Zhao Xue, Ou Yuanyuan, Bai Ruoxue, Yin Jiatai, Wang Ge, Wang Jing, Zhao Xinfeng, Liang Yinku, Li Qian
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China.
School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong, 723001, China.
Biosens Bioelectron. 2025 Aug 1;281:117452. doi: 10.1016/j.bios.2025.117452. Epub 2025 Apr 8.
Surface Plasmon Resonance (SPR) is a pivotal technique for measuring biomolecular interactions, with the sensor surface typically made of gold or silver and requiring proteins to be immobilized in a controlled manner. Traditional methods, such as random crosslinking via covalent amide bonds (EDC/NHS strategy), resulting in diverse protein orientations. Alternatively, site-specific immobilization strategies offer better orientation control, they are still challenged by the purification needs for protein of interests and steric hindrance produced by bulk protein tags. To address these issues, we proposed a novel protein immobilization strategy relying on in situ cleavage and Sortase A (SrtA) to immobilize functional protein on SPR sensor chips. This strategy involves the β-adrenoceptor (βAR) as a model, incorporating an endogenous protease recognition site (EPRS) as a linker to fuse SrtA with βAR, which contains an SrtA recognition sequence (LPXTG) at its C-terminal. When expressed in Escherichia coli (E. coli), the protease cleaves the EPRS, releasing SrtA and βAR. When the lysate is mixed with an oligo-Gly or oligo-Gly-modified SPR chip, transpeptidation occurs, covalently immobilizing βAR. The efficacy of the cleavage and transpeptidation reactions was validated through SDS-PAGE, Western blot, and chromatographic analysis. The SPR chip was characterized by scanning electron microscope (SEM), energy dispersive spectroscopy (EDS) mapping, X-ray photoelectron spectroscopy (XPS), and contact angle analysis, while βAR activity was evaluated by SPR. When compared to the EDC/NHS-based random method and the haloalkane dehalogenase (HaloTag)-mediated site-specific strategy, βAR immobilized through the SrtA-mediated method exhibited higher activity with ligands, demonstrating precision in binding affinity evaluations. This strategy meets the benchmarks for an optimal site-specific immobilization method and holds promise for applications involving the modification of other biological interfaces or biosensors.
表面等离子体共振（SPR）是一种用于测量生物分子相互作用的关键技术，其传感器表面通常由金或银制成，并且需要以可控方式固定蛋白质。传统方法，例如通过共价酰胺键进行随机交联（EDC/NHS策略），会导致蛋白质呈现多种取向。另外，位点特异性固定策略虽能提供更好的取向控制，但仍面临目标蛋白质的纯化需求以及大量蛋白质标签产生的空间位阻的挑战。为了解决这些问题，我们提出了一种新颖的蛋白质固定策略，该策略依靠原位切割和分选酶A（SrtA）将功能性蛋白质固定在SPR传感器芯片上。此策略以β-肾上腺素能受体（βAR）为模型，引入一个内源性蛋白酶识别位点（EPRS）作为连接子，将SrtA与βAR融合，βAR在其C端含有一个SrtA识别序列（LPXTG）。当在大肠杆菌（E. coli）中表达时，蛋白酶切割EPRS，释放出SrtA和βAR。当裂解物与寡聚甘氨酸或寡聚甘氨酸修饰的SPR芯片混合时，会发生转肽作用，从而共价固定βAR。通过SDS-PAGE、蛋白质免疫印迹和色谱分析验证了切割和转肽反应的有效性。通过扫描电子显微镜（SEM）、能量色散光谱（EDS）映射、X射线光电子能谱（XPS）和接触角分析对SPR芯片进行了表征，同时通过SPR评估了βAR活性。与基于EDC/NHS的随机方法和卤代烷脱卤酶（HaloTag）介导的位点特异性策略相比，通过SrtA介导的方法固定的βAR与配体表现出更高的活性，证明了在结合亲和力评估中的精确性。该策略符合最佳位点特异性固定方法的标准，并有望应用于涉及其他生物界面或生物传感器修饰的领域。
