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Homologous Expression of Glycosylphosphatidylinositol-Anchored Glycoproteins in Trypanosoma cruzi

  • Virginia Balouz
  • Andrea C. Mesias
  • Camila Centeno Camean
  • Ivana Ducrey
  • Maite Mabel Lobo
  • Ignacio M. Durante
  • Gaspar E. Cánepa
  • Carlos A. Buscaglia
  • María de los Milagros Cámara
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1955)

Abstract

The surface coat of Trypanosoma cruzi is covered with glycosylphosphatidylinositol (GPI)-anchored glycoproteins (GAGPs) that contribute to parasite protection and to the establishment of a persistent infection in both the insect vector and the mammalian host. Multiple GAGPs that vary by amino acid sequence and/or posttranslational modifications are co-expressed on the parasite surface coat, hence curtailing structural/functional analyses on these molecules. Studies in our lab have indicated that GAGP-tagged variants expressed by transfected parasites undergo analogous posttranslational processing than endogenous ones and therefore constitute suitable tools to overcome these limitations. In this chapter, we detail the entire methodological pipeline for the efficient homologous expression of GAGPs in T. cruzi: from a simple strategy for the simultaneously cloning and tagging of the gene of interest to the biochemical validation of the parasite-expressed product.

Key words

Trypanosoma cruzi Glycosylphosphatidylinositol (GPI) GPI-anchored glycoproteins (GAGPs) Homologous expression Triton X-114 subcellular fractionation 

Notes

Acknowledgments

Investigations in our lab received financial support from the Agencia Nacional de Promoción Científica y Tecnológica, Argentina (www.agencia.mincyt.gob) and the Fundación Bunge y Born, Argentina (www.fundacionbyb.org). A.C.M., I.M.D., and G.E.C. held/hold fellowships from the CONICET, V.B. holds a fellowship from UNSAM-CONICET, and C.A.B. and M.M.C. are career investigators from the CONICET. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Copyright information

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

Authors and Affiliations

  • Virginia Balouz
    • 1
    • 2
  • Andrea C. Mesias
    • 1
    • 2
    • 3
  • Camila Centeno Camean
    • 1
    • 2
  • Ivana Ducrey
    • 1
    • 2
  • Maite Mabel Lobo
    • 1
    • 2
  • Ignacio M. Durante
    • 1
    • 2
    • 4
  • Gaspar E. Cánepa
    • 1
    • 2
    • 5
  • Carlos A. Buscaglia
    • 1
    • 2
  • María de los Milagros Cámara
    • 1
    • 2
    • 6
  1. 1.Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH)Universidad Nacional de San Martín (UNSAM)Buenos AiresArgentina
  2. 2.Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Buenos AiresArgentina
  3. 3.Instituto de Patología ExperimentalCONICET, Universidad Nacional de SaltaSalta-CapitalArgentina
  4. 4.Instituto de Estudios de la Inmunidad Humoral (IDEHU, CONICET-UBA), Cátedra de Inmunología, Facultad de Farmacia y BioquímicaUniversidad de Buenos Aires (UBA)Ciudad Autónoma de Buenos AiresArgentina
  5. 5.Laboratory of Malaria and Vector ResearchNational Institute of Allergy and Infectious Diseases, National Institutes of HealthRockvilleUSA
  6. 6.Instituto de Investigaciones Biotecnológicas ‘Dr Rodolfo Ugalde’, IIB-INTECHUniversidad Nacional de San Martín (UNSAM)Buenos AiresArgentina

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