title: Production of an Arabidopsis halleri foliar defensin in Escherichia coli.
abstract: 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.
authors.affiliation: 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
authors.forename: L
authors.initials: L
authors.lastname: Marquès
authors.forename: R J F J
authors.initials: RJ
authors.lastname: Oomen
authors.forename: A
authors.initials: A
authors.lastname: Aumelas
authors.forename: M
authors.initials: M
authors.lastname: Le Jean
authors.forename: P
authors.initials: P
authors.lastname: Berthomieu
mesh_terms: 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
chemical_list: D023082:Defensins; D010940:Plant Proteins; D011994:Recombinant Proteins