IngentaConnect Production of an Arabidopsis halleri foliar defensin in Escherich...

Authors: Marquès, L.¹; Oomen, R.J.F.J.¹; Aumelas, A.²; Le Jean, M.¹; Berthomieu, P.¹

Source: Journal of Applied Microbiology, Volume 106, Number 5, May 2009 , pp. 1640-1648(9)

Abstract:

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 μmol 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.

Keywords: Arabidopsis halleri; CNBr cleavage; native-like protein; plant defensin; recombinant AhPDF1.1

Document Type: Research article

DOI: 10.1111/j.1365-2672.2008.04131.x

Affiliations: 1: Laboratoire de Biochimie & Physiologie Moléculaire des Plantes, UMR Université Montpellier 2, CNRS, INRA, Montpellier SupAgro, Montpellier Cedex, France 2: Centre de Biochimie Structurale, CNRS UMR 5048, INSERM U554, Université Montpellier 1, Université Montpellier 2, Montpellier Cedex, France

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Production of an Arabidopsis halleri foliar defensin in Escherichia coli