Production of Recombinant Trypanosoma cruzi Antigens in Leishmania tarentolae

  • María José Ferrer
  • Diana Patricia Wehrendt
  • Mariana Bonilla
  • Marcelo Alberto Comini
  • María Teresa Tellez-Iñón
  • Mariana PotenzaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1955)


Trypanosomatids are unicellular organisms that colonize a wide diversity of environments and hosts. For instance, Trypanosoma cruzi is a human pathogen responsible for Chagas diseases, while Leishmania tarentolae infects amphibians and became a biotechnological tool suitable for recombinant protein expression. T. cruzi antigens are needed for the development of improved epitope-based methods for diagnosis and treatment of Chagas disease. Molecular cloning for the production of recombinant proteins offers the possibility to obtain T. cruzi antigens at high yield and purity. L. tarentolae appears as the ideal expression host to obtain recombinant T. cruzi antigens with a structure and posttranslational modifications typical of trypanosomatids. In this chapter, we present a protocol for the analytical to mid-scale production of recombinant T. cruzi antigens, using L. tarentolae as expression host (LEXSY® inducible system).

Key words

Trypanosoma cruzi Recombinant antigen Eukaryotic expression system Leishmania tarentolae 



This work was supported by grants to M.T.T.I. and M.P. (PIP 2015-0937 and PICT 2016-1028). M.A.C. acknowledges the financial support of FOCEM (MERCOSUR Structural Convergence Fund, [COF 03/11]).


