Caracterização bioquímica e funcional da rBnSP-7: uma fosfolipase A2 homóloga recombinante de Bothrops pauloensis

Abstract

CHAPTER II: Phospholipase A2 (PLA2) are among main components of animal venoms able to act on a large spectrum of pharmacological effects, such as anticoagulants, platelet aggregation inhibition, cardiotoxicity, neurotoxicity, hypotension, pro-inflammatory action and myotoxicity. The PLA2s also have different effects such as bactericide, antiparasitary and antitumor. The aim of this work was the expression, purification and biochemical and functional characterization of a Lys-49 PLA2, named BnSP-7. The insert for rBnSP-7 was previously obtained from a cDNA library of the Bothrops pauloensis venom gland. This was inserted into the pPicZA vector and used for transformation and propagation into E. coli (DH5 strain) and then inserted in the genome of Pichia pastoris KM71H. The expression was induced at 200 rpm, 26 °C for 144 hours adding methanol 0.5% (v/v) in the culture on a daily basis. The biochemical characterization (molecular weight, pI and western blotting) of purified rBnSP-7, in Ni-NTA superflow column, confirmed the authenticity of the expressed protein. Furthermore, the secondary structure of rBNSP-7 was analyzed by circular dichroism (CD) showing similarity between the CD spectrum of recombinant and native protein. When evaluated functionally, rBnSP-7 was able to induce myotoxicity in gastrocnemius muscle of mice, however there was no cytotoxic effect on C2C12, HeLa and tEnd cell lines. When analyzed in conjunction, our results demonstrated that the methodology used for the expression of rBnSP7 was satisfactory, however additional experiments should be performed in order to obtain a recombinant toxin form at higher similarity in terms of biological effects when it is compared to wild toxin.