Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli.
The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His(6)-tagged fusion protein was captured by Ni(2+)-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His(6) tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 A resolution was collected at 100 K using synchrotron radiation.
Hultdin
Ulrika W
UW
Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden.
Lindberg
Stina
S
Grundström
Christin
C
Allgardsson
Anders
A
Huang
Shenghua
S
Stier
Gunter
G
Ohman
Anders
A
Uhlin
Bernt Eric
BE
Sauer-Eriksson
A Elisabeth
AE
D018830:Adhesins, Escherichia coli / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D013696:Temperature
D018830:Adhesins, Escherichia coli; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C526544:FocB protein, E coli