鼠伤寒沙门氏菌c-di-GMP结合蛋白YcgR的表达、纯化及活性鉴定

Expression, Purification and Characterization of c-di-GMP Binding Protein YcgR from Salmonella typhimurium

摘要:

【背景】在鼠伤寒沙门氏菌中，c-di-GMP结合蛋白YcgR通过与鞭毛运动蛋白的相互作用，抑制鞭毛运动速度，使细菌由浮游生长向生物膜产生及侵染宿主的静止状态转变。然而，目前关于该蛋白的结构、功能以及与c-di-GMP结合后调控细菌运动状态的分子机制还不清楚。【目的】通过原核表达，获得高纯度的YcgR蛋白，并对其二级结构稳定性以及c-di-GMP结合活性进行表征，为进一步确定YcgR的结构以及阐明c-di-GMP如何通过结合YcgR而减缓细菌运动速度的分子机制奠定基础。【方法】通过构建重组大肠肝菌对YcgR进行原核表达；通过Ni-NTA镍离子亲和层析柱和体积排阻色谱两步纯化获得高纯度的YcgR蛋白；预测YcgR蛋白质结构，利用圆二色光谱仪(CD)测定其二级结构；通过等温滴定量热法(ITC)和热转移技术(Protein Thermal Shift)研究YcgR与c-di-GMP的结合活性。【结果】菌落PCR和质粒测序结果表明，表达YcgR的重组大肠杆菌构建成功；SDS-PAGE和体积排阻色谱显示YcgR的分子量大约为28 kD，在溶液中以二聚体的形式存在；通过预测和对比，发现YcgR的结构与同源蛋白PP4396高度相似，都有一个响应c-di-GMP的PilZ结构域，且CD结果显示YcgR具有稳定的二级结构；ITC和Thermal Shift结果表明，YcgR与c-di-GMP有较强的结合活性，其Kd值为50 nmol/L。【结论】首次实现了鼠伤寒沙门氏菌YcgR蛋白的原核表达及纯化，表征了其c-di-GMP结合活性，为进一步研究YcgR蛋白与细菌信号分子c-di-GMP调控鞭毛运动的分子机制奠定基础，并为鼠伤寒沙门氏菌抗感染药物的研发和治疗提供新的策略。

Abstract:

[Background] In the Salmonella typhimurium, the c-di-GMP binding protein YcgR could inhibit flagellar motor speed by interacting with flagellar protein, which cause the bacteria stop migrate, generate biofilm and infect the host. However, the structure and functions of YcgR as well as the molecular mechanism of YcgR suppressing bacterial motion after binding to c-di-GMP are not clear. [Objective] In this study, YcgR protein was expressed and purified in recombinant E. coli, and then the c-di-GMP binding activity of YcgR were further characterized, which could lay a foundation for determining the structure of YcgR and clarifying how c-di-GMP binding protein YcgR slowing the movement of Salmonella typhimurium. [Methods] The recombinant E. coli was constructed for expressing YcgR protein, which was further purified by Ni-NTA and SEC (Size exclusion chromatography). After that, we predicted the structure of YcgR protein by comparing with other homologous family proteins, and the secondary structure of YcgR was determined by circular dichroism (CD). Finally, the binding parameters of YcgR and c-di-GMP were determined by isothermal titration calorimetry (ITC) and Protein Thermal Shift. [Results] Colony PCR and DNA sequencing results showed that the recombinant E. coli for expressing YcgR was successfully constructed. The size exclusion chromatography and polyacrylamide gel electrophoresis showed that the molecular weight of YcgR was 28 kD and existed as dimer in the supernatant. The structure of YcgR is highly similar to the homologous protein PP4396, which has a PilZ domain for binding c-di-GMP, and CD results showed that YcgR had a stable secondary structure. In addition, ITC and Thermal Shift indicated that YcgR had a strong and specific binding activity with c-di-GMP, and the Kd value was 50 nmol/L. [Conclusion] The YcgR protein from Salmonella typhimurium was successfully expressed in E. coli for the first time, and its c-di-GMP binding activity were also characterized in this study, which is very important for the study of the molecular mechanism of c-di-GMP binding protein YcgR regulating flagellin, and provide a new strategy for bacterial anti-infective therapy.