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

Journal: Methods in molecular biology (Clifton, N.J.) 2019;1955():119-134

PMID: 30868523

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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.

Address: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM), Buenos Aires, Argentina.; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.; Instituto de Patología Experimental, CONICET, Universidad Nacional de Salta, Salta-Capital, Argentina.; Instituto de Estudios de la Inmunidad Humoral (IDEHU, CONICET-UBA), Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires (UBA), Ciudad Autónoma de Buenos Aires, Argentina.; Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.; Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM), Buenos Aires, Argentina. [email protected].; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. [email protected].; Instituto de Investigaciones Biotecnológicas 'Dr Rodolfo Ugalde', IIB-INTECH, Universidad Nacional de San Martín (UNSAM), Buenos Aires, Argentina. [email protected].

DOI: 10.1007/978-1-4939-9148-8_9

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https://doi.org/10.1007/978-1-4939-9148-8_9

MeSH Terms: Chagas Disease, Cloning, Molecular, GPI-Linked Proteins, Gene Expression, Humans, Protozoan Proteins, Recombinant Proteins, Transfection, Trypanosoma cruzi