caa a22 4500 397555172 CHVBK 20180308164759.0 cr unu---uuuuu 161202e199602 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021240 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021240 Purification and Characterization of the DNA-Binding Domain of BTEB, a GC Box-Binding Transcription Factor, Expressed in Escherichia coli [Elektronische Daten] [Yasuo Kikuchi, Kazuhiro Sogawa, Nobuaki Watanabe, Akira Kobayashi, Yoshiaki Fujii-Kuriyama] BTEB is a GC-binding protein that regulates the transcription of genes with a single GC-box or tandemly repeated GC-boxes in the promoter. The DNA-binding domain of BTEB consists of three contiguous Cys2-His2 zinc finger motifs and short segments adjacent to their N- and C-terminal sides [Kobayashi et al. (1995) J. Biochem 117, 91-95]. The truncated BTEB (residues 120 to 244) containing the DNA-binding domain was expressed in Escherichia coli and purified to homogeneity under denaturing conditions. DNA-binding activity of the BTEB was regenerated by refolding in the presence of Zn2+. The efficiency in regeneration was 70±10%, and the dissociation constant (Kd) of the DNA-complex was 4±2 nM. Co2+ also regenerated the DNA-binding affinity of BTEB, albeit with less efficiency than Zn2+. Co-BTEB showed a slightly lower affinity to the specific DNA than Zn-BTEB. Refolding in the presence of Cd2+ resulted in an extremely low efficiency in regeneration of the DNA-binding activity. Zn-BTEB is in a monomer state at concentrations lower than 0.5 μM, and forms a dimer in the concentration range of about 10 to 200 μM. © by the Journal of Biochemistry Regular Papers nationallicence DNA-binding protein nationallicence GC-box nationallicence homodimerization nationallicence transcription factor nationallicence zinc finger nationallicence Kikuchi Yasuo Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut Sogawa Kazuhiro Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut Watanabe Nobuaki Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut Kobayashi Akira Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut Fujii-Kuriyama Yoshiaki Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut The Journal of Biochemistry Oxford University Press 119/2(1996-02), 309-313 0021-924X 119:2<309 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021240 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021240 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kikuchi Yasuo Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut NATIONALLICENCE 700 1- Sogawa Kazuhiro Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut NATIONALLICENCE 700 1- Watanabe Nobuaki Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut NATIONALLICENCE 700 1- Kobayashi Akira Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut NATIONALLICENCE 700 1- Fujii-Kuriyama Yoshiaki Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/2(1996-02), 309-313 0021-924X 119:2<309 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford