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膜蛋白AgrC是金黄色葡萄球菌双组分信号转导系统的感受激酶.其信号转导机制的阐明对于解决细菌耐药问题具有重要意义.目前膜蛋白研究的主要瓶颈是难以获得大量高纯度和功能稳定的蛋白.将目标蛋白表达在大肠杆菌体内,利用表面活性剂将其从细胞膜上溶解,纯化,这一系列步骤容易引起膜蛋白不稳定和功能损失.本文报道了用表面活性剂介导的方法将膜蛋白AgrC镶嵌到脂质体上,即形成蛋白脂质体.脂质体和蛋白脂质体的结构、形貌以及平均粒径分别用透射电镜和动态光散射仪表征.蔗糖密度梯度离心的结果表明蛋白重构效率达80%.硫醇试剂标记实验确定AgrC的细胞质域在脂质体中取向于内侧.体外磷酸化实验表明AgrC蛋白在脂质体中的自我磷酸化活性远远高于表面活性剂中的活性,且其自我磷酸化活性在2周内几乎没有损失.蛋白脂质体的构建不仅解决了膜蛋白的不稳定性问题,也为体外研究AgrC蛋白的结构、功能和信号转导机制提供了新的思路.
Membrane protein AgrC is a sensitive kinase of Staphylococcus aureus two-component signal transduction system.The elucidation of its signal transduction mechanism is of great significance to solve the problem of bacterial resistance.At present, the main bottleneck of membrane protein research is the difficulty of obtaining a large number of high purity and Stable protein.The target protein is expressed in E. coli, the use of surfactant to dissolve and purify the cell membrane, a series of steps easily lead to membrane protein instability and loss of function.This paper reports the use of surfactant-mediated The AgrC membrane was inlaid into liposomes to form protein liposomes.The structure, morphology and average particle size of liposomes and liposomes were characterized by transmission electron microscopy and dynamic light scattering respectively.The sucrose density The results of gradient centrifugation showed that the efficiency of protein reconstitution was up to 80%. The thiol reagent labeling assay confirmed that the cytoplasmic domain of AgrC was oriented in the liposomes in vitro. In vitro phosphorylation assay showed that the autophosphorylation activity of AgrC protein in liposomes was far Far higher than the activity in the surfactant and almost no loss of autophosphorylation activity within 2 weeks.The construction of proteoliposome not only solved the membrane protein The problem of instability also provides a new idea for the study of the structure, function and signal transduction mechanism of AgrC protein in vitro.