Expression and purification of rTs7, a recombinant toxin from <em>Tityus serrulatus</em> scorpion venom

Abstract

Tityus serrulatus venom is composed of several substances, including the neurotoxins that interact with voltage-gated ion channels. These channels are involved in many diseases, such as arrhythmia, asthma, autoimmune diseases, hypertension and immune response to infection and inflammation, making the T. serrulatus venom an important tool to study them. The Ts7, also called TsTx-K-alpha, acts selectively on potassium channels, and can contribute to the treatment of Kv1.3 channel-related diseases, being this channel a potential therapeutic target in the treatment of autoimmune diseases. In this work, we present the heterologous expression of Ts7 in Pichia pastoris yeast and its purification. The toxin gene was synthesized with TEV (tobacco etch virus) protease cleavage site before N-terminal sequence and cloned into pPICZαA vector. The P. pastoris cells (KM71H strain) were transformed with the linearized plasmid rTs7_pPICZαA, by electroporation. Transformation was confirmed by PCR of selected colonies and 1% agarose gel electrophoresis. Positively transformed colonies were submitted to a screening in a 24-wells plate, under standard conditions (pH6, for 144h), in order to determine the maximum expression rate. The colony showing the highest expression level of the recombinant protein was selected for laboratorial-scale expression, and the progress of expression was monitored by SDS-PAGE. The expressed protein was purified through immobilized metal affinity chromatography (IMAC) followed by reversed-phase chromatography on a C-18 column. In the reversed-phase chromatography, three fractions were observed and, after mass spectrometry analysis, the rTs7 was identified in fraction 3, and fractions 1 and 2 were possibly the cleaved toxin. The rTs7 was successfully expressed and purified, with a satisfactory yield of the recombinant toxin, which showed high similarity with the native toxin. The rTs7 immunosuppressive activity in multiple sclerosis model will be further investigated. Financial support: CAPES, CNPq, FAPESP.