Clonagem e expressão da transposase de mos1 em diferentes cepas de escherichia coli bl21 (de3)

Abstract

Transposable elements (TE) are mobile DNA sequences present in most genomes, which are able to move from one locus to another. These elements are distributed in all major branches of life, their diversity and intrinsic biological characteristics makes a considerable source of genetic changes during the evolution of most species. The DNA transposon mariner superfamily is probably the most widespread of transposons in nature, and is characterized by having 2400 base pairs with a single coding region of the enzyme transposase. The transposable element mos1, which is classified within the superfamily of transposons mariner, and these are widely distributed in the family Drosophilidae. The study of transposable elements is still very succinct, and the knowledge about its transposase is little known at the molecular level. This work seeks to determine optimal conditions to optimize the expression and purification system to obtain the transposase of the mariner element MOS1 in different strains of Escherichia coli BL21 D3. It was subcloned into pGEX4T1 expression plasmid vector, the open reading frame (ORF) of the transposase gene. Subsequently, expression and solubilization tests were performed on 3 concentrations of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.1 mM, 0.5 mM and 1.0 mM) and two temperatures of 30°C and 37°C, Where the IPTG functions as an inducer to the promoter for expression of the heterologous protein. Expression of transposable fusion in Glutathione S-transferase (GST), was identified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) having the predicted molecular mass of approximately 67kDa. With the acquisition of these results obtained by SDSPAGE, a lower efficacy was observed in the heterologous expression in the BL21 D3 GROE strain. In addition, there was a decrease in expression in all strains at a temperature of 30ºC. The solubilization tests showed no significant differences in the different temperatures and concentrations of IPTG. With these results from the expressions, we will proceed with the purification and production of antibodies against this protein for further molecular and functional analyzes.