Reversible and oriented immobilization of histidine-tagged protein on silica gel characterized by frontal analysis
Reversible and oriented immobilization of histidine-tagged protein on silica gel characterized by frontal analysis
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DOI：
10.1039/c5ra01012h
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发表时间：
2015-01-01
期刊：
影响因子：
3.9
通讯作者：
Zheng, Xiaohui
中科院分区：
文献类型：
--
作者：
Gao, Xiaokang;Li, Yunzhe;Zheng, Xiaohui
该方法利用N,N'-双（羧甲基）-L-赖氨酸（ANTA）与二价金属阳离子Ni2+配位，为基因重组组氨酸标记的β（2）-肾上腺素受体可逆结合到大孔二氧化硅上留下自由配位位点。通过原子吸收分光光度法测定载体上的瞬态金属镍离子的量。采用正面分析，通过五种 β(2)-肾上腺素受体激动剂对新型蛋白质导向固定 β(2)-AR 柱进行了评估。柱上配体的缔合平衡常数为：沙丁胺醇为 1.98 x 10(4) M-1，克仑特罗为 3.43 x 10(4) M-1，妥洛特罗为 2.09 x 10(4) M-1，特布他林为 1.84 x 10(4) M-1，特布他林为 1.71 x 10(4) M-1甲氧苯那敏和结合位点相应浓度分别为7.46×10(-6)M、1.82×10(-5)M、2.16×10(-5)M、8.29×10(-6)M和3.88×10(-5)M。突破和非线性拟合的结果表明，所有药物在β(2)-肾上腺素受体柱上都具有单一结合位点。本发明的组合组氨酸标记蛋白方法在揭示受体与药物之间的相互作用方面可靠且准确。
This approach utilized N,N'-bis(carboxymethyl)-L-lysine (ANTA) coordinated to bivalent metal cation Ni2+, leaving free coordination sites for the reversible binding of gene recombinant histidine-tagged beta(2)-adrenoceptor onto macropore silica. The amount of transient metal nickel ion on the support was determined by atomic absorption spectrophotometry. The novel protein oriented immobilization beta(2)-AR column was evaluated by five beta(2)-adrenoceptor agonists, applying frontal analysis. The association equilibrium constant for ligands on the column was 1.98 x 10(4) M-1 for salbutamol, 3.43 x 10(4) M-1 for clenbuterol, 2.09 x 10(4) M-1 for tulobuterol, 1.84 x 10(4) M-1 for terbutaline, 1.71 x 10(4) M-1 for methoxyphenamine and corresponding concentrations at binding sites were 7.46 x 10(-6) M, 1.82 x 10(-5) M, 2.16 x 10(-5) M, 8.29 x 10(-6) M and 3.88 x 10(-5) M, respectively. The results obtained from breakthrough and nonlinear fitting indicated that all the drugs have a single binding site on the beta(2)-adrenoceptor column. The present combined histidine-tagged protein method was reliable and exact in revealing interactions between receptor and drugs.