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- Title
One‐step production of fully biotinylated and glycosylated human Fc gamma receptors.
- Authors
Kang, Minhyo; Wang, Zening; Ge, Xin
- Abstract
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.
- Subjects
FC receptors; STREPTAVIDIN; POST-translational modification; TRANSGENE expression; HUMORAL immunity; ENDOPLASMIC reticulum; DRUG target
- Publication
Biotechnology Progress, 2024, Vol 40, Issue 1, p1
- ISSN
8756-7938
- Publication type
Article
- DOI
10.1002/btpr.3392