In situ cleavage-based sortase A-mediated site-specific immobilization of beta2-adrenoceptor on gold surface for surface plasmon resonance measurement

Abstract

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 β₂-adrenoceptor (β₂AR) as a model, incorporating an endogenous protease recognition site (EPRS) as a linker to fuse SrtA with β₂AR, 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 β₂AR. When the lysate is mixed with an oligo-Gly or oligo-Gly-modified SPR chip, transpeptidation occurs, covalently immobilizing β₂AR. 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 β₂AR activity was evaluated by SPR. When compared to the EDC/NHS-based random method and the haloalkane dehalogenase (HaloTag)-mediated site-specific strategy, β₂AR 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.