Purification, crystallization and X-ray diffraction analysis of Trypanosoma congolense insect-stage surface antigen (TcCISSA)

Abstract

Trypanosoma congolense is a major contributor to the vast socioeconomic devastation in sub-Saharan Africa caused by animal African trypanosomiasis. These protozoan parasites are transmitted between mammalian hosts by tsetse-fly vectors. A lack of understanding of the molecular basis of tsetse-trypanosome interactions stands as a barrier to the development of improved control strategies. Recently, a stage-specific T. congolense protein, T. congolense insect-stage surface antigen (TcCISSA), was identified that shows considerable sequence identity (>60%) to a previously identified T. brucei insect-stage surface molecule that plays a role in the maturation of infections. TcCISSA has multiple di-amino-acid and tri-amino-acid repeats in its extracellular domain, making it an especially interesting structure-function target. The predicted mature extracellular domain of TcCISSA was produced by recombinant DNA techniques, purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis; the data were processed to 2.7 Å resolution.

Figure 1

(a) Purification of TcCISSAn by gel-filtration chromatography on a Superdex 75 16/60 HiLoad column. Standards for gel-filtration chromatography (plotted against the secondary axis, dotted curve): peak I, ovalbumin (43 kDa); peak II, carbonic anhydrase (29 kDa); peak III, ribonuclease A (13.7 kDa). TcCISSA (solid blue curve) eluted as a monomer. Inset: SDS–PAGE analysis of a representative fraction showing migration at the expected molecular mass of 26 kDa and residual thioredoxin tag. Lane MW contains molecular-mass markers (labelled in kDa). (b) Further purification of TcCISSAn by cation-exchange chromatography on a 5 ml HiTrap SP FF column. Inset: SDS–PAGE analysis of a representative fraction showing refined sample free of residual thioredoxin tag.

Figure 2

TcCISSAn crystals grown in a sitting drop with 20% PEG 3350, 0.2 M ammonium sulfate as the reservoir solution.

Figure 3

X-ray diffraction image from a TcCISSAn crystal. Inset, high-resolution area; data were processed to 2.7 Å resolution.