Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

  • Yang, Zhaoshou (Department of Parasitology, College of Medicine, The Catholic University of Korea) ;
  • Ahn, Hye-Jin (Department of Parasitology, College of Medicine, The Catholic University of Korea) ;
  • Nam, Ho-Woo (Department of Parasitology, College of Medicine, The Catholic University of Korea)
  • Received : 2014.04.17
  • Accepted : 2014.06.15
  • Published : 2014.08.31
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Abstract

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with $GRA2_{25-105}$, $GRA3_{39-138}$, $ROP2_{324-561}$, and $MIC2_{1-284}$ domains had respectively higher value of IgG avidity. The $rGST-GRA2_{25-105}$ and $rGST-GRA3_{39-138}$ were soluble, while $rGST-ROP2_{324-561}$ and $rGST-MIC2_{1-284}$ were not. $GRA2_{31-71}$, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The $rGST-GRA2_{31-71}-ROP2_{324-561}$, a chimeric protein, appeared to be soluble. Moreover, $rGST-GRA2_{31-71}-MIC2_{1-284}$ was also soluble and had higher IgG avidity comparing to $rGST-MIC2_{1-284}$. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.

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References

  1. Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000; 30: 1217-1258. https://doi.org/10.1016/S0020-7519(00)00124-7
  2. Erratum in: Int J Parasitol 2001; 31: 217-220. https://doi.org/10.1016/S0020-7519(01)00125-4
  3. Wong SY, Remington JS. Toxoplasmosis in pregnancy. Clin Infect Dis 1994; 18: 853-861. https://doi.org/10.1093/clinids/18.6.853
  4. Choi WY, Nam HW, Kwak NH, Huh W, Kim YR, Kang MW, Cho SY. Foodborne outbreaks of human toxoplasmosis. J Infect Dis 1997; 175: 1280-1282. https://doi.org/10.1086/593702
  5. Park YH, Han JH, Nam HW. Clinical features of ocular toxoplasmosis in Korean patients. Korean J Parasitol 2011; 49: 157-171.
  6. Holec-Gasior L. Toxoplasma gondii recombinant antigens as tools for serodiagnosis of human toxoplasmosis: current status of studies. Clin Vaccine Immunol 2013; 20: 1343-1351. https://doi.org/10.1128/CVI.00117-13
  7. Holec-Gasior L, Kur J. Toxoplasma gondii: recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay. Exp Parasitol 2010; 124: 272-278. https://doi.org/10.1016/j.exppara.2009.10.010
  8. Pietkiewicz H, Hiszczynska-Sawicka E, Kur J, Petersen E, Nielsen HV, Paul M, Stankiewicz M, Myjak P. Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis. Parasitol Res 2007; 100: 333-337. https://doi.org/10.1007/s00436-006-0265-1
  9. Hiszczynska-Sawicka E, Brillowska-Dabrowska A, Dabrowski S, Pietkiewicz H, Myjak P, Kur J. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli. Protein Expr Purif 2003; 27: 150-157. https://doi.org/10.1016/S1046-5928(02)00593-4
  10. Huang X, Xuan X, Suzuki H, Sugimoto C, Nagasawa H, Fujisaki K, Mikami T, Igarashi I. Characterization of Toxoplasma gondii SAG2 expressed in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 2002; 9: 1343-1347.
  11. Song KJ, Yang Z, Chong CK, Kim JS, Lee KC, Kim TS, Nam HW. A rapid diagnostic test for toxoplasmosis using recombinant antigenic N-terminal Half of SAG1 linked with intrinsically unstructured domain of gra2 protein. Korean J Parasitol 2013; 51: 503-510. https://doi.org/10.3347/kjp.2013.51.5.503
  12. Ching XT, Lau YL, Fong MY, Nissapatorn V. Evaluation of Toxoplasma gondii recombinant dense granular protein (GRA2) for serodiagnosis by western blot. Parasitol Res 2013; 112: 1229-1236. https://doi.org/10.1007/s00436-012-3255-5
  13. Nam HW, Im KS, Baek EJ, Choi WY, Cho SY. Analysis of antigenic domain of GST fused major surface protein (p30) fragments of Toxoplasma gondii. Korean J Parasitol 1996; 34: 135-141. https://doi.org/10.3347/kjp.1996.34.2.135
  14. Nam HW. GRA proteins of Toxoplasma gondii: maintenance of host-parasite interactions across the parasitophorous vacuolar membrane. Korean J Parasitol 2009; 47 (suppl): S29-S37. https://doi.org/10.3347/kjp.2009.47.S.S29
