Department of Chemical and Environmental Engineering, University of California Riverside, Riverside, California, USA.
Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, Texas, USA.
Biotechnol Prog. 2024 Jan-Feb;40(1):e3392. doi: 10.1002/btpr.3392. Epub 2023 Sep 21.
Initiating and regulating humoral immunity, Fc gamma receptors (FcγRs) have been identified both as therapeutics and as drug targets, and thus production of biologically active FcγRs is highly demanded for biopharmaceutical development. Focusing on low-affinity FcγRs IIA (131H/R allotypes), IIB, and IIIA (176F/V), this study used human 293-F cells to achieve correct post-translational modifications (PTMs) including biotinylation, N-glycosylation, and disulfides. Approaches involving co-expression of FcγR-AviTag and Escherichia coli biotin ligase BirA, endoplasmic reticulum retention, stable and transient transfections, and optimization of transgene ratio were investigated. Protein electrophoresis under reducing and non-reducing conditions, enzymatic deglycosylation, streptavidin pull-down assays, and binding kinetic analysis collectively indicated that the produced FcγR ectodomains were fully biotinylated, N-glycosylated, had formed disulfide bond, and exhibited expected binding affinities toward IgG1 trastuzumab and its Fc mutants. A clear trade-off between production yield and PTM quality was also observed. Achieving multiple types of PTMs completely by one-step cell culture should have applications for the production of a variety of complex proteins of biomedical importance.
启动和调节体液免疫，Fc 受体（FcγRs）已被鉴定为治疗靶点和药物靶点，因此生物活性 FcγRs 的产生对于生物制药的发展有很高的需求。本研究聚焦于低亲和力 FcγRs IIA（131H/R 同种型）、IIB 和 IIIA（176F/V），使用人 293-F 细胞实现了包括生物素化、N-糖基化和二硫键在内的正确翻译后修饰（PTMs）。本研究探讨了涉及 FcγR-AviTag 和大肠杆菌生物素连接酶 BirA 的共表达、内质网保留、稳定和瞬时转染以及转基因比例优化等方法。还原和非还原条件下的蛋白质电泳、酶解糖基化、链霉亲和素下拉实验和结合动力学分析共同表明，产生的 FcγR 胞外结构域完全生物素化、N-糖基化、形成二硫键，并表现出与 IgG1 曲妥珠单抗及其 Fc 突变体的预期结合亲和力。还观察到生产产量和 PTM 质量之间存在明显的权衡。通过一步细胞培养完全实现多种类型的 PTM 应该适用于多种具有重要生物医学意义的复杂蛋白的生产。
