Halo-tagged protein immobilization: Effect of halide linkers on peak profile and drug-protein interaction
Halo-tagged protein immobilization: Effect of halide linkers on peak profile and drug-protein interaction
复制标题
晕环标记蛋白固定：卤化物接头对峰形和药物-蛋白质相互作用的影响
DOI：
10.1016/j.chroma.2021.461946
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发表时间：
2021
影响因子：
4.1
通讯作者：
Zhao Xinfeng
中科院分区：
文献类型：
--
作者：
Fu Xiaoying;Li Linkang;Wen Xin;Xu Ru;Xue Yan;Zuo Haiyue;Liang Qi;Feng Gangjun;Wang Jing;Zhao Xinfeng
在以前的工作中，我们已经建立了一个一步的方法，通过卤素标记和载体表面的卤素连接体之间形成的共价键将卤素标记的蛋白质固定到微球上。我们观察到大多数药物在固定化蛋白质上的峰非常拖尾，这是由于连接子与药物之间的非特异性相互作用。为了证明这一点，本工作设计了五种不同的卤化物连接体用于固定β 2-肾上腺素受体（β2-AR）。我们应用固定化受体，通过分析五种药物的峰形，系统地了解这些卤素连接体对药物-受体相互作用的影响。在6-13个原子范围内，药物的保留时间和半宽似乎与连接基团的原子数呈负相关。随后增加连接原子导致药物的保留时间缩短和峰变宽。应用相同的连接体长度，我们观察到五种药物的保留时间和半宽比不存在氧原子的连接体明显减少。这种改善主要是由氧原子的数量决定的。这些表明接头S-4（2-（2-（2-（2-氯乙氧基）乙氧基）乙氧基）乙酸）对于消除不需要的非特异性相互作用是最佳的。与用LinkerS-1（6-氯己酸）和组氨酸标记的β2-AR制备的柱相比，LinkerS-4柱上的药物具有更大的解离速率常数（如沙丁胺醇为60.3±0.3 s-1），与文献数据更接近。综上所述，我们得出结论，接头结构的优化在减少固定化蛋白质与药物之间的非特异性相互作用方面起着特殊的作用，从而使药物-蛋白质相互作用的测定更可靠。
In previous work, we have established a one-step method to immobilize halo-tagged proteins onto microspheres through the covalent bond formed between the halo-tag and the halide linkers on the support surface. We observe extremely tailed peaks of most of drugs on the immobilized proteins, which is reasoned by the nonspecific interaction between the linkers and the drugs. To prove this, the current work designed five different halide linkers for the immobilization of beta2-adrenoceptor (β2-AR). We applied the immobilized receptor to systematically realize the effects of these halide linkers on drug-receptor interaction by analyzing peak profiles of five drugs. The retention times and the half-widths of the drugs appeared to be negatively correlated to the atom numbers of the linkers in the range of 6–13 atoms. Subsequent increase of linker atoms resulted in reduced retention times and wider peaks of the drugs. Applying identical linker length, we observed clear reduced retention times and half-widths of the five drugs than the linker in the absence of oxygen atom. Such improvement was dominated by the number of oxygen atoms. These indicated that linkerS-4(2-(2-(2-(2-chloroethoxy) ethoxy) ethoxy) acetic acid) was optimal to eliminate the unwanted non-specific interactions. In comparison with the columns prepared by linkerS-1(6-chlorocaproic acid) and histidine tagged β2-AR, the drugs on the linkerS-4column gave greater dissociation rate constants (e.g. 60.3±0.3 s-1for salbutamol), which is closer to the data in literatures. Taking together, we concluded that optimization of the linker structure plays particular role in reducing the non-specific interaction between the immobilized protein and the drugs, thereby making the determination of drug-protein interaction more reliable.