Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies

Protein Expression and Purification
Matthias LechmannAlexander Steinkasserer

Abstract

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, ...Continue Reading

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Citations

Aug 15, 2008·Proceedings of the National Academy of Sciences of the United States of America·Matthias LechmannTak W Mak
Aug 4, 2004·The Journal of Experimental Medicine·Elisabeth ZinserAlexander Steinkasserer
Sep 7, 2007·International Immunology·Yoshihiro KuwanoThomas F Tedder
Dec 27, 2011·Veterinary Immunology and Immunopathology·Sung Hyen LeeYeong Ho Hong
Jun 23, 2010·Protein Expression and Purification·Lin ZhangC Perry Chou
Mar 11, 2008·Trends in Immunology·Minka Breloer, Bernhard Fleischer
Feb 22, 2005·Biochemical and Biophysical Research Communications·Matthias LechmannAlexander Steinkasserer
Oct 16, 2004·Immunobiology·Nicole KotzorAlexander Steinkasserer
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Feb 15, 2017·The Journal of Immunology : Official Journal of the American Association of Immunologists·Joe M HorvatinovichMark A DeBenedette
Nov 17, 2010·Transplantation·Zhu LanHao Wang
Mar 6, 2007·Archives of Dermatological Research·Alexander T Prechtel, Alexander Steinkasserer
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Jun 20, 2002·Trends in Immunology·Matthias LechmannAlexander Steinkasserer

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