Expression, purification, crystallization and preliminary X-ray diffraction analysis of the transcriptional repressor SirR from Mycobacterium tuberculosis H37Rv

Abstract

SirR, a metal-dependent transcriptional repressor from Mycobacterium tuberculosis (Rv2788), was cloned in pQE30 expression vector with an N-terminal His(6) tag for heterologous overexpression in Escherichia coli M15 (pREP4) cells and purified to homogeneity using chromatographic procedures. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. The crystals belonged to the tetragonal space group P4(1)2(1)2/P4(3)2(1)2, with unit-cell parameters a = 105.21, b = 105.21, c = 144.85 A. The X-ray diffraction data were processed to a maximum resolution of 2.5 A. The Matthews coefficient suggests the presence of two (V(M) = 4.01 A(3) Da(-1)) to four (V(M) = 2.0 A(3) Da(-1)) molecules in the asymmetric unit. Calculation of the self-rotation function shows a crystallographic fourfold symmetry axis along the z axis (chi = 90 degrees) and also a twofold symmetry axis around the z axis (chi = 180 degrees).

Figure 1

15% SDS–PAGE analysis. Lane M, molecular-weight markers; lane 1, uninduced M15 cells; lane 2, induced M15 cells; lane 3, cell cytosol; lane 4, Ni-Sepharose eluate; lane 5, Purified SirR after gel-exclusion chromatography.

Figure 2

Molecular-weight determination of SirR by size-exclusion chromatography on a Superdex 200 16/70C column. The protein was eluted as described in §2. The column was calibrated with globular protein molecular-weight markers: β-amylase (B), γ-globulin (G), bovine serum albumin (BSA), soybean trypsin inhibitor (TI) and lysozyme (L). The retention volume of the protein was 90 ml, corresponding to a molecular weight of ∼50 kDa.

Figure 3

Crystals of SirR from 1.5 M NaCl, 10% ethanol at 298 K measuring 0.25 × 0.17 × 0.12 mm.

Figure 4

Self-rotation function of the data collected from the crystal of SirR, showing a crystallographic fourfold symmetry axis along the z axis (χ = 90°), a twofold symmetry axis around the z axis (χ = 180°) and eight other peaks corresponding to crystallographic twofold axes. This figure was generated using the program MOLREP (Collaborative Computational Project, Number 4, 1994 ▶).

Figure 5

Sequence alignment of M. tuberculosis SirR (MtbSirR) with SirR from S. epidermidis (StepSirR) and IdeR from M. tuberculosis. Asterisks indicate fully conserved residues, colons denote strongly conserved residues and dots show weakly conserved residues. Probable metal-binding residues are denoted by triangles. Glu109 is represented by a square, while the DNA-binding residues are denoted by circles.