Acta Parasitologica

, Volume 63, Issue 1, pp 106–113 | Cite as

Measurement of binding strength between prey proteins interacting with Toxoplasma gondii SAG1 and SAG2 using isothermal titration calorimetry (ITC)

  • Meng-Yee Lai
  • Yee-Ling LauEmail author


Following the outcome from a previously performed yeast two-hybrid experiment, the binding strength between T. gondii SAG1 and SAG2 and their respective prey proteins were further confirmed in this study. The sag1, sag2 and their prey genes were amplified and cloned into a pGEMT vector. To express the recombinant proteins, the fragments were then subcloned into a pRSETA vector and transformed into E. coli BL21 (DE3) cells. The recombinant proteins were expressed optimally at 37°C and 1mM of IPTG. The 6X His-tag fusion proteins were purified, dialyzed and concentrated. To confirm the expressed proteins, the recombinant proteins were analysed by SDS-PAGE and Western blot. As expected, the size of SAG1, SAG2, HLY and HZF protein were 32, 23, 28 and 37 kDa, respectively. The purified proteins were loaded onto a MicroCal Auto-iTC200 calorimeter from MicroCal™ to quantify binding strength. ITC results indicated there was a typical binding curve for interactions between SAG1 and HLY protein. However, there was an atypical binding curve obtained for interactions between SAG2 and HZF protein. By observing the data obtained from the ITC assay, both of the human proteins (HLY and HZF) were demonstrated to bind to their respective SAG1 and SAG2 proteins.


