Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis

Abstract

Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein product was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 A resolution and belonged to space group P4(2), with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 A.

Figure 1

Crystals of M. tuberculosis BfrA grew to dimensions of ∼0.3 × 0.3 × 0.2 mm using the hanging-drop method.

Figure 2

X-ray diffraction pattern from a crystal of BfrA from M. tuberculosis. The edge of the frame is at 2.1 Å.

Figure 3

Self-rotation Patterson plots for M. tuberculosis BfrA in the resolution range 7–5 Å with an integration radius of 29 Å. The peaks in the κ = 90° (a), κ = 120° (b) and κ = 180° (c) sections confirm the 432 molecular symmetry. The maximum value was normalized to 100 and the contours are drawn at eight-unit intervals starting at 16. The self-rotation function was calculated using POLARRFN from the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶) with default parameters. Drawings were prepared using PLTDEV.