The search for a mono-valent high-affinity biotin binder (monoavidin)

The avidin and streptavidin were shown to have an ultra-high affinity towards d-biotin. Both proteins are tetramers with four biotin binding sites. The high affinity is extensively utilized as a powerful and diverse biotechnological tool. However, the tetrameric assembly of avidins with multiple biotin binding sites has many advantages but also drawbacks such as unwanted cross-linking and their size. The approach to obtain lower valency high-affinity biotin binder was sought in our lab by screening for new sequences identifying novel avidin-like proteins. We and others discovered a new subfamily of dimeric avidins including 7 members. This new subfamily was essentially a dimer that has the propensity to be manipulated into a monomer. The main goal is to design a monomeric low valency high-affinity biotin binder. Attempts to engineer a monomer by manipulating the dimeric interface resulted in aggregates. Consequently, we used mutagenesis and molecular biology to design and analyze single chain monovalent shwanavidin (one of the dimeric avidins) by introducing a 26-residue linker connecting the C’ and subsequent N’ termini of the monomers and evaluated their structures. We have consequently introduced 4 mutations in the biotin binding site of one monomer. The tetra-mutant single chain shwanavidin appeared to bind biotin but does not tend to crystalize suggesting that differences in the second monomer are significant. The formation of a single chain monovalent shwanavidin with high affinity towards biotin is a valuable approach extending the toolbox of the biotin-based technology.