绿脓杆菌RNA降解蛋白质PNPase和肠沙门氏菌鞭毛蛋白质fliD的初步晶体学研究
发布时间：2019-01-06 17:41
【摘要】：背景RNA降解复合体是RNA在生物体内更新的主要工具。PNPase通过的3′到5′核酸外切酶降解RNA。RraA是RNA降解复合体降解RNA过程中的重要抑制因子。鞭毛是细菌的重要成分也是致病因子之一,鞭毛的组装是一个多蛋白质参与的复杂的过程。前期研究中成功解析了鞭毛FlgD蛋白质结构和纯化了RNase E抑制因子。通过对PNPase和RraA结构研究更能揭示RNA降解及其调节过程,通过对FliD结构研究进一步了解鞭毛的组装机制。 目的本研究将对蛋白质PNPase、RraA和FliD纯化,得到这三个蛋白质的蛋白质晶体,收集X-光衍射数据并解析他们的三维结构。 方法运用相关数据库对目的蛋白质进行生物信息学的分析,通过分子克隆技术,克隆、表达、纯化大量的纯度大于95%的绿脓杆菌RNA降解酶PNPase蛋白质、绿脓杆菌RNase E抑制因子RraA蛋白质和FliD蛋白质。用凝胶层析法检测目的蛋白质在溶液中的聚集状态。用晶体筛选试剂盒对目的蛋白质筛选蛋白质晶体,并对初步得到的晶体进行优化,以获得高质量的晶体。用X衍射仪收集到得到蛋白质晶体的衍射数据。通过Pull-down方法验证了PNPase和RNase E的相互作用,并纯化出该复合物用于晶体筛选。 结果通过分子克隆的技术,成功克隆、表达、纯化了绿脓杆菌PNPase、RNase E和RraA蛋白质,和肠沙门氏菌鞭毛帽FliD蛋白质。PNPase、RraA和FliD过分子筛Superdex 200中显示在溶液中它们分别主要以六聚体、六聚体和多种聚集状态存在。通过Pull-down技术成功获得大量的PNPase-RNase E复合物。通过筛选和优化得到了晶体质量比较好的晶体,并且成功收取PNPase和RraA蛋白质晶体的X-衍射数据,而FliD由于晶体分辨率低未能收到完整衍射数据。成功解析了RraA蛋白质的三维结构,PNPase结构正在修正中。
[Abstract]:Background the RNA degradation complex is the main tool for the renewal of RNA in vivo. The 3'to 5'nucleic acid exonuclease degradation of RNA.RraA by PNPase is an important inhibitory factor in the degradation of RNA by RNA degradation complex. Flagella is an important component of bacteria and one of the pathogenic factors. The assembly of flagella is a complex process involving many proteins. The structure of flagellum FlgD protein and the purification of RNase E inhibitory factor were successfully analyzed in previous studies. The structure of PNPase and RraA can better reveal the degradation and regulation of RNA, and the mechanism of flagellum assembly can be further understood by studying the structure of FliD. Aim in this study, protein PNPase,RraA and FliD were purified, protein crystals of these three proteins were obtained, X-ray diffraction data were collected and their three-dimensional structures were analyzed. Methods the target protein was analyzed by bioinformatics using relevant database. A large number of PNPase proteins of Pseudomonas aeruginosa RNA degrading enzyme were purified by molecular cloning, expression and purification. Pseudomonas aeruginosa RNase E inhibitor RraA protein and FliD protein. The aggregation state of the target protein in solution was detected by gel chromatography. The protein crystals were screened by crystal screening kit and the primary crystals were optimized to obtain high quality crystals. The diffraction data of protein crystals were collected by X-diffractometer. The interaction between PNPase and RNase E was verified by Pull-down method, and the complex was purified for crystal screening. Results PNPase,RNase E and RraA proteins of Pseudomonas aeruginosa and FliD protein of flagella of Salmonella entericus were successfully cloned, expressed and purified by molecular cloning technique. PNPase, RraA and FliD supermolecular sieve Superdex 200 show that they mainly exist in the solution as hexamer, hexamer and a variety of aggregation states, respectively. A large number of PNPase-RNase E complexes were successfully obtained by Pull-down technique. The better crystal quality was obtained by screening and optimizing, and the X- diffraction data of PNPase and RraA protein crystals were successfully collected, while the complete diffraction data could not be obtained by FliD because of its low crystal resolution. The three dimensional structure of RraA protein was successfully analyzed and the PNPase structure was being modified.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
本文编号：2403114
[Abstract]:Background the RNA degradation complex is the main tool for the renewal of RNA in vivo. The 3'to 5'nucleic acid exonuclease degradation of RNA.RraA by PNPase is an important inhibitory factor in the degradation of RNA by RNA degradation complex. Flagella is an important component of bacteria and one of the pathogenic factors. The assembly of flagella is a complex process involving many proteins. The structure of flagellum FlgD protein and the purification of RNase E inhibitory factor were successfully analyzed in previous studies. The structure of PNPase and RraA can better reveal the degradation and regulation of RNA, and the mechanism of flagellum assembly can be further understood by studying the structure of FliD. Aim in this study, protein PNPase,RraA and FliD were purified, protein crystals of these three proteins were obtained, X-ray diffraction data were collected and their three-dimensional structures were analyzed. Methods the target protein was analyzed by bioinformatics using relevant database. A large number of PNPase proteins of Pseudomonas aeruginosa RNA degrading enzyme were purified by molecular cloning, expression and purification. Pseudomonas aeruginosa RNase E inhibitor RraA protein and FliD protein. The aggregation state of the target protein in solution was detected by gel chromatography. The protein crystals were screened by crystal screening kit and the primary crystals were optimized to obtain high quality crystals. The diffraction data of protein crystals were collected by X-diffractometer. The interaction between PNPase and RNase E was verified by Pull-down method, and the complex was purified for crystal screening. Results PNPase,RNase E and RraA proteins of Pseudomonas aeruginosa and FliD protein of flagella of Salmonella entericus were successfully cloned, expressed and purified by molecular cloning technique. PNPase, RraA and FliD supermolecular sieve Superdex 200 show that they mainly exist in the solution as hexamer, hexamer and a variety of aggregation states, respectively. A large number of PNPase-RNase E complexes were successfully obtained by Pull-down technique. The better crystal quality was obtained by screening and optimizing, and the X- diffraction data of PNPase and RraA protein crystals were successfully collected, while the complete diffraction data could not be obtained by FliD because of its low crystal resolution. The three dimensional structure of RraA protein was successfully analyzed and the PNPase structure was being modified.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 林亚静;刘志杰;龚为民;;蛋白质结构研究[J];生命科学;2007年03期
，本文编号：2403114
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