Expression, purification, crystallization and preliminary crystallographic analysis of the mouse transcription factor MafB in complex with its DNA-recognition motif Cmare.
The MafB transcription factor (residues 211-305) has been overexpressed in and purified from Escherichia coli. A protein-DNA complex between the MafB homodimer and the 21 bp Maf-recognition sequence known as Cmare has been successfully reconstituted in vitro and subsequently crystallized. The diffraction properties of the protein-DNA complex crystals were improved using a combination of protein-construct boundary optimization and targeted mutagenesis to promote crystal lattice stability. Both native and mercury-derivatized crystals have been prepared using these optimized conditions. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 94.8, c = 197.9 A. An anomalous difference Patterson map computed using data collected from crystals grown in the presence of HgCl(2) reveals four peaks. This corresponds to two copies of the protein-DNA complex in the asymmetric unit, with a solvent content of 62% and a Matthews coefficient of 3.22 A(3) Da(-1).
EMBL DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
Larissa C
LC
Textor
Matthias
M
Wilmanns
Simon J
SJ
Holton
D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D051258:MafB Transcription Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D051258:MafB Transcription Factor; C490406:Mafb protein, mouse; D011994:Recombinant Proteins; D004247:DNA