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

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DOIResolve DOI: https://doi.org/10.1093/protein/gzu018
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Name affiliation
  1. National Research Council Canada. Human Health Therapeutics
FormatText
TypeArticle
Journal titleProtein Engineering Design and Selection
ISSN1741-0126
1741-0134
Volume27
Issue10
Pages383–390; # of pages: 8
Subjectbinding-site modification; CDR4 loops; histidine and cysteine residues; metal affinity
Abstract
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.
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PublisherOxford University Press
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LanguageEnglish
Peer reviewedYes
NRC numberNRC-HHT-53216
NPARC number21275352
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Record identifier25687992-030e-43ae-ba38-cea14256c0b9
Record created2015-06-15
Record modified2019-03-06
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