Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies.
CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily. It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GST-hCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NMR and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies.
Lechmann
Matthias
M
Department of Dermatology, University of Erlangen-Nuremberg, Hartmannstrasse 14, D-91052 Erlangen, Germany. lechmann@derma.med.uni-erlangen.de
Kremmer
Elisabeth
E
Sticht
Heinrich
H
Steinkasserer
Alexander
A
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015703:Antigens, CD; D003713:Dendritic Cells; D006031:Glycosylation; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007962:Leukocytes; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D000097802:CD83 Antigen
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D000097802:CD83 Antigen