金黄色葡萄球菌Hfq蛋白体外聚体生成和生物学功能的研究

Formation and function study of Hfq polymer of Staphylococcus aureus in vitro

目的:研究金黄色葡萄球菌中Hfq蛋白的聚体形式以及聚体和单体的不同生物学功能。方法:利用pET28-(a)载体克隆表达金葡菌来源的Hfq蛋白,通过天然凝胶电泳和SDS-PAGE检测不同条件下Hfq蛋白的存在形式。通过点突变的方法获得56和63位突变的Hfq蛋白,利用凝胶阻滞试验检测单体和聚体的RNA结合活性。结果:Hfq蛋白以单体、二聚体、四聚体以及六聚体的形式存在,各种聚体比例不同,长时间的高温、强酸和强碱会影响聚体的稳定性。在常规SDS-PAGE条件下,重组蛋白仅以四聚体形式存在。突变体蛋白不能够形成聚体且只有形成聚体的Hfq蛋白才能特异结合RNA。结论:Hfq蛋白形成聚体的过程是二聚体四聚体六聚体,且不同聚体形成的机制不同。56位和63位酪氨酸是Hfq蛋白聚体形成的关键氨基酸。聚体结构对Hfq蛋白发挥RNA伴侣分子生物学功能至关重要。

Objective ：To investigate the formation and function of Hfq polymer of Staphylococcus aureus. Methods： The Hfq protein of S. aureus was expressed in E. coli. The formation of Hfq polymer was tested with the native and denatured gel. The tyrosine 56 and tyrosine 63 of Hfq protein were individually replaced by alanine using site mutation method. The gel-shift was used to examine the interaction of Hfq protein and RNA. Results：It was found that there were different forms of Hfq protein including monomer, dimer, tetramer and hexamer. The formation of Hfq polymer could be destroyed after treatment with high temperature, strong base and acid. The mutants of Hfq protein could not form polymer and the monomer of Hfq protein could not bind RNA. Interestingly, we found that the Hfq protein could form tetramer in SDS-PAGE. Conclusion：The results suggested that the mechanism of formation of the different polymers is different. Both tyrosine 56 and tyrosine 63 are the key amino acids for the formation of polymer whose structure is essential to its RNA chaperone function.