Department of Endoscopy, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy of Tianjin, Tianjin's Clinical Research Center for Cancer, Tianjin, People's Republic of China.
Inner Mongolia, Baotou Teachers College of Inner Mongolia University of Science and Technology, Baotou, People's Republic of China.
Biotechnol Appl Biochem. 2021 Dec;68(6):1372-1385. doi: 10.1002/bab.2059. Epub 2020 Nov 10.
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 allow in vitro transcription and translation using an Escherichia coli lysate 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 IC values 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.
在这项研究中，生成了一种能够特异性结合百草枯（PQ）的抗钙蛋白，PQ 是一种季铵态氮除草剂，作为一组具有抗体样特性的新型工程受体蛋白，用于检测 PQ 浓度。为此，构建并工程化了一个天然和随机文库，以允许在体外使用大肠杆菌裂解物系统进行转录和翻译。同时，合成了一种末端带有活性脂肪族羧酸酯基团的 PQ 衍生物，连接在脂肪族间隔臂上。然后，将该化合物共价偶联到载体蛋白牛血清白蛋白/卵清蛋白和氨基功能化的顺磁珠上。在溶液中进行交替选择，并在微孔板中进行固定化，以筛选 mRNA-核糖体-抗体复合物。经过几轮核糖体展示，从双氢卟啉结合蛋白的随机文库中选择了三个变体。鉴定出能够与高亲和力结合复杂 PQ 并表现出低至 14.039±0.970ng/mL 的 IC 值的变体。此外，检测限达到 0.083±0.011ng/mL。我们的数据表明，抗钙蛋白的产生可能为创建针对生物分析相关半抗原的稳定受体蛋白提供一种有前途的替代重组抗体片段的方法。
