Clonagem e expressão de uma serinoprotease "thrombin-like" em Nicotiana benthamiana

Abstract

The technique of heterologous protein expression in plant species is widely used in the scientific community. As one of the most used methods of transient transformation in plants being agroinfiltration with the bacteria Rhizobium radiobacter, which has the presence of plasmide Ti in its structure, giving the bacteria the capacity of inducing the vegetable to produce heterologous proteins . This process results in a large-scale production of proteins of biotechnological interest that are found in low concentrations in natura. Among the proteins with potential applicability in biotechnology especially for pharmacological proposes, there is the class of thrombin-like serine protease enzymes, which are found in snake venoms as Bothrops pauloensis species, having applicability for future treatments for diseases such as myocardial infarction, bleeding, among others. Therefore, the aim of this work was to clone the gene of a thrombin-like serinoprotease and express it in Nicotiana benthamiana. The cloning technique performed was through the use of digestive enzymes and subsequent binding of the gene of interest on the vector used in the project (pEAQ-HT), having this vector already a silencing gene of the plant, not requiring therefore a coinfiltration . Subsequently the ligation was electroporated into Escherichia coli and R. radiobacter cells. The project resulted in the cloning of the gene of interest in E. coli and R. radiobacter, with the subsequently expression in N. benthamiana, which has not yet been possible to detect protein expression.