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Production of an Arabidopsis halleri foliar defensin in Escherichia coli

2009; Oxford University Press; Volume: 106; Issue: 5 Linguagem: Inglês

10.1111/j.1365-2672.2008.04131.x

1365-2672

L. Pons Marqués, Roelof Oomen, André Aumelas, Marie Le Jean, Pierre Berthomieu,

Animal Nutrition and Physiology

Production of the recombinant Arabidopsis halleri defensin AhPDF1.1 in a native-like form.Mature AhPDF1.1 cDNA was cloned into pET-28-a(+) and expressed in Escherichia coli Rosetta. After a denaturing extraction, purification by metal affinity chromatography and CNBr cleavage of the His-tag, a protein without extra amino acids at the N-terminus was obtained. An oxidative folding step was then required to renature the protein that was then purified to homogeneity by a C18 HPLC separation. Mass spectroscopy and circular dichroism analyses showed that the recombinant AhPDF1.1 has the expected molecular mass and 3D-structure features of a folded defensin with four-disulfide bridges. The recombinant protein is active against the filamentous fungus Fusarium oxysporum with a minimal inhibitory concentration of 0.6 micromol l(-1).The proposed purification protocol produces a native-like defensin suitable for tests of new biological roles.Plant defensins are essentially known as anti-fungal proteins; however, some unexpected actions on plant cells have recently been discovered. AhPDF1.1, for example, has been shown to confer zinc tolerance. Efficient production of native-like defensins is required to explore the different targets and roles of plant defensins.