Laboratoire de Biochimie & Physiologie Moléculaire des Plantes, UMR Université Montpellier 2, CNRS, INRA, Montpellier SupAgro, 2 place Viala, Montpellier Cedex 01, France.
J Appl Microbiol. 2009 May;106(5):1640-8. doi: 10.1111/j.1365-2672.2008.04131.x. Epub 2009 Feb 16.
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.
以天然形式生产拟南芥 Halleri 防御素 AhPDF1.1 的重组体。
成熟的 AhPDF1.1 cDNA 被克隆到 pET-28-a(+)中，并在大肠杆菌 Rosetta 中表达。在变性提取后，通过金属亲和层析和 CNBr 切割 His 标签进行纯化，得到没有 N 末端额外氨基酸的蛋白质。然后需要进行氧化折叠步骤以复性蛋白质，然后通过 C18 HPLC 分离将其纯化为均相。质谱和圆二色性分析表明，重组 AhPDF1.1 具有预期的分子质量和 3D 结构特征，是一种具有四个二硫键的折叠防御素。该重组蛋白对丝状真菌尖孢镰刀菌具有活性，最小抑制浓度为 0.6 μmol l(-1)。
所提出的纯化方案产生了类似于天然的防御素，适合测试新的生物学作用。
植物防御素本质上被认为是抗真菌蛋白；然而，最近发现了一些对植物细胞的意外作用。例如，AhPDF1.1 已被证明赋予锌耐受性。需要高效生产类似于天然的防御素来探索植物防御素的不同靶标和作用。
