Production of Soluble Bioactive NmDef02 Plant Defensin in Escherichia coli

Antibiotic resistance.is one of the biggest challenges sciences faces today. It is extremely urgent to develop new antimicrobial compounds to control infections in the "post-antibiotic era”. Plant defensins belong to a large family of small cationic antimicrobial peptides and are an integral part of the innate immune system of plants. The gene coding for the mature peptide NmDef02 (isolated from Nicotiana megalosiphon ) was cloned into a pSMT3 vector, generating the plasmid pSMT3-DEF02, used to express the protein SUMO-NmDef02 in Escherichia coli , strain Shuffle T7 Express lysY. The soluble chimeric protein was purified by Ni–NTA affinity chromatography and cleaved by the UPL1 protease. The sample was re-applied to a Ni–NTA and approximately 20 mg of NmDef02 was obtained from the fermentation of 1 L of E. coli culture. The purified proteins were analyzed by SDS-PAGE under reduction condition and its identity was confirmed by Western blotting, using anti-histidine and anti-NmDef02 antibodies. NmDef02 defensin showed antimicrobial activity against plant and human pathogens. The recombinant fusion strategy could be an approach to produce bioactive recombinant NmDef02.