Zhao Xinfeng, Li Qian, Xiao Chaoni, Zhang Yajun, Bian Liujiao, Zheng Jianbin, Zheng Xiaohui, Li Zijian, Zhang Youyi, Fan Taiping
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, 195#, No. 229, Taibai North Road, Xi'an, 710069, China.
Anal Bioanal Chem. 2014 May;406(12):2975-85. doi: 10.1007/s00216-014-7723-x. Epub 2014 Mar 15.
A method based on reaction with a diazonium salt was developed to immobilise oriented His-tagged protein onto silica gel. The binding efficiency of the phenylamine-group-coated gel was determined to be 65 %, providing a binding capacity of His-tagged protein up to the gram level. Using His-tagged β2-adrenoceptor (β2-AR) as a probe, we developed a new mathematical model to elucidate the interactions between the receptor and five ligands (methoxyphenamine, terbutaline, salbutamol, tulobuterol and fenoterol). These drugs proved to only have one type of binding site on the immobilised β2-AR, yielding higher association constants and numbers of binding sites than random attachment assays. The association constants determined by the new model positively correlated to the values from a radioligand binding method, with a regression equation of y = 1.75x - 7.18 and a correlation coefficient of 0.9807. The oriented method resulted in a high binding capacity and quantitative immobilisation of the His-tagged protein. The proposed model can be used to determine the interactions between the ligands and the immobilised protein with the advantages of drug and time saving.
一种基于与重氮盐反应的方法被开发出来，用于将定向的His标签蛋白固定到硅胶上。经测定，苯胺基团包被的凝胶的结合效率为65%，His标签蛋白的结合容量可达克级。以His标签的β2肾上腺素能受体（β2-AR）为探针，我们开发了一种新的数学模型来阐明该受体与五种配体（甲氧明、特布他林、沙丁胺醇、妥洛特罗和非诺特罗）之间的相互作用。这些药物在固定化的β2-AR上仅具有一种结合位点，与随机连接测定相比，产生了更高的缔合常数和结合位点数。通过新模型测定的缔合常数与放射性配体结合法得到的值呈正相关，回归方程为y = 1.75x - 7.18，相关系数为0.9807。定向方法导致His标签蛋白具有高结合容量和定量固定化。所提出的模型可用于确定配体与固定化蛋白之间的相互作用，具有节省药物和时间的优点。