  15. Bermudes D, Dubremetz JF, Achbarou A, Joiner KA. Cloning of a cDNA encoding the dense granule protein GRA3 from Toxoplasma gondii. Mol Biochem Parasitol 1994; 68: 247-257. https://doi.org/10.1016/0166-6851(94)90169-4
  16. Mercier C, Lecordier L, Darcy F, Deslee D, Murray A, Tourvieille B, Maes P, Capron A, Cesbron-Delauw MF. Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole in Toxoplasma gondii. Mol Biochem Parasitol 1993; 58: 71-82. https://doi.org/10.1016/0166-6851(93)90092-C
  17. Beckers CJ, Dubremetz JF, Mercereau-Puijalon O, Joiner KA. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J Cell Biol 1994; 127: 947-961. https://doi.org/10.1083/jcb.127.4.947
  18. Carruthers VB, Tomley FM. Microneme proteins in apicomplexans. Subcell Biochem 2008; 47: 33-45. https://doi.org/10.1007/978-0-387-78267-6_2
  19. Dosztanyi Z, Csizmok V, Tompa P, Simon I. The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins. J Mol Biol 2005; 347: 827-839. https://doi.org/10.1016/j.jmb.2005.01.071
  20. Cserzo M, Wallin E, Simon I, von Heijne G, Elofsson GA. Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Prot Eng 1997; 10: 673-676. https://doi.org/10.1093/protein/10.6.673
  21. Kim MH, Choi YK, Park YK, Nam HW. A toxoplasmic uveitis case of a 60-year-old male in Korea. Korean J Parasitol 2000; 38: 29-31. https://doi.org/10.3347/kjp.2000.38.1.29
  22. Son ES, Nam HW. Detection and characterization of excretory/secretory proteins from Toxoplasma gondii by monoclonal antibodies. Korean J Parasitol 2001; 39: 49-56. https://doi.org/10.3347/kjp.2001.39.1.49
  23. Bai Y, He S, Zhao G, Chen L, Shi N, Zhou H, Cong H, Zhao Q, Zhu XQ. Toxoplasma gondii: bioinformatics analysis, cloning and expression of a novel protein TgIMP1. Exp Parasitol 2012; 132: 458-464. https://doi.org/10.1016/j.exppara.2012.09.015
  24. Macedo AG Jr, Cunha JP Jr, Cardoso TH, Silva MV, Santiago FM, Silva JS, Pirovani CP, Silva DA, Mineo JR, Mineo TW. SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts. Parasit Vectors 2013; 6: 163. https://doi.org/10.1186/1756-3305-6-163
  25. Zhao G, Zhou A, Lu G, Meng M, Sun M, Bai Y, Han Y, Wang L, Zhou H, Cong H, Zhao Q, Zhu XQ, He S. Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis. Parasit Vectors 2013; 6: 175. https://doi.org/10.1186/1756-3305-6-175
  26. Shoemaker BA, Portman JJ, Wolynes PG. Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. Proc Natl Acad Sci USA 2000; 97: 8868-8873. https://doi.org/10.1073/pnas.160259697
  27. Tompa P. The interplay between structure and function in intrinsically unstructured proteins. FEBS Lett 2005; 579: 3346-3354. https://doi.org/10.1016/j.febslet.2005.03.072
  28. Uversky VN, Dunker AK. The case for intrinsically disordered proteins playing contributory roles in molecular recognition without a stable 3D structure. F1000 Biol Rep 2013; 5: 1.
  29. Zhou H, Gu Q, Zhao Q, Zhang J, Cong H, Li Y, He S. Toxoplasma gondii: expression and characterization of a recombinant protein containing SAG1 and GRA2 in Pichia pastoris. Parasitol Res 2007; 100: 829-835. https://doi.org/10.1007/s00436-006-0341-6
  30. Kim JY, Ahn HJ, Ryu KJ, Nam HW. Interaction between parasitophorous vacuolar membrane-associated GRA3 and calcium modulating ligand of host cell endoplasmic reticulum in the parasitism of Toxoplasma gondii. Korean J Parasitol 2008; 46: 209-216. https://doi.org/10.3347/kjp.2008.46.4.209
  31. Mercier C, Adjogble KD, Daubener W, Delauw MF. Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites? Int J Parasitol 2005; 35: 829-849. https://doi.org/10.1016/j.ijpara.2005.03.011
  32. Saavedra R, Becerril MA, Dubeaux C, Lippens R, De Vos MJ, Herion P, Bollen A. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii. Infect Immun 1996; 64: 3858-3862.
  33. Martin V, Arcavi M, Santillan G, Amendoeira MRR, De Souza NE, Griemberg G, Guarnera E, Garberi JC, Angel1 SO. Detection of human Toxoplasma-specific immunoglobulins A, M, and G with a recombinant Toxoplasma gondii Rop2 protein. Clin Diagn Lab Immunol 1998; 5: 627-631.

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