Construction of lipid raft-coupled agarose gels as bioaffinity chromatography materials and validation with tropomyosin-related kinase A-targeted drugs

• Published in: Journal of Chromatography A Vol. 1691; p. 463803
• Main Authors: Chi, Hao, Tian, Sheng, Li, Xiu, Chen, Yuchu, Xu, Qiumin, Wang, Qixiao, Shi, Wenwan, Adu-Frimpong, Michael, Tong, Shanshan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 22.02.2023
• Subjects: Affinity chromatography; Chromatography, Affinity - methods; Chromatography, Agarose; Gefitinib; Gels; Lipid Rafts@CNBr-Sepharose 4B; Membrane Microdomains; Reproducibility of Results; Sepharose; Sorption; TrkA; Tropomyosin; Sorption; TrkA; Gefitinib; Affinity chromatography; Lipid Rafts@CNBr-Sepharose 4B
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2023.463803

•The first lipid Rafts@CNBr-Sepharose 4B bioaffinity chromatography model was successfully constructed to screen for TrkA-targeted antitumor drugs with high recovery rates.•Immunofluorescence assay revealed the presence of a large amount of TrkA protein in U251 cell lipid rafts and successfully coupled it to agarose.•Relieved the situation that affinity chromatography mainly uses silica gel as the carrier material, and the better biocompatibility of agarose gel will help the ligand to maintain its activity for a long time. In order to improve the separation process of affinity chromatography that has silica as the main carrier material, we sought to construct Lipid Rafts@CNBr-Sepharose 4B affinity chromatography model. We extracted the lipid rafts from U251 cells with a descaler method and sucrose density gradient centrifugation. Afterwards, it was discovered via immunofluorescence that the lipid rafts contain a large amount of tropomyosin-related kinase A (TrkA) protein. Also, agarose powder in the lyophilised state was pretreated, before the lipid rafts were coupled to the agarose gel in a coupling buffer of alkaline pH. CNBr-Sepharose 4B affinity gel packing was characterised using UV spectrophotometric, immunofluorescence and scanning electron microscopic techniques, wherein and the results showed that the lipid rafts were successfully coupled to the agarose gels. Three compounds were used to verify the specific sorption of Sepharose 4B and CNBr-Sepharose 4B, which showed no specific sorption on the materials. Of note, the prepared Lipid Rafts@CNBr-Sepharose 4B agarose gels packed with TrkA-rich target proteins could be successfully validated for the active drug gefitinib with high affinity sorption efficiency and eluted with good recovery and reproducibility. This study broadens the range of affinity chromatography carrier materials and provides a reference for research in active drug screening.