Protein Labeling and Monovalent Biotin Detection

ABSTRACT: The coupling between the quaternary struc-ture, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnol-ogy applications. For example, the binding pocket of strep-tavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was re-cently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, w