title: Oriented immobilisation of histidine-tagged protein and its application in exploring interactions between ligands and proteins.
abstract: 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.
authors.affiliation: 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.
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Zhao
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.forename: Chaoni
authors.initials: C
authors.lastname: Xiao
authors.forename: Yajun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Liujiao
authors.initials: L
authors.lastname: Bian
authors.forename: Jianbin
authors.initials: J
authors.lastname: Zheng
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zheng
authors.forename: Zijian
authors.initials: Z
authors.lastname: Li
authors.forename: Youyi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Taiping
authors.initials: T
authors.lastname: Fan
mesh_terms: D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000295:instrumentation*; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry*; D058428:Silica Gel / Q000737:chemistry; D013552:Swine
chemical_list: D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D006639:Histidine; D058428:Silica Gel