Electrophilic Affibodies Forming Covalent Bonds to Protein Targets*
AUTOR(ES)
Holm, Lotta
FONTE
American Society for Biochemistry and Molecular Biology
RESUMO
Antibody affinity limits sensitivity of detection in many areas of biology and medicine. High affinity usually depends on achieving the optimal combination of the natural 20 amino acids in the antibody binding site. Here, we investigate the effect on recognition of protein targets of placing an unnatural electrophile adjacent to the target binding site. We positioned a weak electrophile, acrylamide, near the binding site between an affibody, a non-immunoglobulin binding scaffold, and its protein target. The proximity between cysteine, lysine, or histidine on the target protein drove covalent bond formation to the electrophile on the affibody. Covalent bonds did not form to a non-interacting point mutant of the target, and there was minimal cross-reactivity with serum, cell lysate, or when imaging at the cell surface. Electrophilic affibodies showed more stable protein imaging at the surface of mammalian cells, and the sensitivity of protein detection in an immunoassay improved by two orders of magnitude. Thus electrophilic affibodies combined good specificity with improved detection of protein targets.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2781706Documentos Relacionados
- Unsynchronized resonance of covalent bonds in the superconducting state
- Covalent Structure of Bovine Neurophysin-II: Localization of the Disulfide Bonds
- Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA.
- Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired.
- Induced formation of covalent bonds between nucleoprotein components. V. UV or bisulfite induced polynucleotide-protein crosslinkage in bacteriophage MS2.
