Selection of an artificial paraquat-specific binding protein from a ribosome display library based on a lipocalin scaffold
Selection of an artificial paraquat-specific binding protein from a ribosome display library based on a lipocalin scaffold
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基于脂质运载蛋白支架从核糖体展示文库中选择人工百草枯特异性结合蛋白
DOI：
10.1002/bab.2059
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发表时间：
2020-11-10
影响因子：
2.8
通讯作者：
Liu，Jianqing
中科院分区：
文献类型：
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作者：
Li，Hua;Liu，Li;Liu，Jianqing
在这项研究中，产生了一种可以特异性结合百草枯（PQ）的抗运载蛋白，一种季氮除草剂，作为一组新的具有抗体样性质的工程受体蛋白，用于检测PQ浓度。为此，构建了一个天然的随机文库，并将其工程化以允许使用大肠杆菌裂解物系统进行体外转录和翻译。同时，合成了在脂肪族间隔臂末端带有活性脂肪族羧酸酯基团的PQ衍生物。然后，将该化合物共价偶联至载体蛋白牛血清白蛋白/卵清蛋白和氨基官能化的顺磁珠。在溶液中交替选择和在微量滴定威尔斯孔中固定用于淘选mRNA-核糖体-抗体复合物。在几轮核糖体展示后，从胆色素结合蛋白的随机文库中选择三种变体。鉴定了能够以高亲和力结合复合物PQ且表现出低至14.039 ± 0.970 ng/mL的IC 50值的变体。方法的检出限为0.083 ± 0.011 ng/mL。我们的数据表明，产生anticalins可能提供一个有前途的替代重组抗体片段，以创建一个稳定的受体蛋白对半抗原与生物分析的相关性。
In this study, an anticalin that could specifically bind paraquat (PQ), a quaternary nitrogen herbicide, as a new set of engineered receptor protein with antibody‐like properties was generated to detect PQ concentration. To this end, a native and random library was constructed and engineered to allowin vitrotranscription and translation using anEscherichia colilysate system. Meanwhile, a PQ derivative that carries an active aliphatic carboxylate group at the end of an aliphatic spacer arm was synthesized. Then, this compound was coupled covalently to the carrier protein bovine serum albumin/ovalbumin and amino‐functionalized paramagnetic beads. Alternating selection in solution and immobilization in microtiter wells were used to pan mRNA–ribosome–antibody complexes. After several rounds of ribosome display, three variants were selected from a random library of the bilin‐binding protein. The variants that could bind complex PQ with high affinity and exhibit IC50values as low as 14.039 ± 0.970 ng/mL were identified. Moreover, the limits of detection reached 0.083 ± 0.011 ng/mL. Our data suggest that the generation of anticalins may provide a promising alternative to recombinant antibody fragments to create a stable receptor protein against hapten with bioanalytical relevance.