Heterologous expression in Pichia pastoris and purification of the toxin His-TEV-T...
| Grant number: | 19/24980-0 |
| Support type: | Scholarships abroad - Research Internship - Scientific Initiation |
| Effective date (Start): | January 08, 2020 |
| Effective date (End): | March 07, 2020 |
| Field of knowledge: | Health Sciences - Pharmacy - Toxicological Analysis |
| Principal Investigator: | Eliane Candiani Arantes Braga |
| Grantee: | Nádia Mayumi Vilela Bartnick Tanaka |
| Supervisor abroad: | Jan Tytgat |
| Home Institution: | Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil |
| Local de pesquisa : | University of Leuven (KU Leuven), Belgium |
| Associated to the scholarship: | 18/11739-0 - Heterologous expression in Pichia pastoris and purification of the toxin His-TEV-Ts5, a Tityus serrulatus venom component, BP.IC |
Abstract Tityus serrulatus is considered the most dangerous scorpion species in Brazil. Its venom contains several compounds with different application potentials. Among these components, there are ±-neurotoxins, which can interact with voltage-gated sodium channels (Nav channels). These toxins are the most reactive and responsible for the toxic effects on scorpion envenoming. Ts5 is a highly toxic ±-neurotoxin that represents only 2% of T. serrulatus soluble venom. This toxin can be classified as an ±-like toxin, because it interacts with both mammal and insect Nav channels, thus it has a potential to be used as an insecticide. To produce Ts5 in enough amount to enable an in-depth characterization, the heterologous expression technique in Pichia pastoris system can be performed. For that, the recombinant vector "pPICZ±A-His-TEV-Ts5" will be designed with components (Histidine tag and TEV cleavage site) that will facilitate the processes of purification. Recombinant toxin expression will be induced by methanol for 144 hours and purification of final supernatant will be performed by IMAC and reversed-phase chromatography. After these purification processes, the His-tag will be cleaved from the recombinant protein by TEV digestion, releasing a protein more similar to the native one. After that, the recombinant protein must be analyzed to check if it presents the same effects on Nav channels that native toxin does. To perform this analysis, Xenopus laevis oocytes will be used to express Nav channels in cell membrane by cRNAs injection. After that, the toxins will be added to a chamber containing the oocytes and the electrophysiological characterization will be done using the two-microelectrode voltage-clamp technique. Therefore, this project aims to analyze r-Ts5 effects on Nav1.3, Nav1.6, Nav1.7 and DmNav1, and compare them with the effects caused by native Ts5. | |