An engineered redox-switchable streptavidin mutein enables the high-affinity capture and efficient elution of biotinylated ligands.

Although the high affinity and specificity of the interaction between streptavidin and biotin are widely utilized to isolate biotinylated ligands, their elution over practical timescales requires the use of strongly denaturing conditions that are incompatible with many target biomolecules. To overcome this limitation, we previously engineered a disulfide bond into a critical loop of streptavidin to create the redox-sensitive M88 mutein, which releases biotinylated ligands ~19,000-fold faster in the reduced state when compared with the oxidized state. To optimize the speed and efficiency of ligand recovery, we describe how multiple mechanisms like disulfide bond reduction, increasing temperature, elevating pH, and adding organic cosolvents can dramatically increase the rates of dissociation for different types of biotinylated ligands bound to M88 or wild-type streptavidin coupled to magnetic beads. By combining these mechanisms, we demonstrate how a range of biotinylated biomolecules (peptide, oligonucleotide, and protein) can be efficiently recovered under mild conditions that preserve biological activity. M88 magnetic beads thus provide many advantages over currently available methods using either wild-type streptavidin or lower affinity biotin-binding proteins for applications requiring both high affinity capture and efficient release of target biomolecules under non-denaturing conditions.