Production of an Arabidopsis halleri foliar defensin in Escherichia coli.
OBJECTIVE
Production of the recombinant Arabidopsis halleri defensin AhPDF1.1 in a native-like form.


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).


CONCLUSIONS
The proposed purification protocol produces a native-like defensin suitable for tests of new biological roles.


CONCLUSIONS
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.
Marquès
L
L
Laboratoire de Biochimie & Physiologie Moléculaire des Plantes, UMR Université Montpellier 2, CNRS, INRA, Montpellier SupAgro, 2 place Viala, Montpellier Cedex 01, France. laurence.marques@univ-montp2.fr
Oomen
R J F J
RJ
Aumelas
A
A
Le Jean
M
M
Berthomieu
P
P
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D023082:Defensins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D023082:Defensins; D010940:Plant Proteins; D011994:Recombinant Proteins