The intrinsic cysteine and histidine residues of the anti-Salmonella antibody Se155-4: a model for the introduction of new functions into antibody-binding sites
| Autor: | Anna M Cunningham, David C. Watson, N. Martin Young, C. Roger MacKenzie |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Bioengineering Enzyme-Linked Immunosorbent Assay Alkylation Protein Engineering Biochemistry Insert (molecular biology) metal affinity Antigen Salmonella Escherichia coli histidine and cysteine residues Histidine Cysteine Lyase activity Molecular Biology Fluorescent Dyes biology Chemistry Hydrogen-Ion Concentration Fluorescence Antibodies Bacterial binding-site modification Recombinant Proteins biology.protein Binding Sites Antibody Antibody CDR4 loops Copper Biotechnology |
| Zdroj: | Protein engineering, designselection : PEDS. 27(10) |
| ISSN: | 1741-0134 |
| Popis: | New functions can be incorporated into anti-hapten or anti-protein antibodies by mutating selected residues in the binding-site region either to Cys, to allow alkylation with reagents bearing the desired functional groups, or to His, to create metal-binding sites or to make antigen binding pH-sensitive. However, choosing suitable sites for these mutations has been hampered by the lack of antibodies with these features, to serve as models. Remarkably, the anti-carbohydrate antibody Se155-4, specific for the Salmonella group B lipopolysaccharide, already has a Cys and two pairs of His residues close to the antigen-binding pocket in its structure, and shows pH-dependent antigen binding. We therefore investigated modification of its Cys94L in an scFv version of the antibody with the aims of creating a ‘reagentless’ fluorescent sensor and attaching a metal-binding group that might confer lyase activity. These groups were successfully introduced, as judged by mass spectrometry, and had only slightly reduced antigen binding in enzyme-linked immunosorbent assay. The fluorescent product was sensitive to addition of antigen in a solution format, unlike a modification of a more distant Cys introduced into the VH CDR4 loop. Two other routes to modulate antigen binding were also explored, metal binding by the His pair alongside the antigen-binding pocket and insertions into CDR4 to extend the antigen-contact area. His residues adjacent to the antigen-binding pocket bound copper, causing a 5-fold decrease in antigen binding. In CDR4 of the VH domain, the preferred insert length was four residues, which gave stable antigen-binding products but did not improve overall antigen affinity. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|