Voet-van-Vormizeele Jan, Groth Georg
Heinrich-Heine-Universität, Biochemie der Pflanzen, Universitätsstr. 1, Düsseldorf D-40225, Germany.
Protein Expr Purif. 2003 Nov;32(1):89-94. doi: 10.1016/S1046-5928(03)00215-8.
Ethylene responses in plants are mediated by a small family of membrane integral receptors including the ETR1 gene product which are similar to the two-component bacterial histidine kinase regulators. Detailed biochemical and structural analysis of the ethylene-receptor family was hampered by the scarce amount of pure protein. Here, we report the construction, expression, and single-step purification of the ETR1 receptor protein from Arabidopsis thaliana in a bacterial expression system. The DNA fragment encoding the mature ETR1 receptor protein was subcloned into the pET15b expression vector and highly expressed in derivatives C41(DE3) and C43(DE3) of the Escherichia coli strain BL21(DE3). The recombinant protein was solubilised from the bacterial cells using mild non-denaturing detergents and purified to homogeneity by Ni-NTA affinity chromatography, yielding approximately 2-3 mg pure protein per litre of cells. The molecular mass of the purified protein was estimated to be 78 kDa by SDS-PAGE. The expression and purification of recombinant ETR1 reported here provide a basis for detailed functional and structural studies of the receptor protein, which might help to understand signal perception and signal transduction of the phytohormone ethylene on the molecular level.
植物中的乙烯反应由一小类膜整合受体介导，包括ETR1基因产物，它们类似于双组分细菌组氨酸激酶调节因子。乙烯受体家族详细的生化和结构分析因纯蛋白量稀少而受阻。在此，我们报道了在细菌表达系统中从拟南芥构建、表达和一步纯化ETR1受体蛋白的过程。编码成熟ETR1受体蛋白的DNA片段被亚克隆到pET15b表达载体中，并在大肠杆菌菌株BL21(DE3)的衍生物C41(DE3)和C43(DE3)中高效表达。使用温和的非变性去污剂从细菌细胞中溶解重组蛋白，并通过Ni-NTA亲和层析纯化至同质，每升细胞产生约2-3毫克纯蛋白。通过SDS-PAGE估计纯化蛋白的分子量为78 kDa。本文报道的重组ETR1的表达和纯化，为受体蛋白的详细功能和结构研究提供了基础，这可能有助于在分子水平上理解植物激素乙烯的信号感知和信号转导。
