Cell-free synthesis of membrane receptors for preparation of NKG2A monolithic micro-affinity chromatography

Membrane receptor affinity chromatography is a practical strategy for screening compounds targeting membrane receptors and determining their binding affinity. The key of these methods is to create a biomimetic cell membrane environment for the immobilization of specific receptors on chromatographic stationary phase. However, its large-scale applications are limited by the labor-intensive and time-consuming procedures including cell culture, recombinant protein expression and column packing. Furthermore, traditional affinity chromatography columns are prone to drawbacks such as heterogeneous composition of the stationary phase and low permeability. In this study, a novel membrane receptor biological affinity chromatographic method based on cell-free protein synthesis (CFPS) and Bis(sulfosuccinimidyl)suberate modified monolithic stationary phase was developed for fast preparation of monolithic micro-affinity column in batches, which realized efficient synthesis and immobilization of immune checkpoint natural killer group 2 family of receptor A (NKG2A) with controlled orientation. Coupling the prepared NKG2A micro-affinity column with an offline-2D-UPLC-QTOF/MS system, two new NKG2A inhibitors, baicalin and wogonoside, were screened out with the K_D values of 30.23 and 13.01 μM respectively, significantly upregulating the gene expression of granzyme B, tumor necrosis factor-α and interferon-γ and the protein expression of CD107a in natural killer (NK) cells. Moreover, the cytotoxic activity of NK cells against tumor cells was enhanced by these two compounds. The proposed CFPS-based monolithic micro-affinity chromatography realizes rapid synthesis and immobilization of transmembrane receptors within one day, achieving homogeneity, good permeability and orientation-controlled high expression. This approach could be extended to any interested transmembrane receptors for rapid drug screening and affinity determination.