MersV1, Main, Exploration, bibRecord, 003469

Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function

Identifieur interne : 003469 ( Main/Exploration ); précédent : 003468; suivant : 003470

Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function

Auteurs : Sarah E. Henson [États-Unis] ; Donald Smith [États-Unis] ; Susan A. Boackle [États-Unis] ; V. Michael Holers [États-Unis] ; David R. Karp [États-Unis]

Source :

Journal of Immunological Methods [ 0022-1759 ] ; 2001.

RBID : ISTEX:A2F9063EE99F729CFC8B057D4EDAD8E055FD95CF

English descriptors

Teeft :
- Amino, Amino terminus, Assay, Bamhi site, Becton dickinson, Bglii site, Bind, Biotin, Biotin acceptor peptides, Biotin ligase, Biotinylated, Biotinylated c3dg, Biotinylated protein, Biotinylation, Biotinylation signal peptide, Bira gene, Boackle, C3dg, C3dg binding, C3dg molecules, C3dg tetramers, Calcium flux, Carboxyl terminus, Cell activation, Cell line, Cell lines, Cell lymphoma, Cell responses, Cell surface, Cell surface receptors, Colorado health sciences center, Comparable experiments, Complement receptor, Complement receptor type, Cytometry, Enzymatic biotinylation, Escherichia coli, Fearon, Flow cytometry, Follicular dendritic cells, Henson, High avidity, High levels, Holers, Human c3dg, Human plasma, Immune, Immune complexes, Immune response, Immunol, Immunological, Immunological methods, Intracellular, Intracellular calcium, Intracellular calcium flux, Karp, Kinase, Kinase activation, Kinase assay, Ligand, Loading buffer, Lymphocyte, Mgrml, Molecular adjuvant, Monoclonal, Monoclonal antibodies, Monoclonal antibody, Monomeric, Monomeric c3dg, Mouse splenic, Peptide, Peripheral blood, Physiological ligand, Plasmid, Polyclonal, Polyclonal antibody, Previous studies, Raji, Raji cells, Rc3dg, Receptor, Recombinant, Recombinant c3dg, Recombinant c3dg tetramers, Recombinant protein, Recombinant proteins, Relative intensity, Rheumatoid factor, Room temperature, Short consensus, Streptavidin, Terminus, Tetrameric form, Tetramers, Thick line, Thin line, Third component, Tyrosine kinases, Virus receptor, Vivo biotinylation, Western blot.

Abstract

Abstract: CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3′ end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.

Url:

https://api.istex.fr/ark:/67375/6H6-0X7S409Q-H/fulltext.pdf

DOI: 10.1016/S0022-1759(01)00471-9

Affiliations:

États-Unis

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Le document en format XML

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<profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Amino</term>
<term>Amino terminus</term>
<term>Assay</term>
<term>Bamhi site</term>
<term>Becton dickinson</term>
<term>Bglii site</term>
<term>Bind</term>
<term>Biotin</term>
<term>Biotin acceptor peptides</term>
<term>Biotin ligase</term>
<term>Biotinylated</term>
<term>Biotinylated c3dg</term>
<term>Biotinylated protein</term>
<term>Biotinylation</term>
<term>Biotinylation signal peptide</term>
<term>Bira gene</term>
<term>Boackle</term>
<term>C3dg</term>
<term>C3dg binding</term>
<term>C3dg molecules</term>
<term>C3dg tetramers</term>
<term>Calcium flux</term>
<term>Carboxyl terminus</term>
<term>Cell activation</term>
<term>Cell line</term>
<term>Cell lines</term>
<term>Cell lymphoma</term>
<term>Cell responses</term>
<term>Cell surface</term>
<term>Cell surface receptors</term>
<term>Colorado health sciences center</term>
<term>Comparable experiments</term>
<term>Complement receptor</term>
<term>Complement receptor type</term>
<term>Cytometry</term>
<term>Enzymatic biotinylation</term>
<term>Escherichia coli</term>
<term>Fearon</term>
<term>Flow cytometry</term>
<term>Follicular dendritic cells</term>
<term>Henson</term>
<term>High avidity</term>
<term>High levels</term>
<term>Holers</term>
<term>Human c3dg</term>
<term>Human plasma</term>
<term>Immune</term>
<term>Immune complexes</term>
<term>Immune response</term>
<term>Immunol</term>
<term>Immunological</term>
<term>Immunological methods</term>
<term>Intracellular</term>
<term>Intracellular calcium</term>
<term>Intracellular calcium flux</term>
<term>Karp</term>
<term>Kinase</term>
<term>Kinase activation</term>
<term>Kinase assay</term>
<term>Ligand</term>
<term>Loading buffer</term>
<term>Lymphocyte</term>
<term>Mgrml</term>
<term>Molecular adjuvant</term>
<term>Monoclonal</term>
<term>Monoclonal antibodies</term>
<term>Monoclonal antibody</term>
<term>Monomeric</term>
<term>Monomeric c3dg</term>
<term>Mouse splenic</term>
<term>Peptide</term>
<term>Peripheral blood</term>
<term>Physiological ligand</term>
<term>Plasmid</term>
<term>Polyclonal</term>
<term>Polyclonal antibody</term>
<term>Previous studies</term>
<term>Raji</term>
<term>Raji cells</term>
<term>Rc3dg</term>
<term>Receptor</term>
<term>Recombinant</term>
<term>Recombinant c3dg</term>
<term>Recombinant c3dg tetramers</term>
<term>Recombinant protein</term>
<term>Recombinant proteins</term>
<term>Relative intensity</term>
<term>Rheumatoid factor</term>
<term>Room temperature</term>
<term>Short consensus</term>
<term>Streptavidin</term>
<term>Terminus</term>
<term>Tetrameric form</term>
<term>Tetramers</term>
<term>Thick line</term>
<term>Thin line</term>
<term>Third component</term>
<term>Tyrosine kinases</term>
<term>Virus receptor</term>
<term>Vivo biotinylation</term>
<term>Western blot</term>
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<front><div type="abstract" xml:lang="en">Abstract: CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3′ end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.</div>
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Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function

Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function

Source :

English descriptors

Abstract

Links toward previous steps (curation, corpus...)

Le document en format XML

Pour manipuler ce document sous Unix (Dilib)

Pour mettre un lien sur cette page dans le réseau Wicri