Cloning and heterologous expression of hyaluronidase and/or novel toxins obtained from the transcriptome of Tityus serrulatus' venom gland

Abstract

Currently, the interest in the study of chemical and functional characteristics of toxins isolated from animal venoms is not restricted to its relevance in envenoming, but also due to their potential use as drugs in the treatment of several diseases. With the advent of "omics" techniques, such as transcriptome, it was possible not only to characterize these toxins, but also to identify new components with potential biotechnological applications. However, despite the diverse physiological actions and pharmacological potential of animal toxins, there is a large gap between the number of proteins already isolated and characterized and those that are effectively applied as drugs, which can be explained by limitations in obtainment of sufficient toxin for detailed structural and functional characterization. Thus, the heterologous expression of proteins is an viable alternative for obtaining these molecules, both in a laboratory scale, for the study of their functional and enzymatic activities, and in industrial scale, aimed at the large scale production of pharmaceuticals. Facing this challenge, this project has two main objectives, namely: the construction of a cDNA library from the venom gland of Tityus serrulatus to identify new toxins with biotechnological potential, and the cloning and expression in heterologous system of hyaluronidase and/or new toxins obtained from the transcriptome, and also to characterize them structurally and functionally to validate the heterologous expression process. Therefore, molecular biology techniques will be applied for the preparation of venom gland transcriptome, and the interesting sequences will be cloned into a vector for heterologous expression. To validate the expression, native proteins obtained from the venom, such as hyaluronidase, will be purified by chromatographic procedures and subjected to structural characterization such as mass spectrometry, N-terminal sequencing, electrophoresis, among others. In addition, native and recombinant proteins will be characterized the biochemical (enzyme kinetics, determination of isoelectric point and activity on different substrates) and functional (in vivo assays) parameters. Therefore, in this project we will validate the process of heterologous expression which may enable us to produce novel toxins bearing high biotechnological potential and others which are usually difficult to purify from venom. (AU)