Expressão de uma serinoprotease de Bothrops pauloensis em Pichia pastoris e avaliação da sua atividade citotóxica sobre diferentes linhagens tumorais.

Abstract

Brazil has a great biotechnological potential, since there is a great diversity of fauna and flora. The beneficial, safe and conscious exploration of these resources leads to the discovery of molecules and models that can be used for therapeutic purposes. Many studies have revealed the biotechnological potential of toxins isolated from snake venoms, so they are studied both to understand issues related to poisoning and to act as therapeutic models. Serinoproteases derived from snake venoms (SVSPs) are glycosylated proteases known to have the ability to interfere with hemostasis, but their antitumor activity is still poorly explored. From this, the objective of this work is to express a recombinant serinoprotease cloned from Bothrops pauloensis venom (rBpSP-II) and to evaluate its cytotoxic activity in different tumoral strains. First the protein of interest was expressed in Pichia pastoris for 96h. The first chromatographic step used for the purification of rBpSP-II was Ni-NTA Superflow nickel resin affinity chromatography. After verification that the recombinant protein was not pure, the second chromatographic step was performed in reverse phase HPLC system, but the protein yield at the end of the purification was not satisfactory, obtaining only 0.30mg / L. Cytotoxic assays were then performed on tumor cells (MCF-7 and HepG2) and on non-tumor cells (MCF10A). RBpSP-II showed its highest cytotoxic activity in the MCF-7 line, killing approximately 40% of the cells at a concentration of 50 μg / mL, while in the HepG2 line the recombinant protein was cytotoxic to 20% of the cells. However, rBpSP-II did not show significant cytotoxicity against the MCF10A non-tumor line, demonstrating its preference for tumor cells. Once confirmed its cytotoxic potential, an apoptosis / necrosis induction assay was performed. At the concentration of 6.25μg / mL of recombinant protein, approximately 50% of MCF-7 cells were in apoptotic process, demonstrating rBpSP-II can cause death of tumor cells via necrosis / apoptosis. Thus, the recombinant rBpSP-II protein has an antitumor potential, however, further studies need to be developed to elucidate the mechanism of action of these proteases against tumor cells.