  1. 1.
    Watanabe Costa R, da Silveira JF, Bahia D (2016) Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways. Front Microbiol 7:388. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Pérez-Molina JA, Molina I (2018) Chagas disease. Lancet 391(10115):82–94. CrossRefPubMedGoogle Scholar
  3. 3.
    Balouz V, Aguero F, Buscaglia CA (2017) Chagas disease diagnostic applications: present knowledge and future steps. Adv Parasitol 97:1–45. CrossRefPubMedGoogle Scholar
  4. 4.
    Thomas MC, Fernández-Villegas A, Carrilero B et al (2012) Characterization of an immunodominant antigenic epitope from Trypanosoma cruzi as a biomarker of chronic Chagas’ disease pathology. Clin Vaccine Immunol 19(2):167–173CrossRefGoogle Scholar
  5. 5.
    Schnaidman BB, Yoshida N, Gorin PA et al (1986) Cross-reactive polysaccharides from Trypanosoma cruzi and fungi (especially Dactylium dendroides). J Protozool 33(2):186–191CrossRefGoogle Scholar
  6. 6.
    Moure Z, Sulleiro E, Iniesta L et al (2018) The challenge of discordant serology in Chagas disease: the role of two confirmatory techniques in inconclusive cases. Acta Trop 185:144–148. CrossRefPubMedGoogle Scholar
  7. 7.
    Balouz V, Melli LJ, Volcovich R et al (2017) The Trypomastigote small surface antigen from Trypanosoma cruzi improves treatment evaluation and diagnosis in pediatric Chagas disease. J Clin Microbiol 55:3444–3453. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Jones K, Versteeg L, Damania A et al (2018) Vaccine-linked chemotherapy improves Benznidazole efficacy for acute Chagas disease. Infect Immun 86(4):e00876–e00817. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Biter AB, Weltje S, Hudspeth EM et al (2018) Characterization and stability of Trypanosoma cruzi 24-C4 (Tc24-C4), a candidate antigen for a therapeutic vaccine against Chagas disease. J Pharm Sci 107(5):1468–1473. CrossRefPubMedGoogle Scholar
  10. 10.
    Cerny N, Sánchez Alberti A, Bivona AE et al (2016) Coadministration of cruzipain and GM-CSF DNAs, a new immunotherapeutic vaccine against Trypanosoma cruzi infection. Hum Vaccin Immunother 12(2):438–450. CrossRefPubMedGoogle Scholar
  11. 11.
    Bivona AE, Sánchez Alberti A, Matos MN et al (2018) Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a novel immunogen for Chagas disease vaccine. PLoS Negl Trop Dis 12(3):e0006384. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Umezawa ES, Silveira JF (1999) Serological diagnosis of Chagas disease with purified and defined Trypanosoma cruzi antigens. Mem Inst Oswaldo Cruz 94(1):285–288CrossRefGoogle Scholar
  13. 13.
    Scharfstein J, Rodrigues MM, Alves CA et al (1983) Trypanosoma cruzi: description of a highly purified surface antigen defined by human antibodies. J Immunol 131(2):972–976PubMedGoogle Scholar
  14. 14.
    Berrizbeitia M, Ndao M, Bubis J et al (2006) Purified excreted-secreted antigens from Trypanosoma cruzi trypomastigotes as tools for diagnosis of Chagas’ disease. J Clin Microbiol 44(2):291–296. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Santos FL, Celedon PA, Zanchin NI et al (2017) Accuracy of chimeric proteins in the serological diagnosis of chronic Chagas disease – a Phase II study. PLoS Negl Trop Dis 11(3):e0005433. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    De Marchi CR, Di Noia JM, Frasch AC et al (2011) Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas’ disease. Clin Vaccine Immunol 18(11):1850–1855. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Seid CA, Jones KM, Pollet J et al (2017) Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human Chagas disease. Hum Vaccin Immunother 13(3):621–633. CrossRefPubMedGoogle Scholar
  19. 19.
    Matos MN, Sánchez Alberti A, Morales C et al (2016) A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection. Vaccine 34(28):3243–3251. CrossRefPubMedGoogle Scholar
  20. 20.
    Almeida IC, Covas DT, Soussumi LM et al (1997) A highly sensitive and specific chemiluminescent enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma cruzi infection. Transfusion 37(8):850–857CrossRefGoogle Scholar
  21. 21.
    Lingg N, Zhang P, Song Z et al (2012) The sweet tooth of biopharmaceuticals: importance of recombinant protein glycosylation analysis. Biotechnol J 7(12):1462–1472. CrossRefPubMedGoogle Scholar
  22. 22.
    Quanquin NM, Galaviz C, Fouts DL et al (1999) Immunization of mice with a TolA-like surface protein of Trypanosoma cruzi generates CD4(+) T-cell-dependent parasiticidal activity. Infect Immun 67(9):4603–4612PubMedPubMedCentralGoogle Scholar
  23. 23.
    Tate CG, Haase J, Baker C et al (2003) Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter. Biochim Biophys Acta 1610(1):141–153. CrossRefPubMedGoogle Scholar
  24. 24.
    Jenkins N, Murphy L, Tyther R (2008) Post-translational modifications of recombinant proteins: significance for biopharmaceuticals. Mol Biotechnol 39(2):113–118. CrossRefPubMedGoogle Scholar
  25. 25.
    Miranda MR, Sayé M, Reigada C et al (2015) Phytomonas: a non-pathogenic trypanosomatid model for functional expression of proteins. Protein Expr Purif 114:44–47. CrossRefPubMedGoogle Scholar
  26. 26.
    Tetaud E, Lecuix I, Sheldrake T et al (2002) A new expression vector for Crithidia fasciculata and Leishmania. Mol Biochem Parasitol 120(2):195–204CrossRefGoogle Scholar
  27. 27.
    Lee MG, Van der Ploeg LH (1997) Transcription of protein-coding genes in trypanosomes by RNA polymerase I. Annu Rev Microbiol 51:463–489CrossRefGoogle Scholar
  28. 28.
    Kushnir S, Gase K, Breitling R et al (2005) Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expr Purif 42(1):37–46CrossRefGoogle Scholar
  29. 29.
    Klatt S, Konthur Z (2012) Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae. Microb Cell Fact 11:97. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Klatt S, Rohe M, Alagesan K, Kolarich D et al (2013) Production of glycosylated soluble amyloid precursor protein alpha (sAPPalpha) in Leishmania tarentolae. J Proteome Res 12(1):396–403CrossRefGoogle Scholar
  31. 31.
    Ben-Abdallah M, Bondet V, Fauchereau F et al (2011) Production of soluble, active acetyl serotonin methyl transferase in Leishmania tarentolae. Protein Expr Purif 75(1):114–118. CrossRefPubMedGoogle Scholar
  32. 32.
    Hemayatkar M, Mahboudi F, Majidzadeh-A K et al (2010) Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae. Biotechnol J 5(11):1198–1206. CrossRefPubMedGoogle Scholar
  33. 33.
    Breitling R, Klingner S, Callewaert N et al (2002) Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr Purif 25:209–218CrossRefGoogle Scholar
  34. 34.
    Puigbo P, Guzmen E, Romeu A et al (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 35:126–131CrossRefGoogle Scholar
  35. 35.
    Vieira J, Messing J (1991) New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • María José Ferrer
    • 1
  • Diana Patricia Wehrendt
    • 1
  • Mariana Bonilla
    • 2
  • Marcelo Alberto Comini
    • 2
  • María Teresa Tellez-Iñón
    • 1
  • Mariana Potenza
    • 1
    Email author
  1. 1.Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, “Dr. Héctor Torres” (INGEBI-CONICET)Buenos AiresArgentina
  2. 2.Group Redox Biology of TrypanosomesInstitut Pasteur de MontevideoMontevideoUruguay

Personalised recommendations