利用表面活性剂介导的方法制备AgrC蛋白脂质体

Construction of AgrC Proteoliposomes by Detergent-Mediated Method

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.

To respond appropriately to different environmental changes, bacteria have evolved two-component signal transduction systems （TCSTs）, which are absent in mammals （including human beings）. A typical TCST consisted of a sensor kinase （histidine kinase, HK） and a response regulator （RR）. HK is capable of autophosphorylation in response to an environmental signal, while RR interacts with the phosphorylated HK. Membrane protein AgrC is a sensor kinase of a TCST from Staphylococcus aureus. Illumination of signal transduction mechanism is of great significance to solve the problem of bacte- rial resistance. At present, the main bottleneck of membrane protein is the difficulties in obtaining large quantities of suffi- ciently pure and functional protein. The target protein was overexpressed in Escherichia coli, solubilized from cell membranes and purified in detergent micelles. This series of steps tend to lead to destabilizations of membrane protein and loss of function. In this study, AgrC was incorporated into liposomes by a detergent-mediated method. For standard incorporation, protein solution was added to detergent-lipid suspension containing lipid at 2.5 mmol/L at a lipid-to-protein ratio of 300 （mol/mol）. For slow detergent removal, successive additions of small amounts of beads at Bio-bead-to-detergent ratios of 2 （w/w） will allow the removal of the detergent, resulting in formation ofproteoliposome. The structure, morphology and average diameter of liposomes and proteoliposomes were characterized by transmission electron microscopy （TEM） and dynamic light scattering （DLS）, respectively. Sucrose density gradient centrifugation was employed to separate proteoliposomes. The result showed efficiency of protein incorporation and liposomes recovery reached 80% and 90%, respectively. Thiol reagent labeling test showed the cytoplasmic domain of AgrC was almost exclusively oriented towards the inside of the liposome vesicles. In vitro phosphorylation experiments showed that kinase activity of AgrC in proteoliposomes was significantly higher than in detergent micelles. Proteoliposomes could be stored for two weeks with little loss of function. Preparation of proteoliposome not only solves the instability problem of membrane proteins, but also provides a new approach of the study of membrane protein structure, function and signal transduction mechanism in vitro.