Surface plasmon resonance (SPR) biosensor has emerged as a transformative tool in high-throughput drug screening and label-free analysis of biomolecular interactions. However, a critical limitation of SPR lies in its stringent requirement for highly purified proteins to ensure reliable quantification of binding affinities and kinetic parameters. In order to address the limitations, the lentiviral particle and styrene-maleic acid polymer have been previously developed to extract and stabilize transmembrane proteins (TMs) indirectly and thus to detect ligands interaction with TMs by SPR biosensor. The present study proposes a high-throughput SPR-based drug screening system that utilizes cell-free protein synthesis (CFPS) to achieve in situ purification and immobilization of TMs on SPR biosensors. First, C−X−C chemokine receptor 4 (CXCR4) protein with His-tag was prepared by CFPS. Then, two types of nickel-nitrilotriacetic acid (Ni-NTA) biosensors, modified with carboxymethylated dextran (CMD) and coated with polycarboxylate hydrogel coating (HC) matrix, were compared with classical carboxymethylated dextran 5 (CM5) biosensor in order to determine the optimal strategy for coupling the CXCR4-CFPS protein. The CMD-NTA/SPR biosensor was next applied to screen for CXCR4 ligands from 96 natural products. Finally, glycyrrhizic acid and ginsenoside Re were proved to be function of CXCR4 inhibitors by affinity test, molecular docking, and cell migration assay. The combination of CFPS with SPR technology facilitates in-situ purification and immobilization of target proteins in a single step, thereby significantly enhancing the efficiency of SPR assay procedures. The system has broad applicability for targeting various challenging TMs and provides potential candidates for subsequent drug development. • CFPS technology was utilized to in situ construct SPR biosensors targeting transmembrane proteins. • The NTA sensor has been proven to be capable of purification and immobilization of CFPS proteins in one step. • Glycyrrhizic acid and ginsenoside Re were screened out to be potential inhibitors of CXCR4.
Wang et al. (Sun,) studied this question.