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Selection of active ScFv to G-protein-coupled receptor CCR5 using surface antigen-mimicking peptides.
- Source :
-
Biochemistry [Biochemistry] 2004 Oct 05; Vol. 43 (39), pp. 12575-84. - Publication Year :
- 2004
-
Abstract
- This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library. Antibodies reactive with CCR5 on cells were detected using a homogeneous high throughput assay. Of 19 isolated ScFv antibodies that bound to CCR5+ cells, three inhibited CCR5-mediated but not CXCR4-mediated HIV infection. Only ScFvs selected by binding to cyclic constrained peptides exhibited inhibitory activity. Our results demonstrate that surface-antigen mimetics of a GPCR are effective tools for selecting active, site-specific ScFv antibodies that hold promise as immunological reagents and therapeutics.
- Subjects :
- Amino Acid Sequence
Anti-HIV Agents chemistry
Anti-HIV Agents metabolism
Antigens, Surface chemistry
Antigens, Surface immunology
Cell Line, Tumor
Cross Reactions
Cysteine chemistry
Enzyme-Linked Immunosorbent Assay
Esters
Growth Inhibitors chemistry
Growth Inhibitors metabolism
HIV-1 growth & development
HIV-1 immunology
HeLa Cells
Humans
Immunoglobulin Variable Region chemistry
Ligands
Molecular Sequence Data
Peptide Library
Peptides, Cyclic chemical synthesis
Receptors, CCR5 chemistry
Receptors, CCR5 immunology
Sulfhydryl Compounds chemistry
Antigens, Surface metabolism
Binding Sites, Antibody
Immunoglobulin Variable Region metabolism
Molecular Mimicry immunology
Peptides, Cyclic metabolism
Receptors, CCR5 metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 43
- Issue :
- 39
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15449947
- Full Text :
- https://doi.org/10.1021/bi0492152