铜绿假单胞菌寡核苷酸酶的纯化、结晶和活性验证

Purification, crystallization and activity validation of oligoribonuclease from Pseudomonas aeruginosa

目的:研究致病菌铜绿假单胞菌寡核苷酸酶(PaOrn)的三维结构和体外水解活性,验证PaOrn活性位点并探讨PaOrn对铜绿假单胞菌(P.aeruginosa)胞内3',5'-环二鸟苷(c-di-GMP)水平和致病毒性的影响。方法:构建重组质粒pET 32M3C-PaOrn,在大肠杆菌(E.coli)BL21中诱导表达目的蛋白,获得组氨酸(His)标签和谷胱甘肽(GST)标签标记。采用多级纯化方法纯化蛋白,经Ni亲和层析、GST亲和层析和阴离子交换层析后获得高纯度和高均一性PaOrn目的蛋白。采用商业化结晶试剂和坐滴法筛选晶体,根据晶体状态进一步优化。采用X射线衍射法验证晶体质量,分辨率高于3Å时用于结构解析。采用高分辨质谱技术Q TOF验证PaOrn蛋白水解底物二鸟苷磷酸(pGpG)活性,鸟苷单磷酸(GMP)产物作为验证活性的指标。采用尿素聚丙烯酰胺(Urea-PAGE)法验证PaOrn蛋白和点突变PaOrn蛋白(Orn^(D11A)、Orn^(E13A)、Orn^(D111A)、Orn^(H157A)和Orn^(D162A))对10 nt长度寡核苷酸RNA的水解活性,降解条带作为活性正常的分析指标。结果:经原核系统表达和多级纯化,得到纯度高达95%的目的蛋白PaOrn。于结晶试剂WizardⅠ#12[1.0 mmol·L^(-1)(NH4)2HPO4,0.1 mmol·L^(-1) Imidazole]pH 8.0条件下生长出质量较好的单晶,晶体PaOrn的分辨率为1.85Å,复合物PaOrn-GMP和PaOrnD11A-pGpG的分辨率分别为1.90Å和1.70Å。高分辨质谱,PaOrn将底物pGpG水解为GMP。尿素聚丙烯酰胺凝胶电泳,PaOrn可降解长达10 nt的RNA,当活性位点Asp11、Glu13、Asp111、His157和Asp162分别突变后,PaOrn丧失活性,无法降解底物pGpG和RNA(10 nt)。结论:PaOrn通过降解寡核苷酸参与调控胞内c-di-GMP和RNA水平,影响转录起始和细胞多种生物学行为。

Objective:To investigate the three-dimensional structure of oligoribonuclease from Pseudomonas aeruginosa(P. aeruginosa)Orn(PaOrn),and to verify the active sites of PaOrn,and explore the effect of PaOrn on the intracellular Bis-(3’-5’)cyclic diguanylic acid(c-di-GMP)levels and virulence of P. aeruginosa. Methods:A fusion protein with a histidine(His)tag and a glutathione(GST)tag was obtained by constructing the recombinant plasmid pET 32M3C-PaOrn that was expressed by the Escherichia coli(E. coli) BL21. The protein was purified with multistage purifiation method;after Ni affinity chromatography,GST affinity chromatography,and anion exchange chromatography,and finally yielding the PaOrn target protein with high purity and high homogeneity were obtained. The crystals were screened with commercial crystallization reagents and sitting drops,and they were further optimized according to the crystal state. The quality of crystal was verified by X-ray diffraction method,and resolution above 3 ? was used for structure resolution. The activity of PaOrn degradation5’-phosphoguanylyl-(3’,5’)-guanosine(pGpG) was verified by using high resolution mass spectrometry Q TOF,and the GMP product was used as an indicator to verify the activity. The hydrolytic activities of the PaOrn and the mutant PaOrn proteins(Orn^(D11A)、Orn^(E13A)、Orn^(D111A)、Orn^(H157A),and Orn^(D162A))against the10 nt RNA was verified by using Urea-PAGE,and the degradation bands were used as analytical indicators for the normal activity. Results:The target protein PaOrn was obtained with purity up to 95% after prokaryotic expression and multistage purification. Good quality single crystals were grown at Wizard Ⅰ # 12[1. 0 mmol·L^(-1)(NH_(40)_(2))HPO_(4),0. 1 mmol·L^(-1)Imidazole]pH 8. 0,the resolution of crystal PaOrn was 1. 85 A,and the resolutions of PaOrn-GMP complex and PaOrnD11A-pGpG complex were1. 90 A and 1. 70A,respectively. The Urea-PAGE results showed that PaOrn hydrolyzed the substrate pGpG to GMP. The urea polyacrylamide gel electrophoresis results revealed that PaOrn could degrade up to 10 nt of RNA,and after mutation of the active sites of PaOrn(Asp11,Glu13,Asp111,His157,and Asp162),the activity of PaOrn was lost,and failed to degrade the substrate pGpG and RNA(10 nt).Conclusion:PaOrn is involved in the regulation of intracellular c-di-GMP and RNA levels through degradation of oligonucleotides,and affects transcription initiation and the multiple cellular biological behaviors.