构建蜕皮激素受体(Ecdysteroid receptor,EcR)和超气门蛋白(Ultraspiracle,USP)的表达载体,使两者在大肠杆菌内高效共表达,获得具有活性的目的蛋白,即EcR和USP的复合蛋白(EcR-USP)。将N端带有6×His tag的EcR目的基因片段克隆于p ET...构建蜕皮激素受体(Ecdysteroid receptor,EcR)和超气门蛋白(Ultraspiracle,USP)的表达载体,使两者在大肠杆菌内高效共表达,获得具有活性的目的蛋白,即EcR和USP的复合蛋白(EcR-USP)。将N端带有6×His tag的EcR目的基因片段克隆于p ET21b MDX12(+)载体内,将N端带有6×His tag的USP目的基因片段克隆于p ET28a(+)载体内,并将两个重组质粒同时转化入同一宿主菌BL21(DE3)内利用IPTG低温诱导双基因共表达,选用"超声破碎法"提取共表达蛋白,色谱柱纯化,SDS-PAGE和Western Blot分析纯化结果,Micro Cal i TC200法与小分子化合物20-羟基蜕皮激素(20-hydroxyecdysone,20E)相互作用进行活性分析。SDS-PAGE与Western Blot分析结果显示宿主菌中表达产物存在蛋白复合体,大小与预期相符,Micro Cal i TC200实验显示EcR-USP与20E有结合,即EcR-USP有活性。得到有活性的家蚕的EcR-USP的可溶性表达产物,为进一步对其进行结构与功能的研究奠定了基础。展开更多
Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatme...Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatment processes because of their inherent roles in biofilm development,bacterial aggregation and so on.The widely QS signals was Antoinducer-2(AI-2),primarily involved to allow the possibility of interspecies communication.However,the cellular components that mediate the response of Streptococcus agalactiae to AI-2 have not been fully characterized.Analysis of the complete genome sequence of S.agalactiae indi-cated that its RbsB protein has similarity to Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2.We hypothesized that RbsB protein mediates quorum sensing signal uptake via interaction with AI-2.To evaluate the regulatory effect of RbsB on QS system,the recombinant plasmid pGEX-6p-1-RbsB was constructed and RbsB protein was purified with GST-tag.To further elucidate the role of RbsB protein binding to DPD(AI-2 precursor dihydroxypentanedione),the systemati-cally throughput circular dichroism(CD)spectroscopy,isothermal titration calorimetry200(ITC200)and molecular docking methods were employed.The high expression of soluble RbsB protein with molecular weight of 33 kDa was obtained.The thermodynamics results(ΔH<0,ΔS<0,ΔG<0)with ITC determination indicated that the binding process between DPD and RbsB was exothermic and spontaneous,with hydrogen bonds and van der Waals forces as the main binding forces.Obviously,DPD can be more easily combined with RbsB in a dose-dependent manner,suggesting that RbsB was changed in the microenvironment of DPD when the DPD concentration was between 0.8-1.0mmolL−1 and reaching the maximum binding amount.According to molecular docking,3 hydrophobic residues involved in DPD and RbsB protein stable binding were be found,and also hydrogen bonding plays a key role in the formation of the new complex.RbsB efficiently inhibited V.展开更多
文摘构建蜕皮激素受体(Ecdysteroid receptor,EcR)和超气门蛋白(Ultraspiracle,USP)的表达载体,使两者在大肠杆菌内高效共表达,获得具有活性的目的蛋白,即EcR和USP的复合蛋白(EcR-USP)。将N端带有6×His tag的EcR目的基因片段克隆于p ET21b MDX12(+)载体内,将N端带有6×His tag的USP目的基因片段克隆于p ET28a(+)载体内,并将两个重组质粒同时转化入同一宿主菌BL21(DE3)内利用IPTG低温诱导双基因共表达,选用"超声破碎法"提取共表达蛋白,色谱柱纯化,SDS-PAGE和Western Blot分析纯化结果,Micro Cal i TC200法与小分子化合物20-羟基蜕皮激素(20-hydroxyecdysone,20E)相互作用进行活性分析。SDS-PAGE与Western Blot分析结果显示宿主菌中表达产物存在蛋白复合体,大小与预期相符,Micro Cal i TC200实验显示EcR-USP与20E有结合,即EcR-USP有活性。得到有活性的家蚕的EcR-USP的可溶性表达产物,为进一步对其进行结构与功能的研究奠定了基础。
基金This study was supported by the National Natural Sci-ence Foundation of China(Nos.31702386,31660251,31860245 and 31960203)the International Cooperation Science&Technology Planning Project of Guangdong Province of China(No.2017A050501037)+2 种基金the Natural Science Foundation of Guangxi Province(Nos.2018 GXNSFAA281019,2017GXNSFAA198010)the Central Government Directs Special Funds for Local Science and Technology Development Projects(No.ZY1949015)E.Wangkahart was supported financially by the Ministry of Science and Technology of Thailand and Mahasarakham University.
文摘Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatment processes because of their inherent roles in biofilm development,bacterial aggregation and so on.The widely QS signals was Antoinducer-2(AI-2),primarily involved to allow the possibility of interspecies communication.However,the cellular components that mediate the response of Streptococcus agalactiae to AI-2 have not been fully characterized.Analysis of the complete genome sequence of S.agalactiae indi-cated that its RbsB protein has similarity to Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2.We hypothesized that RbsB protein mediates quorum sensing signal uptake via interaction with AI-2.To evaluate the regulatory effect of RbsB on QS system,the recombinant plasmid pGEX-6p-1-RbsB was constructed and RbsB protein was purified with GST-tag.To further elucidate the role of RbsB protein binding to DPD(AI-2 precursor dihydroxypentanedione),the systemati-cally throughput circular dichroism(CD)spectroscopy,isothermal titration calorimetry200(ITC200)and molecular docking methods were employed.The high expression of soluble RbsB protein with molecular weight of 33 kDa was obtained.The thermodynamics results(ΔH<0,ΔS<0,ΔG<0)with ITC determination indicated that the binding process between DPD and RbsB was exothermic and spontaneous,with hydrogen bonds and van der Waals forces as the main binding forces.Obviously,DPD can be more easily combined with RbsB in a dose-dependent manner,suggesting that RbsB was changed in the microenvironment of DPD when the DPD concentration was between 0.8-1.0mmolL−1 and reaching the maximum binding amount.According to molecular docking,3 hydrophobic residues involved in DPD and RbsB protein stable binding were be found,and also hydrogen bonding plays a key role in the formation of the new complex.RbsB efficiently inhibited V.
