Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry
Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry
复制标题
使用生物层干涉测量法进行多价鼠李糖结合凝集素的无细胞表达和表征
DOI：
10.1093/glycob/cwad018
复制
发表时间：
2023
期刊：
影响因子：
4.3
通讯作者：
Imberty, Anne
中科院分区：
文献类型：
--
作者：
Warfel, Katherine F.;Laigre, Eugénie;Sobol, Sarah E.;Gillon, Emilie;Varrot, Annabelle;Renaudet, Olivier;Dejeu, Jerome;Jewett, Michael C.;Imberty, Anne
凝集素是结合聚糖的重要生物工具，但重组蛋白表达对某些凝集素类构成挑战，限制了发现和表征的步伐。为了发现和设计具有新功能的凝集素，需要能够快速表达和随后表征的工作流程。在这里，我们提出细菌无细胞表达作为有效的手段，小范围表达多价，二硫键丰富，鼠李糖结合凝集素。此外，我们证明了无细胞表达的凝集素可以直接与生物层干涉分析相结合，无论是在溶液中还是固定在传感器上，都可以测量与碳水化合物配体的相互作用，而无需纯化。该工作流程能够确定凝集素底物特异性和估计结合亲和力。总的来说，我们相信这种方法将使高通量表达、筛选和表征新的和工程化的多价凝集素在合成糖生物学中的应用成为可能。
Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.