肺炎链球菌血红素结合脂蛋白PiuA的表达、纯化和表征

【背景】肺炎链球菌是社区获得性肺炎最常见的致病菌之一,它也会引起脑膜炎、鼻窦炎、中耳炎、菌血症等一系列疾病,对人类(特别是儿童、老人、免疫缺陷患者)健康造成重大威胁。铁是肺炎链球菌生存和感染所必需的元素之一,其中血红素转运系统PiuABCD是肺炎链球菌最重要的铁转运系统。【目的】克隆、表达和纯化肺炎链球菌血红素转运系统脂蛋白PiuA,并在体外表征PiuA蛋白的血红素结合特性。【方法】将肺炎链球菌D39菌株中的piuA(spd_1652)基因连接到载体pBAD-HisA上,在大肠杆菌Top10菌株中进行异源表达,然后运用Ni-NTA亲和层析纯化PiuA-His蛋白,并用肠激酶切掉His标签获得不含标签的PiuA蛋白,最后运用圆二色谱、紫外光谱和荧光光谱表征PiuA蛋白的血红素结合特性。【结果】构建了pBAD/HisA-PiuA重组表达载体,获得了纯度大于95%的PiuA蛋白,圆二色谱显示PiuA蛋白与Hemin结合后,其二级结构不发生改变;紫外光谱结果显示PiuA蛋白具有血红素结合能力;荧光光谱结果显示apo-PiuA蛋白与Hemin结合常数K=3.4×10~5 L/mol。【结论】肺炎链球菌血红素转运系统脂蛋白PiuA能够特异地结合血红素,为肺炎链球菌的生存和感染提供必需的铁源,PiuA蛋白的体外表征结果为针对PiuABCD血红素转运系统设计抗菌药物奠定了基础。

Expression, purification and characterization of hemin-binding lipoprotein PiuA from Streptococcus pneumoniae

Background] Streptococcus pneumoniae is one of the most common pathogens of community-acquired pneumonia, and causes a variety of other serious diseases including bacterial meningitis, sinusitis, otitis media and bacteremia, and poses health threats to humans, especially among children and the elderly, or individuals with immature/compromised immune systems. Iron is essential for Streptococcal survival and infection, and haem ABC uptake system PiuABCD is the most iron ABC uptake system for S. pneumoniae. Objective] Clone, express and purify hemin-binding lipoprotein PiuA belonging to haem ABC uptake system PiuABCD, and characterize its hemin-binding properties in vitro. Methods] The piuA (spd_1652) gene from S. pneumoniae D39 strain was ligated into pBAD-HisA vector, and expressed in E. coli Top 10 strain, then the PiuA-His fusion protein was purified using Ni-NTA affinity chromatography, and the His tag free PiuA protein was obtained by cutting off the His tag with enterokinase. Finally, the hemin-binding properties of PiuA protein were characterized by circular dichroism, UV spectroscopy and fluorescence spectroscopy. Results] We successfully constructed recombinant vector pBAD/HisA-piuA, and acquired the PiuA protein with the purity higher than 95%. CD spectra showed that hemin binding did not induce any significant structural change in PiuA, UV spectroscopy revealed that PiuA protein can specifically bind hemin, and fluorescence spectroscopy demonstrated that the binding constant K between the PiuA and hemin is 3.4×105 L/mol. Conclusion] Hemin-binding lipoprotein PiuA can specifically bind to hemin and provide the iron for Streptococcal survival and infection, these characterization results of PiuA protein provide basis data for design of antibacterial drug targeted for haem ABC uptake system PiuABCD.