Heterologous expression in Pichia pastoris and purification of the toxin His-TEV-Ts5, a Tityus serrulatus venom component

Abstract

The heterologous expression of proteins is a useful tool that allows studies of components which are hard to obtain from their natural sources. One of these components is the ±-neurotoxin Ts5 from Tityus serrulatus (Ts) venom. This scorpion is the most dangerous in Brazil because it's the most related in accidents with scorpions in the country, including those that let patients to death. Scorpionism in Brazil is a public health issue. Therefore, studies about Ts and its venoms are necessary to better understand these animals and their toxins as well as to develop more efficient antivenoms with less side effects. The venom total volume extracted by each Ts is very low, and only 2% corresponds to Ts5. Therefore, the heterologous expression of Ts5 has proved to be a great and indispensable tool for the study of this toxin. The purpose of this project is the heterologous expression of Ts5 in Pichia pastoris system and its purification to use in future studies concerning pharmaceutical potentials of this toxin. In this regard, the synthetic gene of Ts5 will be designed with a N-terminal poli-histidine head (as a facilitator to the purification through affinity chromatography) followed by a cleavage site with the TEV (Tobacco Etch Virus) protease (which will remove the poli-histidine head, making the recombinant Ts5 more similar to the native Ts5). The purification of recombinant Ts5 will be performed through different chromatography techniques and its structural characterization includes the determination of molecular mass by mass spectrometry and the confirmation of its production and cleavage site by TEV through the N-terminal sequencing through the Edman degradation method. This work will allow the realization of new studies with Ts5, especially those that needs great amounts of proteins. This will be the initial step to make feasible its biotechnological applications, such as its potential use as an insecticide or as a molecular tool for the study of voltage-dependent sodium channels, its target.