Expressão de uma osmotina de Calotropis procera (Ait) R. Br (CpOsm) em Escherichia coli como proteína de fusão com tiorredoxina (Trx-CpOsm): purificação e avaliação da atividade antifúngica da proteína recombinante Trx-CpOsm

Abstract

In previous works, an osmotin-like protein isolated from Calotropis procera latex (CpOsm) was purified and characterized and exhibited antifungal activity. In this work, Escherichia coli cells ArcticExpress (DE3), BL21 (DE3) and Shuffle strains, used for heterologous expression, and the DH5α strain used for cloning were transformed by thermal shock with four synthetic plasmids (GenOne). Each plasmid had the sequence of a fusion tag (Trx, SUMO, GST and Nus), in an attempt to express the osmotin in a soluble form. The transformed DH5α cells were cultured and undergo a plasmidial DNA extraction. The extracted plasmid DNA was digested with restriction enzymes BamHI and XhoI and the digestion was confirmed by agarose gel electrophoresis (0.8%). ArcticExpress (DE3), BL21 (DE3) and SHuffle expression cells were cultured and recombinant osmotin expression was performed by the addition of IPTG. Then, cell lysis was performed and the soluble and insoluble fractions were analyzed by SDS-PAGE. It was found that none of the cells had produced soluble recombinant osmotin and that the BL21 (DE3) strain, transformed with the synthetic plasmid pET 32a, obtained a higher production of protein (Trx-CpOsm). Thus, this strain was selected for further attempt to solubilize with CTAB. Inclusion bodies were solubilized with CTAB at three concentrations (1.0%, 2.5% and 5%), in addition to the control without CTAB. The best result was obtained with CTAB at 1% concentration. With the fraction solubilized with 1% CTAB an immobilized nickel affinity chromatography was performed. A stepwise gradient of the imidazole concentration was used and the recombinant protein was eluted in the buffer containing 100 mM imidazole. The purified protein fraction was concentrated in Vivaspin 3,000 MWCO (GE Healthcare) to a concentration of 1,39 mg/mL and used for antifungal tests against Lasiodiplodia sp. The agar diffusion assay was performed with four treatments: fungicide Carbendazim (2mL/L), deionized water, TrxCpOsm and the buffer in which the protein was diluted (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1% CTAB, 15% Glycerol). The inhibition halo formed by the protein was observed from 48h of assay and remained up to 168h, noting the activity of the protein. Trx-CpOsm was used in assays against Candida albicans ATCC 10231, Candida tropicalis ATCC 13803 and Candida parapsilosis ATCC 90018. By the microdilution technique in 96-well plates the Minimum Inhibitory Concentrations (MIC) were defined as being 62,50 ng / mL for strains C. parapsilosis ATCC 90018 and C. albicans ATCC 10231 and 31,20 ng / mL for C. tropicalis ATCC 13803 strain. Through Plate-Count agar (Merck) plating the Minimum LethalConcentrations (MLC) were determined to be 62,50 ng/mL, 31,20 ng/mL and 125 ng/mL for the strains C. albicans ATCC 10231, C. tropicalis ATCC 13803 and C. parapsilosis ATCC 90018, respectively.