Predeina Olga, Atkinson Misha, Wissett Oliver, Ali Montader, Visentin Cristina, Ricagno Stefano, Keeble Anthony H, Howarth Mark R, Sormanni Pietro
Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge CB2 1EW Cambridge UK
Department of Biosciences, University of Milan Milan Italy.
RSC Chem Biol. 2025 Jun 23. doi: 10.1039/d5cb00079c.
Accurate measurements of binding kinetics, encompassing equilibrium dissociation constant ( ), association rate ( ), and dissociation rate ( ), are critical for the development and optimisation of high-affinity binding proteins. However, such measurements require highly purified material and precise ligand immobilisation, limiting the number of binders that can be characterised within a reasonable timescale and budget. Here, we present the SpyBLI method, a rapid and cost-effective biolayer interferometry (BLI) pipeline that leverages the SpyCatcher003-SpyTag003 covalent association, eliminating the need for both binder purification and concentration determination. This approach allows for accurate binding-kinetic measurements to be performed directly from crude mammalian-cell supernatants or cell-free expression blends. We also introduce a linear gene fragment design that enables reliable expression in cell-free systems without any PCR or cloning steps, allowing binding kinetics data to be collected in under 24 hours from receiving inexpensive DNA fragments, with minimal hands-on time. We demonstrate the method's broad applicability using a range of nanobodies and single-chain antibody variable fragments (scFvs), with affinity values spanning six orders of magnitude. By minimising sample preparation and employing highly controlled, ordered sensor immobilisation, our workflow delivers reliable kinetic measurements from crude mixtures without sacrificing precision. We expect that the opportunity to carry out rapid and accurate binding measurements in good throughput should prove especially valuable for binder engineering, the screening of next-generation sequencing-derived libraries, and computational protein design, where large numbers of potential binders for the same target must be rapidly and accurately characterised to enable iterative refinement and candidate selection.
结合动力学的准确测量，包括平衡解离常数（ ）、结合速率（ ）和解离速率（ ），对于高亲和力结合蛋白的开发和优化至关重要。然而，此类测量需要高度纯化的材料和精确的配体固定，这限制了在合理的时间尺度和预算内能够表征的结合物数量。在此，我们介绍了SpyBLI方法，这是一种快速且经济高效的生物层干涉术（BLI）流程，它利用SpyCatcher003-SpyTag003共价结合，无需进行结合物纯化和浓度测定。这种方法允许直接从粗制的哺乳动物细胞上清液或无细胞表达混合物中进行准确的结合动力学测量。我们还引入了一种线性基因片段设计，能够在无细胞系统中可靠表达，无需任何PCR或克隆步骤，从接收廉价的DNA片段开始，在不到24小时内即可收集结合动力学数据，且实际操作时间最少。我们使用一系列纳米抗体和单链抗体可变片段（scFv）证明了该方法的广泛适用性，其亲和力值跨越六个数量级。通过最小化样品制备并采用高度可控、有序的传感器固定，我们的工作流程能够从粗混合物中提供可靠的动力学测量，而不牺牲精度。我们预计，在高通量下进行快速准确的结合测量的机会对于结合物工程、下一代测序衍生文库的筛选以及计算蛋白质设计尤其有价值，在这些领域中，必须快速准确地表征大量针对同一靶点的潜在结合物，以实现迭代优化和候选物选择。
