Homologous expression of glycosylphosphatidylinositol-anchored glycoproteins in trypanosoma cruzi

MESIAS, ANDREA C.; LOBO, MAITE MABEL; BUSCAGLIA, CARLOS A.; CAMEAN, CAMILA CENTENO; DURANTE, IGNACIO M.; DE LOS MILAGROS CÁMARA, MARÍA; BALOUZ, VIRGINIA; DUCREY, IVANA; CÁNEPA, GASPAR E.

METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.)

Humana Press Inc.

Año: 2019 vol. 1955 p. 119 - 134

1064-3745

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.