Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura

Abstract

Despite their toxicity, snake venoms are composed of a series of organic and non-organic components that show activities against virus and bacteria, as well as therapeutic activities in thrombotic disorders. Thus, the characterization of biomolecules present in venoms has shown to be important to develop alternative therapies. Bhalternin, a toxin from Bothrops alternatus, has been described as a thrombin-like enzyme and has a therapeutic potential in cases of thrombotic disorders. So, the present study had as objective to perform cloning and expression of Bhalternin in bacterial and yeast systems. The mature Bhalternin coding sequence was cloned into pGS-21a e pPICZα A vectors and, after confirmation of the nucleotide sequences by Sanger Method, the plasmids that were constructed, pPGSBH2 e pPαABH9, were utilized in the transformation of Escherichia coli (strain BL21-Codon-Plus(DE3)-RIPL) and the Pichia pastoris (strain X-33), respectively. Different colonies, which showed presence of plasmid DNA by Polymerase Chain Reaction, were purified, submitted to the expression process and, then, evaluated for Bhalternin expression by protein eletrophoresis and Western blotting. In the bacterial system, a protein band corresponding to the expected molecular weight of Bhalternin expressed as fusion protein with glutathione S-transferase (GST) was observed in the supernatant and in the precipitate of bacterial lysate. However, in yeast system, it was not possible to identify a protein band corresponding to the secreted form of Bhalternin. However, in ELISA it was observed levels of expression in yeast system. Still, the coagulation test was carried out and, although a bacterial sample obtained by treating the lysate precipitate with 3 M urea showed activity, the result was inconclusive, since a sample obtained with the control clone (transformed with the vector) also showed activity. In the azocaseinolytic test, all results samples showed activity, including samples obtained with control clones. In the case of yeast expression system, the results seem to indicate low expression of Bhalternin, even though codons optimized for P. pastoris expression were used. On the other hand, in bacterial system, solubilization of Bhalternin with urea was necessary and the treatment may have caused changes in its activities. Therefore, further tests should be performed in order to obtain Bhalternin as soluble form and to evaluate its therapeutic potential in thrombotic disorders, as well as, its antiviral, antibacterial, and antiparasitic activities.