PuSH - Publication Server of Helmholtz Zentrum München: Overexpression, Purification and Biochemical Characterization of the Extracellular Human CD83 Domain and Generation of Monoclonal Antibodies.

Navigation

Home

Deutsch

Research

Advanced Search

Browse by ...

... Journal

... Publication Type

... Research Data

... Publication Year

Publication overview

Support & Contact

Contact persons

Help

Data protection

Lechmann, M.* ; Kremmer, E. ; Sticht, H.* ; Steinkasserer, A.*

Overexpression, Purification and Biochemical Characterization of the Extracellular Human CD83 Domain and Generation of Monoclonal Antibodies.

Protein Expr. Purif. 24, 445-452 (2002)

Open Access Green as soon as Postprint is submitted to ZB.

Abstract
Metrics
Extra information

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. GSThCD83ext 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 NI M and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies.

Additional Metrics?

[➜Log in]

Edit extra informations Login

Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords CD83; dendritic cells; recombinant expression; monoclonal antibodies

ISSN (print) / ISBN 1046-5928

e-ISSN 1046-5928

Journal Protein Expression and Purification

Quellenangaben Volume: 24, Pages: 445-452

Publisher Elsevier

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Molecular Immunology (IMI)