A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein
Creators: name: Mukherjee, Sonia
name: Leblanc, Pierre
name: Poznansky, Mark C
name: Sluder, Ann E
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Statistics
subject: FOS: Mathematics
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4′-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins. The avidin-biotin interaction is a robust protein-ligand interaction utilized for several applications. We have developed a miniaturized assay protocol using the small molecule dye HABA to quantify the available biotin binding sites in avidin-containing fusion proteins. This method also serves as a quality control assay for different batches of assembled avidin-biotin fusion proteins, reducing technical complexities and time required for batch release. Application of the HABA dye assay to determine the number of biotin binding sites in an avidin-containing fusion protein and to evaluate the saturation of an avidin fusion-based vaccine with biotinylated peptides. Proof-of-concept study was conducted by biotin competition assay using commercially purchased avidin. HABA dye and binding competition assay was validated for MtbHSP70 avidin (MAV) fusion protein with biotin monomer. The impact of increasing PBS concentrations on the HABA dye assay was analyzed. HABA dye and biotinylated peptide competition assay was evaluated for MAV. The number of biotin binding sites was estimated in the MtbHSP70-avidin fusion protein using a colorimetric one-step assay requiting no wash steps or complex instrumentation. Increasing the ionic concentration of the reaction buffer had no impact on assay performance from a range of 0.25X–2X PBS. The assay was also utilized with biotinylated peptide ligands to assess whether peptides with complex structures showed evidence of steric hinderance while binding to the single biotin binding site in the fusion protein. This assay can be employed as a quality control assay for different batches of the MAV fusion protein and to evaluate vaccine assembly as a final step before preclinical evaluation in animal models.