Cloning and expression of Trypanosoma Cruzi giant protein fragment to perform interaction with the cytoskeletal proteins

• Grant number: 19/11047-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2019
• End date: December 31, 2022
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Munira Muhammad Abdel Baqui
• Grantee: Aylla Salomão Krebs Von Ermland
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Chagas' disease is caused by Trypanosoma cruzi and affects 8 million people, mostly in Latin America. In trypanosomatids, including T. cruzi, the presence of a giant protein (PGs) family with high molecular mass (e 500 kDa) has been demonstrated and located at the Flagellar Adhesion Zone (FAZ). This region has the function of connecting the cell body to the flagellum, playing numerous roles in the parasite survival. A mass spectrometry (MS) analysis of proteins with high molecular mass present in the T. cruzi cytoskeleton identified a calpain-like cysteine protease (CLP). This CLP is considered a giant protein since the molecular weight is e519 kDa; however, the function of this protein remains unknown. This project aims to clone a CLP fragment in the pGEX-4T1 expression vector in fusion with the N-terminal GST (Glutathione S-Transferase) in order to express and obtain a recombinant protein. The recombinant protein will be used to produce specific polyclonal antibodies as a tool for western blotting, immunofluorescence and co-immunoprecipitation assays. In addition, pull-down assays using the fusion protein will be performed to find elements that interact with PLC in the T. cruzi cytoskeleton. The project is aligned to the PhD project of Lays A. M. Trajano Silva, who analyzes the function of the giant T. cruzi protein by genome editing using CRISPR / Cas9. This project will support the studies being developed in the laboratory to characterize PGs in T. cruzi, which may have an important role in the process of division and differentiation of the parasite. (AU)