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

被引:38
作者
Lechmann, M
Kremmer, E
Sticht, H
Steinkasserer, A
机构
[1] Univ Erlangen Nurnberg, Dept Dermatol, D-91052 Erlangen, Germany
[2] GSF Forschungszentrum Umwelt & Gesundheit GMBH, Inst Mol Immunol, D-81377 Munich, Germany
[3] Univ Bayreuth, Lehrstuhl Biopolymere, D-95447 Bayreuth, Germany
关键词
CD83; dendritic cells; recombinant expression; monoclonal antibodies;
D O I
10.1006/prep.2001.1594
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2002 Elsevier Science (USA).
引用
收藏
页码:445 / 452
页数:8
相关论文
共 15 条
  • [1] Dendritic cells as vectors for therapy
    Banchereau, J
    Schuler-Thurner, B
    Palucka, AK
    Schuler, G
    [J]. CELL, 2001, 106 (03) : 271 - 274
  • [2] Dendritic cells and the control of immunity
    Banchereau, J
    Steinman, RM
    [J]. NATURE, 1998, 392 (6673) : 245 - 252
  • [3] Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood
    Bender, A
    Sapp, M
    Schuler, G
    Steinman, RM
    Bhardwaj, N
    [J]. JOURNAL OF IMMUNOLOGICAL METHODS, 1996, 196 (02) : 121 - 135
  • [4] Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation
    Berchtold, S
    Mühl-Zürbes, P
    Heufler, C
    Winklehner, P
    Schuler, G
    Steinkasserer, A
    [J]. FEBS LETTERS, 1999, 461 (03) : 211 - 216
  • [5] Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites
    Dieu, MC
    Vanbervliet, B
    Vicari, A
    Bridon, JM
    Oldham, E
    Aït-Yahia, S
    Brière, F
    Zlotnik, A
    Lebecque, S
    Caux, C
    [J]. JOURNAL OF EXPERIMENTAL MEDICINE, 1998, 188 (02) : 373 - 386
  • [6] WATER SUPPRESSION THAT WORKS - EXCITATION SCULPTING USING ARBITRARY WAVE-FORMS AND PULSED-FIELD GRADIENTS
    HWANG, TL
    SHAKA, AJ
    [J]. JOURNAL OF MAGNETIC RESONANCE SERIES A, 1995, 112 (02) : 275 - 279
  • [7] Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity
    Kruse, M
    Rosorius, O
    Krätzer, F
    Stelz, G
    Kuhnt, C
    Schuler, G
    Hauber, J
    Steinkasserer, A
    [J]. JOURNAL OF VIROLOGY, 2000, 74 (15) : 7127 - 7136
  • [8] Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with nuclear export of CD83 mRNA
    Kruse, M
    Rosorius, O
    Krätzer, F
    Bevec, D
    Kuhnt, C
    Steinkasserer, A
    Schuler, G
    Hauber, J
    [J]. JOURNAL OF EXPERIMENTAL MEDICINE, 2000, 191 (09) : 1581 - 1589
  • [9] Steinman RM., 2003, ANNU REV IMMUNOL, V9, P271
  • [10] Vaccination with Mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma
    Thurner, B
    Haendle, I
    Röder, C
    Dieckmann, D
    Keikavoussi, P
    Jonuleit, H
    Bender, A
    Maczek, C
    Schreiner, D
    von den Driesch, P
    Bröcker, EB
    Steinman, RM
    Enk, A
    Kämpgen, E
    Schuler, G
    [J]. JOURNAL OF EXPERIMENTAL MEDICINE, 1999, 190 (11) : 1669 - 1678