Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Dec 1;68(Pt 12):1503-6.
doi: 10.1107/S1744309112042686. Epub 2012 Nov 14.

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

Michelle L Tonkin  1 , Sean D Workman, Brett A Eyford, Bianca C Loveless, Jessica L Fudge, Terry W Pearson, Martin J Boulanger
Affiliations

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

Michelle L Tonkin et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

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.

PubMed Disclaimer

Figures

Figure 1
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
Figure 2
TcCISSAn crystals grown in a sitting drop with 20% PEG 3350, 0.2 M ammonium sulfate as the reservoir solution.
Figure 3
Figure 3
X-ray diffraction image from a TcCISSAn crystal. Inset, high-resolution area; data were processed to 2.7 Å resolution.
See this image and copyright information in PMC

References

    1. Aslett, M. et al. (2010). Nucleic Acids Res. 38, D457–D462. - PMC - PubMed
    1. Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. (2011). Acta Cryst. D67, 271–281. - PMC - PubMed
    1. Dargie, J. (2011). Editor. Tsetse and Trypanosomosis Information, Vol. 34, pp. 1–132. Rome: Food and Agriculture Organization of the United Nations.
    1. Eyford, B. A., Sakurai, T., Smith, D., Loveless, B., Hertz-Fowler, C., Donelson, J. E., Inoue, N. & Pearson, T. W. (2011). Mol. Biochem. Parasitol. 177, 116–125. - PMC - PubMed
    1. Fragoso, C. M., Schumann Burkard, G., Oberle, M., Renggli, C. K., Hilzinger, K. & Roditi, I. (2009). PLoS One, 4, e7074. - PMC - PubMed

Publication types

MeSH terms