PDF(Pubmed)
Abstract：
G protein-coupled receptors (GPCRs) are a large family of cell membrane proteins that are important targets for drug discovery. Nanobodies, also known as VHH (variable domains of heavy chain-only antibodies, HcAbs) antibodies, are small antibody fragments derived from camelids that have gained significant attention as potential therapeutics for targeting GPCRs due to their advantages over conventional antibodies. However, there are challenges in developing nanobodies targeting GPCRs, among which epitope accessibility is the most significant because the cell membrane partially shields the GPCR surface. We developed a universal protocol for making nanobodies targeting GPCRs using the cell membrane extract of GPCR-overexpressing HEK293 cells as the llama/alpaca immunization antigen. We constructed an immune VHH library and identified nanobodies by phage display bio-panning. The monoclonal nanobodies were recombinantly expressed in Escherichia coli (E. coli) and purified to characterize their binding potency.
摘要：
G蛋白偶联受体（GPCRs）是细胞膜蛋白的一大家族，是药物发现的重要靶标。纳米抗体,也称为VHH（仅重链抗体的可变结构域，HcAbs）抗体，是源自骆驼科动物的小抗体片段，由于其优于常规抗体的优势，其作为靶向GPCRs的潜在治疗剂而获得了极大的关注。然而,在开发靶向GPCRs的纳米抗体方面存在挑战，其中表位的可及性是最重要的，因为细胞膜部分屏蔽了GPCR表面。我们开发了一种通用方案，用于使用GPCR过表达HEK293细胞的细胞膜提取物作为美洲驼/羊驼免疫抗原来制备靶向GPCR的纳米抗体。我们构建了免疫VHH文库，并通过噬菌体展示生物淘选鉴定了纳米抗体。单克隆纳米抗体在大肠杆菌中重组表达(E.大肠杆菌)并纯化以表征它们的结合效力。
