Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenicEscherichia coli

Abstract

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.

Structure of FocB – a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli

“…As anticipated from CD measurements and secondary structure prediction software [27] (Fig. 2), the amino acid chain of FocB forms an all α‐helical structure that comprises five α‐helices: α1 (Asp12‐Ser21), α2 (30Glu‐Ser40), α3 (Asp45‐Gly58), α4 (Arg61‐Tyr68), and α5 (Asn71‐Tyr95) (Fig.…”

“…Cross‐linking and size exclusion chromatography studies showed that FocB forms predominantly dimers in solution [27]. Furthermore, the packing of molecules in the crystal structure suggests that FocB is dimeric.…”

“…The culture was harvested by centrifugation at 53 000 g for 30 min. Purification of Se‐Met‐FocB was performed in the same way as described for the native protein [27]. Pure fractions of the protein in 10 m m Hepes (pH 7.9), 500 m m NaCl, 5 m m EDTA and 0.1%β‐mercaptoethanol were pooled and concentrated to 13.5 mg·mL −1 and stored at 4 °C.…”

“…Both native [27] and Se‐Met FocB were crystallized by hanging‐drop vapor diffusion of a mixture of equal volumes of protein solution and well solution. The well solution contained typically 0.1 m Mes (pH 6.5) and 1.5–1.75 m MgSO 4 .…”

“…The overexpression and purification of native FocB (109 amino acid residues) has been described previously [27]. The full-length FocB protein was cloned into pETM11 and overexpressed in E. coli strain BL21 (DE3).…”