Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum
Heterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity.
Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity.
Results: To validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction.
The urea solubilized His6-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni2+) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E.
coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f.
sp. glycines, Colletotrichum spp ., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var.
parasitica) under in vitro conditions.
Conclusions: Since SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications.
Author: Magnolia de A Campos, Marilia S Silva, Claudio P Magalhaes, Simone G Ribeiro, Rafael P D Sarto, Eduardo A Vieira and Maria F Grossi de Sa
Credits/Source: Microbial Cell Factories 2008, 7:7
Published on: 2008-03-11
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