by the fusion protein can be to in on and on The fusion were using the for glutaredoxin A. J. Biol. Chem. Full Text PDF PubMed Google and by of the fusion protein with and on and yields significant quantities of protein a as by as well as by gel The for the of the fusion protein were to the of protein and the of the dichroism of that the protein to the for the to in a in but a of the of in a of of of fusion in a of of for a a the N-terminal sequence and of the N-terminal and were on N-terminal the N-terminal amino acids but the of the amino a of in with the of for the 143-amino acid and of the used for the to with a containing of expression of the fusion protein were and the protein be the for the fusion of the fusion protein with in of the of this of the protein N terminus on the of the protein in the the and residues and of The of amino acids residues and are and which the sequence for that at these this a between the and the of fusion protein are and can be the used with of the protein to be of a region in the of the this with be to to the of on is as for followed by on to the the full-length The is as by as well as gel A of of is 1 of have that the 141 amino acids of CBFβ were for with CBFα (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar,15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 16Niki M. Okada H. Takano H. Kuno J. Tani K. Hibino H. Asano S. Ito Y. Satake M. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5697-5702Crossref PubMed Scopus (164) Google Scholar). and the full-length have similar for CBFα, to of proteins for a CBFα·DNA The of CBFα used in these of a acid containing the DNA binding domain the murine which and as B.E. Lewis A.F. Bushweller J.H. Speck N.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the complex associated with binding of CBFβ has been to equilibrium for the binding of CBFβ to a complex (15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). shows gel these and the for the of the of and at were for the binding of and to a are very that the for binding to the domain in the N-terminal 141 amino well with the homology between CBFβ and its is a of homology in the N-terminal amino acids but very homology in the C-terminal region of the The function of the C-terminal region is at this the of and CBFβ(187), equilibrium measurements have been and as single with of of the and of the are in with the of the proteins and that proteins are these the proteins to to the for the equilibrium proteins be for structure determination by NMR spectroscopy. The for the proteins have been to of the of using the procedure of 1990; PubMed Scopus Google and and P. PubMed Scopus Google Scholar). and are mixed α/β proteins with and and The of the residues in proteins are between and with proteins a similar of residues in but a of residues in on the similar of residues in the similar and the very similar NMR of the proteins it that the N-terminal 141 amino acids the in and the full-length on the in the of residues for for to residues in the C-terminal of that this C-terminal is P. T. J. Chem. Scopus Google of and have been to the of the proteins and to their for structure determination by NMR spectroscopy. shows of the proteins be to the for NMR spectroscopy and for proteins well of mixed α/β The show a to one in additional of the of the of the N-terminal 141 amino acids in In the residues with that to amino acids in in 1990; PubMed Scopus Google to one that the proteins at the for NMR have 1 and on of the of the can be the of 1 and A. PubMed Scopus Google Scholar). shows the of this for of the in the of and of the in the of on these of for and for The for is in with for a of proteins of similar A. PubMed Scopus Google Scholar, M. J. Mol. Biol. 1995; PubMed Scopus Google Scholar, 1990; PubMed Scopus Google Scholar, J. Mol. Biol. 1993; PubMed Scopus Google Scholar, A. PubMed Scopus Google is at the for NMR spectroscopy. the of be to its Scholar, PubMed Scopus Google and by its the in the for is with its in We that at these The core binding factor proteins a of transcription factors containing a DNA-binding subunit and a β subunit N.A. Stacy T. Gene 1995; PubMed Scopus Google Scholar). The domain of CBFα is for DNA binding and heterodimerization with CBFβ binds to the domain and is to a that results in in the affinity of the domain for DNA of (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar). E. X. J. J. A. and J. H. in The of CBFβ and the domain are and the show homology to DNA-binding or of the genes encoding and CBFβ in mice are embryonic and to a in hematopoiesis (15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 16Niki M. Okada H. Takano H. Kuno J. Tani K. Hibino H. Asano S. Ito Y. Satake M. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5697-5702Crossref PubMed Scopus (164) Google Scholar, 17Okuda T. van Deursen J. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1606) Google Scholar, 18Sasaki K. Yagi H. Bronson R.T. Tominaga K. Matsunashi T. Deguchi K. Tani Y. Kishimoto T. Komori T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12359-12363Crossref PubMed Scopus (328) Google Scholar, 19Wang Q. Stacy T. Binder M. Marı́n-Padilla M. Sharpe A.H. Speck N.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3444-3449Crossref PubMed Scopus (1029) Google that CBF a in in the genes encoding and CBFβ are associated with a of leukemias (15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, Wang Q. Zhang P. Sharpe A.H. Binder M. M. Speck N.A. Zhang 1997; PubMed Scopus Google Scholar). and structure determination of these proteins and their with as well as with DNA the of novel to the leukemias associated with the forms of these We have developed a procedure for the and of full-length CBFβ, CBFβ(187), as well as a truncated form containing the N-terminal 141 amino acids, CBFβ(141), using a novel protein fusion E. coli glutaredoxin as the fusion The of the glutaredoxin protein as the fusion results in very of expression of the proteins in a of the CBFβ the fusion protein has been either or in the fusion of for the full-length protein of the of sites of for in the C-terminal of sites have been by of the and show a to the sequence of The of glutaredoxin for be to We have a of to the biophysical of and gel have been to the equilibrium binding for the binding of and to a We have that the binding of proteins is that the for binding to the domain in the N-terminal 141 amino Sedimentation equilibrium NMR measurements show proteins to be in for structure determination by NMR spectroscopy. Circular dichroism spectroscopy shows these proteins are mixed α/β proteins with the additional C-terminal residues in is the that the fusion protein by the chromosomal that disrupts the CBFB inv(16)(p13;q22), results in the fusion of the N-terminal amino acids of CBFβ to a protein encoded by the (10Liu P. Tarle S.A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Crossref PubMed Scopus (651) Google Scholar, P.P. A. Collins F.S. 1995; PubMed Google Scholar). The of the to CBFβ corresponds to the in a for this of the fusion protein as NMR spectroscopy has been on the proteins to their and their for structure determination by NMR spectroscopy. A structure determination by NMR spectroscopy of is We of the for the measurements and for the NMR We for of the