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Article Dans Une Revue Journal of Applied Microbiology Année : 2009

Production of an Arabidopsis halleri foliar defensin in Escherichia coli.

Résumé

AIMS: Production of the recombinant Arabidopsis halleri defensin AhPDF1.1 in a native-like form. METHODS AND RESULTS: 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). CONCLUSION: The proposed purification protocol produces a native-like defensin suitable for tests of new biological roles. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.
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Dates et versions

hal-00409218 , version 1 (06-08-2009)

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L. Marquès, R. J. F. J. Oomen, A. Aumelas, M. Le Jean, P. Berthomieu. Production of an Arabidopsis halleri foliar defensin in Escherichia coli.. Journal of Applied Microbiology, 2009, 106 (5), pp.1640-8. ⟨10.1111/j.1365-2672.2008.04131.x⟩. ⟨hal-00409218⟩
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