Balouz Virginia, Mesias Andrea C, Camean Camila Centeno, Ducrey Ivana, Lobo Maite Mabel, Durante Ignacio M, Cánepa Gaspar E, Buscaglia Carlos A, de Los Milagros Cámara María
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.
Methods Mol Biol. 2019;1955:119-134. doi: 10.1007/978-1-4939-9148-8_9.
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.
克氏锥虫的表面被糖基磷脂酰肌醇（GPI）锚定糖蛋白（GAGP）所覆盖，这些糖蛋白有助于寄生虫的保护，并有助于在昆虫媒介和哺乳动物宿主中建立持续感染。多种因氨基酸序列和/或翻译后修饰不同的GAGP在寄生虫表面共同表达，因此限制了对这些分子的结构/功能分析。我们实验室的研究表明，转染寄生虫表达的GAGP标记变体经历与内源性变体类似的翻译后加工，因此是克服这些限制的合适工具。在本章中，我们详细介绍了在克氏锥虫中高效同源表达GAGP的整个方法流程：从同时克隆和标记感兴趣基因的简单策略到寄生虫表达产物的生化验证。
