Homologous expression of glycosylphosphatidyl inositol-anchored glycoproteins in Trypanosoma cruzi

VIRGINIA BALOUZ , ANDREA C. MESIAS1,2, CAMILA CENTENO CAMEAN1 , IVANA DUCREY1 , MAITE MABEL LOBO1 , IGNACIO M. DURANTE1,3, GASPAR E. CÁNEPA1,4, CARLOS A. BUSCAGLIA1 AND MARÍA DE LOS MILAGROS CÁMARA

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

Humana Press

Año: 2018

1064-3745

The surface coat of Trypanosoma cruzi is covered with glycosylphosphatidyl inositol (GPI)-anchored glycoproteins (GAGPs) that contribute to parasite protection and to the establishmentof a persistent infection in both the insect vector and the mammalian host. In every parasitedevelopmental stage, multiple members of each GAGP family that vary by amino acidsequence and by post-translational modifications are concurrently expressed on the surfacecoat, hence curtailing structural/functional analyses on these molecules. Studies in our lab haveindicated that GAGP tagged-variants expressed by transfected parasites undergo analogouspost-translational processing than endogenous ones and therefore constitute suitable surrogatemodels. In this chapter, we detail the entire methodological pipeline for the efficienthomologous expression of GAGPs in T. cruzi: from a simple strategy for the simultaneouslycloning and tagging of the gene of interest to the biochemical validation of the parasiteexpressed product