Clonagem e expressão Datransposase mos1 de Drosophila simulans e o desenho in silico de um vetor para expressão heteróloga de proteínas

Abstract

The importance of heterologous protein expression has surprisingly grown in view of the progressive need for its production for applications in the pharmaceutical industry, for therapeutic use, or commercial use, and study of structure and function. However, the expression of the proteins may be limited by several factors, and may present several difficulties such as the formation of inclusion bodies or proteins with incorrect conformation, for example. Host studies for expression of heterologous proteins have been developed in order to optimize factors such as temperature and concentration of inducer in order to improve the quality of proteins obtained. In this study, we initially aimed to optimize transposase expression of the mariner element mos1 in different strains of Escherichia coli BL21 (DE3), E. coli BL21 (DE3) pLysS, E. coli BL21CodonPlus (DE3) -RP; E. coli BL21-CodonPlus (DE3) -RIL; E. coli BL21 (DE3) pTGROE, using for subcloning the pGEX-4T1 expression plasmid vector. Analyzing the best expression system for this particular element, comparing the temperatures of 30°C and 37°C for expression and using concentrations of 0.1 mM; 0.5mM and 1.0mM IPTG. Even with expression success, with all IPTG conditions and at both temperatures tested, the expression system was not effective in obtaining proteins in a soluble form. In view of this problem we have tried to develop an expression vector of heterologous proteins in order to make this process faster and more efficient, using only one event of cloning and transformation of the expression bacterium. The vector for expression was drawn in silico from an IPTG responsive Lac promoter, followed by a cell export signal (peptide-signal), a Strep-tagII fragment for purification and a site for thrombin, allowing cleavage of the protein from Interest of merged tags. Two sites for restriction endonucleases (BamHI and XbaI) were inserted which allow the cleavage of the cloned gene in the construct. In this way, we demonstrated that all strains were effective in the expression of the transposase mos1, with the most suitable temperature being 37 ° C in all concentrations of IPTG, since there was no difference in the change of this factor. We present a new expression vector of heterologous proteins, in order to facilitate the obtaining of purified heterologous proteins, such problems encountered during the attempt to express the transposase.