Halo-tagged protein immobilization: Effect of halide linkers on peak profile and drug-protein interaction

• Published in: Journal of Chromatography A 2021-03, Vol.1640, p.461946, Article 461946
• Main Authors: Fu, Xiaoying, Li, Linkang, Wen, Xin, Xu, Ru, Xue, Yan, Zuo, Haiyue, Liang, Qi, Feng, Gangjun, Wang, Jing, Zhao, Xinfeng
• Format: Article
• Language: English
• Subjects: Drug-protein interaction; Halide linker; Halo-tagged protein; Peak profile
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2021.461946

•We studied the effect of halide linkers on peak profile and drug-protein binding.•S-4 (2-(2-(2-(2-chloroethoxy) ethoxy) ethoxy) acetic acid) is the optimal linker.•S-4 can minimize the non-specific interaction between the drugs and the receptor. 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 linker S-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 linker S-1 (6-chlorocaproic acid) and histidine tagged β2-AR, the drugs on the linker S-4 column gave greater dissociation rate constants (e.g. 60.3±0.3 s-1 for 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.