Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombinant protein expression,which are time-consuming for cell culture or purification,and also difficult to ensure the purity and consistent orientation of MPs in the chromato-graphic stationary phase.In this study,a novel in situ synthesis membrane protein affinity chromatog-raphy(iSMAC)method was developed utilizing cell-free protein expression(CFE)and covalent immobilized affinity chromatography,which achieved efficient in situ synthesis and unidirectional inser-tion of MPs into liposomes in the stationary phase.The advantages of iSMAC are:1)There is no need to culture cells or prepare recombinant proteins;2)Specific and purified MPs with stable and controllable content can be obtained within 2 h;3)MPs maintain the transmembrane structure and a consistent orien-tation in the chromatographic stationary phase;4)The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites.iSMAC was successfully applied to screen PDGFRβinhibitors from Salvia miltiorrhiza and Schisandra chinensis.Micro columns prepared by in-situ synthe-sis maintain satisfactory analysis activity within 72 h.Two new PDGFRβ inhibitors,salvianolic acid B and gomisin D,were screened out with KD values of 13.44 and 7.39 μmol/L,respectively.In vitro exper-iments confirmed that the two compounds decreased α-SMA and collagen I mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38,AKT and ERK.In vivo,Sal B could also attenuate CCl4-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels.The iSMAC method can be applied to other general MPs,and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

Department of Pharmacy,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University,School of Medicine,Shanghai 201999,China;School of Pharmacy,Naval Medical University(Second Military Medical University),Shanghai 200433,China;Shanghai Key Laboratory for Pharmaceutical Metabolite Research,Shanghai 200433,China

[1]Yanqiu Gu;Rong Wang;Panpan Chen;Shengnan Li;Xinyi Chai;Chun Chen;Yue Liu;Yan Cao;Diya Lv;Zhanying Hong;Zhenyu Zhu;Yifeng Chai;Yongfang Yuan;Xiaofei Chen-.In situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography)[J].药学学报（英文版）,2022(09):3682-3693

microaffinity,chromatog,iSMAC,gomisin

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