植物免疫激酶AtCERK1的重组表达与结晶研究

Expression and Crystallization of Recombinant Plant Immune Kinase AtCERK1

植物免疫激酶中的CERK1(chitin elicitor receptor kinase 1)家族是能够感知细菌及真菌侵染的模式识别受体。此类受体蛋白可以通过识别多糖类的病原微生物相关分子,引起病原微生物相关分子模式触发的免疫反应,是植物产生先天性免疫应答以应对微生物感染的重要元件,同时也是诱导植物-微生物共生的关键蛋白质。为推进CERK1激酶区真实蛋白质三维空间构象的解析,本研究针对拟南芥源的CERK1蛋白构建了野生型及突变型AtCERK1的E.coli-pRSF原核表达系统,经原核表达得到带有6个His标签的融合蛋白,经镍柱亲和层析、离子交换层析和凝胶过滤层析等一系列纯化过程得到高纯度蛋白质样品后对其进行结晶条件筛选。最终,AtCERK1^(F460V)突变型成功在0.2 mol/L氟化铵和20%PEG3350溶液环境中析出蛋白质晶体。经上海光源BL17U1线站采集,蛋白质晶体X射线衍射数据的分辨率为3.2A。综上所述,本研究成功获得了高纯度的AtCERK1蛋白样品,且突变型蛋白质成功析出晶体;同时,利用X射线收取了一套中等分辨率的衍射数据,为AtCERK1介导的植物免疫的结构机理进行了实验探索。

The chitin elicitor receptor kinase 1(CERK1)family of plant immune kinases are pattern recognition receptors(PRRs)capable of sensing bacterial and fungal infections.By recognizing pathogen-associated molecular patterns(PAMPs),such as some pathogen polysaccharides,or symbiotic factors,these receptor proteins involve in induction of PAMP-triggered immunity(PTI)or plant-microbe symbiosis.To determine the three-dimensional structure of CERK1 kinase domain,the E.coli-pRSF prokaryotic expression systems of wild-type and mutant AtCERK1 proteins were constructed for overexpression of CERK1 from Arabidopsis.The AtCERK1 proteins with a 6×His tag were expressed and subsequently purified by nickel column affinity chromatography.Based on the biophysical and biochemical properties of AtCERK1,ion exchange chromatography and gel filtration chromatography were performed for further purification of the AtCERK1 proteins.The resulting AtCERK1 proteins,up to 95%purity,were subjected to screening of crystallization conditions.The AtCERK1^(F460V) mutant protein was finally crystallized in the solution containing 0.2 mol/L ammonium fluoride and 20%PEG3350.X-ray diffraction data of these crystals were collected at the highest resolution of 3.2Å.In conclusion,the AtCERK1 protein was successfully obtained and crystallized.The preliminary crystallographic analysis of this protein will facilitate future investigation of the structural and functional basis of AtCERK1 mediated plant immunity.