Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinase

Abstract

In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His(6) tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75 A resolution indicated that the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.84, b = 121.50, c = 154.59 A. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (V(M)) of 2.5 A(3) Da(-1), corresponding to 50% solvent content.

Figure 1

12.5% SDS–PAGE gel stained with Coomassie Brilliant Blue R-250 showing the apparent homogeneity of the purified rTbgGK. Lane 1, molecular-weight markers (kDa); lane 2, rTbgGK purified by Ni–NTA affinity chromatography and Superdex 200 gel filtration.

Figure 2

A typical X-ray diffraction pattern of an rTbgCK crystal. The detector edge corresponds to 2.4 Å resolution and an enlarged image of the indicated area around 2.75 Å resolution is shown. The exposure time was 1 s, with an oscillation angle of 1.0°.

Figure 3

Crystals of rTbgGK obtained by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant.