In silico Analysis and Expression of Osmotin-EAAAK-LTP Fused Protein

Abstract

Antifungal agents are causing different problems in the agriculture industry. Plants are using various defense mechanisms for resistance against fungal pathogens. Some examples of these mechanisms are making physical barriers, producing chemical components and pathogenesis-related proteins such as lipid transfer protein (LTP) and Osmotin which can inhibit the growth of fungi at micro-molar concentrations. In this study, <em>Osmotin </em>and<em> LTP</em> genes were fused by the EAAAK linker to produce a single-fused gene construct. An in silico approach was used to predict and analyze Osmotin-EAAAK-LTP fused protein. Secondary and tertiary structure and mRNA formation of fused protein were predicted using bioinformatics tools. The designed construct was chemically synthesized and cloned in the pUC57 cloning vector. To express the fused protein gene was subcloned in expression vector pET-21b (+) with a hexahistidine tag. This gene was used for prokaryotic expression in <em>E</em>. coliBL21 (DE3) host. Different expression conditions were examined for expressing of fused protein. The fused protein was expressed with 1 mM IPTG after 3 hours of incubation at 28°C. The expression of 36.5 kDa protein was confirmed by western blotting. The study of antifungal activity of expressed fused protein was achieved by radial diffusion assay. This protein was able to exhibit antifungal activity towards experimented plant pathogenic fungi under <em>in vitro </em>conditions.