title: Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.
abstract: Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.
authors.affiliation: School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Seijsing
authors.forename: Malin
authors.initials: M
authors.lastname: Lindborg
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Torbjörn
authors.initials: T
authors.lastname: Gräslund
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000235:genetics; D007231:Infant, Newborn; D016086:Lentivirus / Q000235:genetics; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012709:Serum Albumin / Q000737:chemistry / Q000276:immunology; D012995:Solubility
chemical_list: D055910:Antibodies, Immobilized; D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D049452:Green Fluorescent Proteins; C100688:Fc receptor, neonatal