Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

Abstract

Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 angstroms. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6 degrees and a high-resolution limit of 1.8 angstroms. After flash-annealing, the apparent mosaicity decreased to 0.9 degrees and the high-resolution limit of usable data increased to 1.6 angstroms. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

Figure 1

SDS–PAGE analysis of fractions from nickel-affinity chromatography. Lane 1 is the flowthrough indicated as peak A on the chromatogram. Lane 2 represents a sample from peak B, which resulted from a washing step at 100 mM imidazole. Lanes 3, 4 and 5 correspond to the first, middle and last fractions from peak C. Peak C eluted at 100–200 mM imidazole and contained the desired enzyme. Lane 6 shows molecular-weight standards.

Figure 2

A crystal of B. anthracis class C NSAP grown by sitting-drop vapor diffusion over a reservoir containing 0.1 M HEPES pH 7.0, 30%(w/v) Jeffamine ED-2001 pH 7.0.