具核梭杆菌CarRS双组分...的表达、纯化及生化活性测定_李竺婷.pdf

李竺婷 等/具核梭杆菌 CarRS 双组分系统组氨酸激酶 CarS 的表达、纯化及生化活性测定 Chinese Journal of Biotechnology http:/ Apr.25,2023,39(4):1596-1608 DOI:10.13345/j.cjb.220779 2023 Chin J Biotech,All rights reserved 资助项目：辽宁省自然科学基金民族创新联合基金(2020-MZLH-07)This work was supported by the Innovation Joint Fund of Natural Science Foundation of Liaoning Province(2020-MZLH-07).*Corresponding authors.E-mail:QUAN Chunshan,;ZHENG Wei, Received:2022-09-28;Accepted:2023-01-07 1596 生物工程学报 具核梭杆菌CarRS双组分系统组氨酸激酶CarS的表达、纯化及生化活性测定 李竺婷1,2，师舷1,2，范若辰3，王路路3，卜婷婷1,2，郑维1,2*，张旭强1,2，权春善1,2*1 大连民族大学 生物技术与资源利用教育部重点实验室，辽宁 大连 116600 2 大连民族大学生命科学学院，辽宁 大连 116600 3 大连理工大学生物工程学院，辽宁 大连 116081 李竺婷,师舷,范若辰,王路路,卜婷婷,郑维,张旭强,权春善.具核梭杆菌 CarRS 双组分系统组氨酸激酶 CarS 的表达、纯化及生化活性测定J.生物工程学报,2023,39(4):1596-1608.LI Zhuting,SHI Xian,FAN Ruochen,WANG Lulu,BU Tingting,ZHENG Wei,ZHANG Xuqiang,QUAN Chunshan.Expression,purification,and characterization of the histidine kinase CarS from Fusobacterium nucleatumJ.Chinese Journal of Biotechnology,2023,39(4):1596-1608.摘 要：具核梭杆菌(Fusobacterium nucleatum)是一种条件致病菌，能够在结直肠癌组织中富集，影响结直肠癌发生发展的多个阶段。双组分系统在病原菌耐药性、致病性相关基因的调控和表达中起重要作用。本文以具核梭杆菌 CarRS 双组分系统为研究对象，重点对其组氨酸激酶蛋白CarS 进行重组表达和性质研究。利用在线软件 SMART、CCTOP 和 AlphaFold2 对 CarS 二级结构和三级结构进行预测，其结果表明 CarS 蛋白具有 2 个跨膜螺旋区，包含 9 个 螺旋和 12 个 折叠结构；由两个结构域构成，一是位于 N 末端的跨膜域(氨基酸 1170)，另一个是位于 C 末端的胞内域，胞内域由信号接收域(histidine kinases,adenylyl cyclases,methyl-accepting proteins,prokaryotic signaling proteins,HAMP)、磷酸受体结构域(histidine kinase domain,HisKA)和组氨酸激酶催化结构域(histidine kinase-like ATPase catalytic domain,HATPase_c)组成。由于全长的 CarS 蛋白未能在宿主细胞中表达，因此根据其二级、三级结构特点，构建了 CarS 胞内蛋白的融合表达载体 pET-28a(+)-MBP-TEV-CarScyto，并在大肠杆菌(Escherichia coli)BL21-CodonPlus(DE3)-RIL 中进行过表达。经亲和层析、离子交换层析和凝胶过滤层析，最终获得纯度较高的 CarScyto-MBP蛋白，终浓度达 20 mg/mL。活性实验结果表明，CarScyto-MBP具有蛋白激酶和磷酸转移酶双活性，麦芽糖结合蛋白(maltose binding protein,MBP)标签对 CarScyto蛋白的生物活性无影响。上述结果为深入解析 CarRS 双组分系统在具核梭杆菌中的生物学功能提供了一定的理论基础。关键词：具核梭杆菌；CarRS 双组分系统；蛋白激酶活性；磷酸转移酶活性 医药生物技术 李竺婷 等/具核梭杆菌 CarRS 双组分系统组氨酸激酶 CarS 的表达、纯化及生化活性测定 ：010-64807509： 1597 Expression,purification,and characterization of the histidine kinase CarS from Fusobacterium nucleatum LI Zhuting1,2,SHI Xian1,2,FAN Ruochen3,WANG Lulu3,BU Tingting1,2,ZHENG Wei1,2*,ZHANG Xuqiang1,2,QUAN Chunshan1,2*1 Key Laboratory of Biotechnology and Bioresources Utilization,Ministry of Education,Dalian Minzu University,Dalian 116600,Liaoning,China 2 School of Life Sciences,Dalian Minzu University,Dalian 116600,Liaoning,China 3 School of Bioengineering,Dalian University of Technology,Dalian 116081,Liaoning,China Abstract:Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues,affecting multiple stages of colorectal cancer development.The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity.In this paper,we focused on the CarRS two-component system of F.nucleatum,and the histidine kinase protein CarS was recombinantly expressed and characterized.Several online software such as SMART,CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein.The results showed that CarS is a membrane protein with two transmembrane helices and contains 9-helices and 12-folds.CarS protein is composed of two domains,one is the N-terminal transmembrane domain(amino acids 1170),the other is the C-terminal intracellular domain.The latter is composed of a signal receiving domain(histidine kinases,adenylyl cyclases,methyl-accepting proteins,prokaryotic signaling proteins,HAMP),a phosphate receptor domain(histidine kinase domain,HisKA),and a histidine kinase catalytic domain(histidine kinase-like ATPase catalytic domain,HATPase_c).Since the full-length CarS protein could not be expressed in host cells,a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures,and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL.CarScyto-MBP protein was purified by affinity chromatography,ion-exchange chromatography,and gel filtration chromatography with a final concentration of 20 mg/ml.CarScyto-MBP protein showed both protein kinase and phosphotransferase activities,and the MBP tag had no effect on the function of CarScyto protein.The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F.nucleatum.Keywords:Fusobacterium nucleatum;CarRS two-component system;kinase activity;phosphotransferase activity 致病菌在进化过程中，逐渐形成了一系列信号感应和调节网络，可以快速识别人体生态环境的变化，调控病原菌的入侵和定殖策略，突破人体的多重免疫屏障。因此，对致病菌信号通路的研究显得尤为重要。随着基因组学、蛋白质组学等多种组学技术的快速发展和对基因组序列的深入挖掘，近年在结直肠癌组织中发现了一种“肿 瘤 细 菌”具 核 梭 杆 菌(Fusobacterium nucleatum)1。具核梭杆菌最早发现于口腔中，但最近的大量研究表明具核梭杆菌是结直肠癌 ISSN 1000-3061 CN 11-1998/Q 生物工程学报 Chin J Biotech http:/ 1598 发生和发展的驱动因素或致病因子。具核梭杆菌外膜上的黏附素能够促进肿瘤细胞的增殖与转移，协助肿瘤细胞抵抗抗生素，保护具核梭杆菌及附近的肿瘤细胞不被免疫细胞杀死2-3。具核梭杆菌可在结直肠癌组织中大量富集，直接影响结直肠癌进展的多个阶段。双组分系统(two-component system,TCS)存在于大多数病原细菌中，是细菌细胞非常重要的感应外界环境刺激的调控机制，被喻为细菌的“神经系统”。TCS 由组氨酸激酶(histidine kina