Aplicação de diferentes cepas de Escherichia coli na otimização da expressão de uma aglutinina de leguminosa

Abstract

The technology of recombinant DNA aims at the heterologous production of proteins in different systems, including Escherichia coli, of optimized proteins with high biotechnological and medicinal potential, with low cost and laboratory time. Among the heterologous systems, the most common is the bacterial system Escherichia coli. There are different strains of E. coli available on the market for the most diverse applications, depending on the objective pursued with the protein in question. When it is not known for sure which is the best host and under what conditions the protein will be better expressed, the most suitable is to perform expression tests. Lectins are proteins capable of binding in a specific and reversible way to free carbohydrates or coupled to glycoconjugates, the latter being normally found in cell membranes. This makes them molecules of high biotechnological interest. The lectin extracted from the seeds of Canavalia virosa (ConV) is a ConA-like lectin, with valuable antiglioma, anti-tumor, inflammatory and vasorelaxant activities. However, lectins purified from natural sources can present isolectins or subunits resulting from post-translational processing, generating changes in the biological activities of these proteins. Thus, the present study aimed to optimize the heterologous expression of the gene corresponding to the ɑ chain of lectin ConV in Escherichia coli system in different expression strains. The recombinant ConV (rConV) was assembled in a pET-SUMO vector and expressed in the strains E. coli BL21 (DE3), BL21-CodonPlus(DE3)-RIL, BL21 (DE3) pLysS, Tuner (DE3) pLysS and ArcticExpress (DE3) under different conditions of temperature, concentration and time of action of the inductor. In the soluble fraction, rConV was expressed in two expression strains, with a high specific hemagglutinating activity in the bacterial protein extract obtained from the strain ArcticExpress (DE3). The results presented here show the possibility of heterologous production of rConV in the soluble fraction of at least two strains of E. coli expression, characterizing an efficient protocol. This makes it possible to obtain and use it in future structural and biological studies, as well as its biotechnological application.