鼠伤寒沙门菌小分子热休克蛋白AgsA的N与C末端在溶液中暴露比较

Comparison of the exposure of the N-and the C-terminal of Salmonella enterica serovar typhimurium small heat shock protein AgsA in solution

目的探讨鼠伤寒沙门菌小分子热休克蛋白AgsA的N末端在其寡聚体中所处的位置,为临床治疗提供参考依据。方法通过分别在小分子热休克蛋白AgsA的N、C末端添加多聚组氨酸标签(His-tag),检测二者与镍螯合琼脂糖凝胶(Ni-NTA Agarose)的结合能力,比较目标蛋白的N、C末端在溶液中的暴露情况。结果 Histag的添加既不影响AgsA蛋白的寡聚状态,也不影响其类分子伴侣活性;通过比较分别在N、C末端添加Histag的AgsA蛋白与Ni-NTA Agarose的结合能力,发现AgsA蛋白的C末端在溶液中的暴露情况远大于其N末端。结论 AgsA-N-His6与Ni-NTA Agarose的结合能力远远小于AgsA-C-His6,暗示了AgsA蛋白的N末端被包裹在其寡聚体内部。

OBJECTIVE To investigate the location of the N-terminal of the Salmonella enterica serovar typhimurium small heat shock protein AgsA in its oligomers,in order to provide clinical reference for the treatment.METHODS A His-tag was added on the N or the C-terminal of AgsA protein,then the binding capacities of the two recombinant proteins with Ni-NTA agarose were measured to compare how much the N-and the C-terminal of AgsA was exposed in solution.RESULTS The gel exclusion chromatography analysis and chaperone-like activity assay showed that the added His-tag neither affected the oligomeric state of AgsA,nor affected the chaperone-like activity of AgsA.By comparing the binding capacity of the two His-tag added proteins with Ni-NTA agarose,we found that the C-terminal of AgsA protein was far greater exposed in solution than its N-terminal.CONCLUSION AgsA-N-His6 with Ni-NTA agarose binding capacity is far less than AgsA-C-His6,which suggests that the Nterminal of AgsA is buried within its oligomers.