Homologous Expression of Glycosylphosphatidylinositol-Anchored Glycoproteins in Trypanosoma cruzi.
Balouz, V., Mesias, A.C., Centeno Camean, C., Ducrey, I., Lobo, M.M., Durante, I.M., Cánepa, G.E., Buscaglia, C.A. and Cámara, M.M.
Instituto de Investigaciones Biotecnologicas-Instituto Tecnologico de Chascomus (IIB-INTECH), Universidad Nacional de San Martin (UNSAM), Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Buenos Aires, Argentina.
Instituto de Patologia Experimental, CONICET, Universidad Nacional de Salta, Salta-Capital, Argentina.
Instituto de Estudios de la Inmunidad Humoral (IDEHU, CONICET-UBA), Catedra de Inmunologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires (UBA), Ciudad Autonoma 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 Biotecnologicas-Instituto Tecnologico de Chascomus (IIB-INTECH), Universidad Nacional de San Martin (UNSAM), Buenos Aires, Argentina. milagritos.camara@gmail.com.
Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Buenos Aires, Argentina. milagritos.camara@gmail.com.
Instituto de Investigaciones Biotecnologicas 'Dr Rodolfo Ugalde', IIB-INTECH, Universidad Nacional de San Martin (UNSAM), Buenos Aires, Argentina. milagritos.camara@gmail.com.
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.
Methods in Molecular Biology 1955: 135-146 (2019)