綠膿桿菌RNA降解蛋白質(zhì)PNPase和腸沙門氏菌鞭毛蛋白質(zhì)fliD的初步晶體學(xué)研究
發(fā)布時間：2019-01-06 17:41
【摘要】：背景RNA降解復(fù)合體是RNA在生物體內(nèi)更新的主要工具。PNPase通過的3′到5′核酸外切酶降解RNA。RraA是RNA降解復(fù)合體降解RNA過程中的重要抑制因子。鞭毛是細(xì)菌的重要成分也是致病因子之一,鞭毛的組裝是一個多蛋白質(zhì)參與的復(fù)雜的過程。前期研究中成功解析了鞭毛FlgD蛋白質(zhì)結(jié)構(gòu)和純化了RNase E抑制因子。通過對PNPase和RraA結(jié)構(gòu)研究更能揭示RNA降解及其調(diào)節(jié)過程,通過對FliD結(jié)構(gòu)研究進一步了解鞭毛的組裝機制。 目的本研究將對蛋白質(zhì)PNPase、RraA和FliD純化,得到這三個蛋白質(zhì)的蛋白質(zhì)晶體,收集X-光衍射數(shù)據(jù)并解析他們的三維結(jié)構(gòu)。 方法運用相關(guān)數(shù)據(jù)庫對目的蛋白質(zhì)進行生物信息學(xué)的分析,通過分子克隆技術(shù),克隆、表達、純化大量的純度大于95%的綠膿桿菌RNA降解酶PNPase蛋白質(zhì)、綠膿桿菌RNase E抑制因子RraA蛋白質(zhì)和FliD蛋白質(zhì)。用凝膠層析法檢測目的蛋白質(zhì)在溶液中的聚集狀態(tài)。用晶體篩選試劑盒對目的蛋白質(zhì)篩選蛋白質(zhì)晶體,并對初步得到的晶體進行優(yōu)化,以獲得高質(zhì)量的晶體。用X衍射儀收集到得到蛋白質(zhì)晶體的衍射數(shù)據(jù)。通過Pull-down方法驗證了PNPase和RNase E的相互作用,并純化出該復(fù)合物用于晶體篩選。 結(jié)果通過分子克隆的技術(shù),成功克隆、表達、純化了綠膿桿菌PNPase、RNase E和RraA蛋白質(zhì),和腸沙門氏菌鞭毛帽FliD蛋白質(zhì)。PNPase、RraA和FliD過分子篩Superdex 200中顯示在溶液中它們分別主要以六聚體、六聚體和多種聚集狀態(tài)存在。通過Pull-down技術(shù)成功獲得大量的PNPase-RNase E復(fù)合物。通過篩選和優(yōu)化得到了晶體質(zhì)量比較好的晶體,并且成功收取PNPase和RraA蛋白質(zhì)晶體的X-衍射數(shù)據(jù),而FliD由于晶體分辨率低未能收到完整衍射數(shù)據(jù)。成功解析了RraA蛋白質(zhì)的三維結(jié)構(gòu),PNPase結(jié)構(gòu)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346
【參考文獻】
相關(guān)期刊論文 前1條
1 林亞靜;劉志杰;龔為民;;蛋白質(zhì)結(jié)構(gòu)研究[J];生命科學(xué);2007年03期
，本文編號：2403114
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