Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Technological Institute E136, Evanston, IL 60208, United States.
Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL 60208, United States.
Glycobiology. 2023 Jun 3;33(5):358-363. doi: 10.1093/glycob/cwad018.
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.
凝集素是结合糖的重要生物工具，但对于某些凝集素类别来说，重组蛋白表达存在挑战，限制了发现和表征的速度。为了发现和工程具有新功能的凝集素，需要易于快速表达和后续表征的工作流程。在这里，我们提出了细菌无细胞表达作为一种有效的、小规模表达多价、富含二硫键、鼠李糖结合凝集素的方法。此外，我们证明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。该工作流程可用于确定凝集素的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能够实现高通量表达、筛选和表征新型和工程化的多价凝集素，用于合成糖生物学中的应用。
