YlyA和RNA聚合酶的反应性鉴定

Identification of Interaction between YlyA and RNA Polymerase

目的:用亲和层析法鉴定YlyA与RNA聚合酶(RNAP)的结合性能。方法:将YlyA分别上样于以Affigel15为亲和介质制备的空白柱、牛血清白蛋白(BSA)柱和RNAP柱;以GreA和绿色荧光蛋白(GFP)为阳性和阴性对照蛋白分别上样于同一RNAP柱,洗涤和洗脱缓冲液(pH均为7.9)的盐离子浓度分别为30 mmol/L和400 mmol/L;用免疫印迹法对洗涤和洗脱流出液中的YlyA进行检测。结果:在空白柱和BSA柱的洗脱收集液中,没有检测到YlyA,大量的YlyA出现在了洗涤收集液中;而在RNAP柱的洗脱收集液中,检测到了YlyA和GreA,没有检测到GFP。结论:YlyA与RNAP之间具有特异性结合能力,为YlyA极有可能是一种转录因子的生物信息学分析结果提供了实验证据。

Objective. Using affinity chromatography assay to identify the binding ability of YlyA to RNA polymerase （RNAP）. Methods： YlyA was applied separately to blank column, bovine serum albumin（ BSA ） column and RNAP column filled by affinity matrix-Affi-gel 15. The positive （GreA） and negative control protein（ Green fluorescence protein, GFP） was applied to the same RNAP column. The salt concentration of washing buffer （ pH 7.9 ） and elution buffer（ pH 7.9） was 30 mM and 400 mM respectively, YlyA in the washing fraction and elution fraction was analyzed by western blotting. Results：While applied to blank column and BSA column, YlyA wasnI detected in the elution fraction, but detected in great amount in the washing fraction. YIyA and GreA, but not GFP, were detected in the elution fraction while they were applied to the RNAP column. Conclusion：YlyA can bind to RNAP specifically, which approved the bioinformatics prediction that YlyA is a putative transcriptional factor.