| Reference | ||
|---|---|---|
| Reference Type | Literature | IEDB_Reference:1030088 |
| Title | Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. | |
| Authors | Michael B Braun; Bjoern Traenkle; Philipp A Koch; Felix Emele; Frederik Weiss; Oliver Poetz; Thilo Stehle; Ulrich Rothbauer | |
| Affiliations | Interfaculty Institute of Biochemistry, Eberhard-Karls University Tuebingen, Germany; Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany; Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany. | |
| Journal | Sci Rep | |
| Year | 2016 | |
| Abstract | Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a -sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. | |
| Curation Last Updated | 2023-08-18 22:38:56 | |
| Epitope | ||
|---|---|---|
| Epitope ID | 534778 | IEDB_epitope:534778 |
| Chemical Type | Linear peptide | |
| Linear Sequence | PDRKAAVSHWQQ | |
| Source Molecule Name | Catenin beta-1 | |
| Source Organism | Homo sapiens (human) | |
| Starting Position | 16 | |
| Ending Position | 27 | |
| Epitope Reference Details | ||
|---|---|---|
| Epitope Structure Defines | Epitope containing region/antigenic site | |
| Epitope Name | BC2 peptide | |
| Reference Starting Position | 16 | |
| Reference Ending Position | 27 | |
| Location of Data in Reference | Supplementary Figure 2 and Supplementary Methods | |
| Immunization | ||
|---|---|---|
| Host Organism | Vicugna pacos (alpaca) | |
| 1st In Vivo Process | ||
|---|---|---|
| In Vivo Process Type | Administration in vivo | |
| 1st Immunogen | ||
|---|---|---|
| Epitope Relation | Source Antigen | |
| Chemical Type | Protein | |
| Molecule Name | Catenin beta-1 | |
| Organism | Homo sapiens (human) | |
| Immunization Comments | ||
|---|---|---|
| Immunization Comments | Generation of the monoclonal antibody was previously described in the cited reference: Traenkle et al. (2015) Mol Cell Proteomics 14(3):707-23. doi: 10.1074/mcp.M114.044016. [PMID: 25595278]. Two alpacas were immunized with purified β-catenin protein, either GST-β-catenin or C-terminal histidine-tagged β-catenin (β-catenin-His6). Six weeks after immunization, blood was collected and total RNA was extracted from lymphocytes. A VHH phage display library was constructed and selected using full-length β-catenin. The selected VHH (nanobody) BC2 was cloned and expressed in bacteria. | |
| B Cell Assay | ||
|---|---|---|
| Qualitative Measurement | Positive | |
| Method/Technique | surface plasmon resonance (SPR) | |
| Measurement of | dissociation constant KD | |
| Assay Type Units | nM | |
| Measurement Details | ||
|---|---|---|
| Quantitative measurement | 1.4 | |
| Assayed Antibody | ||
|---|---|---|
| Assayed Antibody Source Material | Purified Immunoglobulin | |
| Assayed Antibody Immunoglobulin Domain | VH Domain | |
| Assayed Antibody Purification Status | Display Library (monoclonal) | |
| Assayed Antibody Name | BC2-Nb | |
| Assayed Antibody Object | ||
|---|---|---|
| Chemical Type | Multi-Chain protein | |
| Chain 1 Accession Name | Chain A, Bc2 Nanobody In Complex With The Bc2 Peptide Tag | |
| Source Organism | Vicugna pacos (alpaca) | |
| Molecule Name | BC2-Nb | |
| Source Organism | Vicugna pacos (alpaca) | |
| Antigen | ||
|---|---|---|
| Epitope Relation | Epitope | |
| Chemical Type | Linear peptide | |
| Linear Sequence | PDRKAAVSHWQQ | |
| Source Molecule Name | Catenin beta-1 | |
| Source Organism | Homo sapiens (human) | |
| Starting Position | 16 | |
| Ending Position | 27 | |
| Assay Reference Details | ||
|---|---|---|
| Assay Comments by IEDB Curator | Antigen carrier: Protein conjugate: Green fluorescent protein (Aequorea victoria). The affinity of BC2-Nb bound to the BC2 tag peptide (BC2T) was determined by surface plasmon resonance. Mutation R106E or R106S in BC2-Nb reduced the affinity almost 10-fold, KD = 12 nM and 9.7 nM, respectively. | |
| Location of Assay Data in Reference | Table 1 and Supplementary Figure 3 | |
| Receptor(s) |
|---|
1 receptor (Click to view) |