摘要
目的采用Biacore T200分析系统建立高通量检测蛋白与组蛋白多肽相互作用的表面等离子共振(SPR)方法。方法将生物素标记的牛血清白蛋白BSA和YEATS2蛋白分别固定在SA芯片的参比和样品通道上,将不同修饰的组蛋白多肽H3K27cr(巴豆酰化)、H3K27ac(乙酰化)和H3K27(未修饰)稀释成3.9,7.8,15.6,31.2,62.4,125,250μmol/L,依次注入芯片表面,所得数据经Biacore T200分析软件处理,计算YEATS2蛋白与多肽相互作用的亲和力。结果参比通道固定BSA有效地降低了H3K27cr多肽与芯片基质的非特异性吸附。BSA与H3K27cr无特异性结合。YEATS2与组蛋白多肽的亲和力为H3K27cr>H3K27ac>H3K27,与等温滴定微量热(ITC)技术所得结果相吻合。结论优化的SPR方法适用于高通量筛选与特定蛋白相互作用的组蛋白多肽,并可进行亲和力的检测。
Objective To establish a surface plasmon resonance(SPR)method for high throughput measurement of protein-peptide interaction by Biacore T200 unit.Methods Biotinylated BSA and YEATS2 were respectively immobilized on Fc1 and Fc2 channels of a SA chip.H3K27cr,H3K27ac and H3K27 peptides were diluted at 3.9,7.8,15.6,31.2,62.4,125 and 250μmol/L,and sequentially flowed over the sensor surface.Data were analyzed using Biacore T200 evaluation software.Results Non-specific interaction between H3K27cr peptide and matrix on the chip was reduced by immobilized BSA.BSA did not interact with H3K27cr peptide.YEATS2 bound to H3K27 peptides in a preference order of H3K27cr>H3K27ac>H3K27,which was in accordance with that obtained by ITC method.Conclusion Optimized SPR method can be utilized for high throughput screening of histone peptides binding to specific proteins and measurement of binding affinity.
作者
杨颖
胡家
李蕾
殷爱红
李慎涛
YANG Ying;HU Jia;LI Lei;YIN Aihong;LI Shentao(Proteomics Research Platform,Core Facilities Center,Capital Medical University,Beijing 100069,China)
出处
《山西医科大学学报》
CAS
2019年第12期1760-1763,共4页
Journal of Shanxi Medical University
基金
北京市自然科学基金资助项目(7182016)
