title: Eukaryotic expression of functionally active recombinant soluble CD83 from HEK 293T cells.
abstract: The cell surface protein CD83 belongs to the immunoglobulin super family and is highly expressed on mature dendritic cells (DCs). A membrane bound and a soluble form of CD83 (sCD83) have been described. Previously, the isolation of a purified recombinant sCD83 molecule from bacterial cultures using high pressure liquid chromatography was reported. This recombinant protein reduced DC-mediated T cell proliferation in vitro and displayed an inhibitory effect in the experimental autoimmune encephalomyelitis (EAE) model. When purifying sCD83 from bacteria, however, a lipopolysaccharide fraction is frequently co-isolated with the recombinant sCD83 protein. Moreover, the subsequent separation of sCD83 from contaminating LPS is usually accompanied by a considerable loss of soluble CD83. A further disadvantage of soluble CD83 expression in prokaryotic cells is the lack of functional glycosylation. To overcome these problems, we developed an alternative strategy to express sCD83 in eukaryotic human embryonic kidney (HEK) 293 T cells. Using this system, we showed that recombinant sCD83 was LPS-free and effectively glycosylated with all three asparagine residues at least partially involved. The functionality of the expressed sCD83 protein was examined using the mixed lymphocyte reaction (MLR) assay, demonstrating a reduced DC-mediated T cell proliferation as previously reported for the sCD83 protein purified from E. coli. Thus, a new protocol for efficient eukaryotic expression and purification of sCD83 was established, which might have several advantages compared to prokaryotic expression systems.
authors.affiliation: Department of Dermatology, University Hospital Erlangen, Erlangen, Germany. christine.staab@uk-erlangen.de
authors.forename: Christine
authors.initials: C
authors.lastname: Staab
authors.forename: Petra
authors.initials: P
authors.lastname: Mühl-Zürbes
authors.forename: Alexander
authors.initials: A
authors.lastname: Steinkasserer
authors.forename: Mirko
authors.initials: M
authors.lastname: Kummer
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D003713:Dendritic Cells / Q000378:metabolism; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000188:drug therapy / Q000276:immunology*; D005057:Eukaryotic Cells; D015870:Gene Expression / Q000276:immunology*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D019076:Transgenes / Q000235:genetics; D000097802:CD83 Antigen
chemical_list: D015703:Antigens, CD; D007136:Immunoglobulins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000097802:CD83 Antigen