Multiplexed Affinity Characterization of Protein Binders Directly from a Crude Cell Lysate by Covalent Capture on Suspension Bead Arrays.
Anal Chem
; 93(4): 2166-2173, 2021 02 02.
Article
em En
| MEDLINE
| ID: mdl-33397084
ABSTRACT
The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (Kd) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Proteínas Recombinantes
/
Proteínas Fúngicas
/
Proteínas Imobilizadas
/
Anticorpos de Cadeia Única
/
Microesferas
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Reino Unido