| 稻瘟菌CYP51蛋白表达、纯化及其与抑制剂的复合体的结晶条件筛选 |
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| 引用本文：张秀娟,张峰.稻瘟菌CYP51蛋白表达、纯化及其与抑制剂的复合体的结晶条件筛选.植物保护学报,2023,50(4):873-882 |
| DOI：10.13802/j.cnki.zwbhxb.2023.2022045 |
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| 中文摘要:为阐明稻瘟菌Magnaporthe oryzae甾醇14α-脱甲基酶（sterol 14α-demethylase，CYP51）与抑制剂的互作机制，首先通过分子生物学软件预测并分析稻瘟菌CYP51蛋白的跨膜域、二级结构以及氨基酸序列保守性，对CYP51蛋白的氮端跨膜域序列进行截除处理并以此构建蛋白表达质粒；其次对表达质粒进行原核表达，并使用亲和层析、酶切、透析和凝胶过滤层析等多种蛋白纯化手段得到目的蛋白；最后利用坐滴法对CYP51蛋白与抑制剂的复合体的结晶进行筛选。结果表明，稻瘟菌CYP51A和CYP51B蛋白均有两段跨膜域，在不破坏CYP51蛋白底物和抑制剂结合区域的前提下，将稻瘟菌CYP51A蛋白氮端1~100位氨基酸以及稻瘟菌CYP51B蛋白氮端1~110位氨基酸截除，构建蛋白表达质粒pET28a-His6-MBP-TEV-CYP51A和pET28a-MBP-TEV-CYP51B-His6。经原核表达与多种纯化方法成功获得质量佳且纯度高的稻瘟菌CYP51A和CYP51B-His6单体蛋白，并分别与氯氟醚菌唑、烯唑醇及戊唑醇等抑制剂孵育获得复合体，稻瘟菌CYP51B蛋白与烯唑醇复合体在0.1 mol/L酒石酸钾钠、0.1 mol/LTris-HCl （pH 8.5）、0.4 mol/L水合硫酸镁结晶条件下长出晶体。 |
| 中文关键词:稻瘟菌 CYP51蛋白 抑制剂 表达 纯化 结晶 |
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| Expression and purification of CYP51 proteins from rice blast fungus Magnaporthe oryzae and optimization of crystallization conditions for the CYP51-inhibitors complexes |
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| Abstract:In order to reveal the interaction mechanism between sterol 14α-demethylase (namely CYP51) from rice blast fungus Magnaporthe oryzae and the inhibitors, the transmembrane domains, secondary structure and conserved amino acid sequences of CYP51 protein from M. oryzae were predicted and analyzed by molecular biology software. Deleting the N-terminal transmembrane domain of CYP51 and constructing the protein-expression plasmid were performed based on the above information. Then prokaryotic expression was induced and the target protein purified with various means such as affinity chromatography, enzyme digestion, dialysis and gel filtration chromatography. High-throughput crystallization of CYP51 proteins complexes with their inhibitors were performed with sitting drop method. The results showed that both CYP51A and CYP51B proteins from M. oryzae had two transmembrane domains, amino acids 1-100 at the N-terminus of CYP51A and amino acids 1-110 at the N-terminus of CYP51B were removed without damaging the substrate and inhibitor binding regions of CYP51 protein. The protein expression plasmids pET28a-His6-MBP-TEV-CYP51A and pET28a-MBP-TEV-CYP51B-His6 were constructed. Monomer proteins of M. oryzae CYP51A and CYP51B-His6 with high quality and high purity were obtained by using prokaryotic expression and various purification methods. By incubating monomer CYP51 proteins from M. oryzae the complexes formed with mefentrifluconazole, diniconazole and tebuconazole, respectively, and the complexes were used to perform high throughput crystallization screening. The complex for CYP51B protein from M. oryzae with diniconazole grew crystals with the crystallization condition of 0.1 mol/L potassium sodium tartrate tetrahydrate, 0.1 mol/L Tris hydrochloride (pH 8.5), and 0.4 mol/L magnesium sulfate hydrate. |
| keywords:Magnaporthle oryzae CYP51 protein inhibitor expression purification crystallization |
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