Toxoplasma gondii isothermal titration calorimetry ITC protein-protein interaction surface antigens protein measurement binding strength 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahn H., Kim S., Kim S., Nam H. 2006. Interactions between secreted GRA proteins and host cell proteins across the parasitophorous vacuolar membrane in the parasitism of Toxo-plasma gondii. Korean Journal of Parasitology, 44, 303–312. DOI: 10.3347/kjp.2006.44.4.303CrossRefGoogle Scholar
  2. Coleman R. A., Pugh B. F. 1997. Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding. Proceedings of the National Academy Sciences United States America, 94, 7221–7226CrossRefGoogle Scholar
  3. Fernandez-Robledo J.A., Vasta G.R. 2010. Production of recombinant proteins from protozoan parasites. Trends Parasitology, 5, 244–254. DOI: 10.1016/ Scholar
  4. Golds E. E., Braun P. E. 1978. Protein associations and basic protein conformation in the myelin membrane. The Journal of Biological Chemistry, 253, 8162–8170PubMedGoogle Scholar
  5. Grimwood J., Smith J.E. 1996. Toxoplasma gondii: The role of parasite surface and secreted proteins in host cell invasion. International Journal of Parasitology, 26, 169–173. DOI: 10.1016/0020-7519(95)00103-4CrossRefGoogle Scholar
  6. Heaslip A.T., Nishi M., Stein B., Hu K. 2011. The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase. PLOS Pathogen, 7, e1002201. DOI:10.1371/journal.ppat.1002201Google Scholar
  7. Jones E.J., Korcsmaros T., Carding S.R. 2017. Mechanisms and pathways of Toxoplasma gondii transepithelial migration. Tissue Barriers, 5, e1273865. DOI: 10.1080/21688370.2016.1273865CrossRefGoogle Scholar
  8. Lai M. Y., Lau Y. L. 2017. Screening and identification of host proteins interacting with Toxoplasma gondii SAG2 by yeast two-hybrid assay. Parasites & Vectors, 10, 456. DOI: 10.1186/ s13071-017-2387-yCrossRefGoogle Scholar
  9. Laity J.H., Lee B.M., Wright P.E. 2001. Zinc finger proteins: new insights into structural and functional diversity. Current Opinion Structural Biology, 11, 39–46. DOI: 10.1016/S0959-440X(00)00167-6CrossRefGoogle Scholar
  10. Laliberte J., Carruthers V.B. 2008. Host cell manipulation by the human pathogen Toxoplasma gondii. Cell Molecular Life Sciences, 5, 1900–1915. DOI: 10.1007/s00018-008-7556-xCrossRefGoogle Scholar
  11. Leavitt S., Freire E. 2001. Direct measurement of protein binding energetics by isothermal titration calorimetry. Current Opinion in Structural Biology, 11, 560–566. DOI: 10.1016/S0959-440X(00)00248-7CrossRefGoogle Scholar
  12. Lekutis C., Ferguson D.J.P., Grigg M.E., Camps M., Boothroyd J.C. 2000. Surface antigens of Toxoplasma gondii: Variations on a theme. International Journal of Parasitology, 31, 1285–1292. DOI: 10.1016/S0020-7519(01)00261-2CrossRefGoogle Scholar
  13. Lin L. N., Hasumi H., Brandts J. F. 1988. Catalysis of proline isomerization during protein-folding reactions. Biochimica et Biophysica Acta, 956, 256–266. DOI: 10.1016/0167-4838(88)90142-2CrossRefGoogle Scholar
  14. Liu J., Walsh C. T. 1990. Peptidyl-prolyl cis-transisomerase from Escherichia coli: a periplasmic homolog of cyclophilin that is not inhibited by cyclosporin A. Proceedings of the National Academy Sciences United States America, 87, 4028–4032CrossRefGoogle Scholar
  15. Liu Q., Li F.C., Elsheikha H.M., Sun M.M., Zhu X.Q. 2017. Identification of host proteins interacting with Toxoplasma gondii GRA15 (TgGRA15) by yeast two-hybrid system. Parasites & Vectors, 10, 1. DOI: 10.1186/s13071-016-1943-1CrossRefGoogle Scholar
  16. Mineo J.R., Kasper L.J. 1994. Attachment of Toxoplasma gondii to host cells involves major surface protein, SAG1-1 (P30). Experimental Parasitology, 79, 351–361. DOI: 10.1006/expr. 1994.1054CrossRefGoogle Scholar
  17. Mineo J.R., Mcleod R., Mack D., Smith J.E., Khan I.A., Ely K.H., Kasper L.H., 1993. Antibodies to Toxoplasma gondii major surface protein (SAG-1, P30) inhibit infection of host cells and are produced in mapical membrane antigen 1 (AMA1) governs ligand binding selectivity. Plos One, 10, e0126206. DOI: 10.1371/journal.pone.0126206Google Scholar
  18. Pierce M.M., Raman C.S., Nall B.T. 1999. Isothermal titration calorimetry of protein-protein interactions. Methods, 19, 213–221. DOI: 10.1006/meth.1999.0852CrossRefGoogle Scholar
  19. Rajarathnam K., Rosgen J. 2014. Isothermal titration calorimetry of membrane proteins-progress and challenges. Biochemica et Biophysica Acta, 1838, 69–77. DOI: 10.1016/j.bbamem. 2013.05.023CrossRefGoogle Scholar
  20. Robinson S.A., Smith J.E., Miller P.A. 2004. Toxoplasma gondii major surface antigen (SAG1): in vitro analysis of host cell binding. Parasitology, 128, 391–396. DOI: 10.1017/S00311 82003004736CrossRefGoogle Scholar
  21. Sevinc A., Witek M.A., Fung L.W.M. 2001. Yeast two-hybrid and ITC studies of alpha and beta spectrin interaction at the tetramerization site. Cell Molecular Biology Letter, 16, 452–461. DOI: 10.2478/s11658-011-0017-9Google Scholar
  22. Sibley L.D., Boothroyd J.C. 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature, 359, 82–85. DOI: 10.1038/359082a0CrossRefGoogle Scholar
  23. Su C., Evans D., Cole R.H., Kissinger J.C., Ajioka J.W., Sibley L.D. 2003. Recent expansion of Toxoplasma through enhanced oral transmission. Science, 299, 414–416. DOI: 10.1126/science.1078035CrossRefGoogle Scholar
  24. Takemae H., Sugi T., Kobayashi K., Gong H., Ishiwa A., Recuenco F.C., et al. 2013. Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin. Scientific Reports, 3, 3199. DOI: 0.1038/srep03199CrossRefGoogle Scholar
  25. Vanchinathan P., Brewer J.L., Harb O.S., Boothroyd J.C., Singh U. 2005. Disruption of a locus encoding a nucleolar zinc finger protein decreases tachyzoite-to-radyzoite differentiation in Toxoplasma gondii. Infection and Immunology, 73, 6680–6688. DOI: 10.1128/IAI.73.10.6680-6688.2005CrossRefGoogle Scholar
  26. Velge-Roussel F., Dimer-Poisson I., Buzoni-Gatel D., Bout D. 2001. Anti-SAG1peptide antibodies inhibit the penetration of Toxoplasma gondii tachyzoites into enterocyte cell lines. Parasitology, 123: 225–233. DOI: 10.1017/S0031182001008460CrossRefGoogle Scholar
  27. Velmurugan G.V., Tewari A.K., Rao J.R., Baidya S., Kumar M.U., Mishra A.K. 2008. High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis. Veterinay Parasitology, 154, 185–192. DOI: 10.1016/j.vetpar.2008. 03.032CrossRefGoogle Scholar
  28. Wang Y.H., Yin H. 2014. Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii. Parasites & Vectors, 7, 180. DOI: 10.1186/1756-3305-7-180CrossRefGoogle Scholar
  29. Yadav R., Pathak P.P., Shukla V., Jain A., Srivastava S., Tripathi S., et al. 2011. Solution structure and dynamics of ADF from Toxoplasma gondii. Journal of Structural Biology, 176, 97–111. DOI: 10.1016/j.jsb.2011.07.011CrossRefGoogle Scholar
  30. Yan H., Li H., Zhou X.H., Wu K., Chen X.G. 2004. Efficient soluble expression, purification and identification of the truncated SAG1 gene of Toxoplasma gondii in Escherichia coli. Journal of First Military Medical University, 4, 412–414. DOI: 1000-2588(2004)04-0412-03Google Scholar

Copyright information

© Witold Stefański Institute of Parasitology, Polish Academy of Sciences 2018

Authors and Affiliations

  1. 1.Department of Parasitology, Faculty of MedicineUniversity of MalayaKuala LumpurMalaysia

Personalised recommendations