Monomeric streptavidin with reversible biotin binding capability has many potential applications. Because a complete biotin binding site in each streptavidin subunit requires the contribution of tryptophan 120 from a neighboring subunit, monomerization of the natural tetrameric streptavidin can generate streptavidin with reduced biotin binding affinity. Three residues, valine 55, threonine 76, and valine 125, were changed to either arginine or threonine to create electrostatic repulsion and steric hindrance at the interfaces. The double mutation (T76R,V125R) was highly effective to monomerize streptavidin. Because interfacial hydrophobic residues are exposed to solvent once tetrameric streptavidin is converted to the monomeric state, a quadruple mutein (T76R,V125R,V55T,L109T) was developed. The first two mutations are for monomerization, whereas the last two mutations aim to improve hydrophilicity at the interface to minimize aggregation. Monomerization was confirmed by four different approaches including gel filtration, dynamic light scattering, sensitivity to proteinase K, and chemical cross-linking. The quadruple mutein remained in the monomeric state at a concentration greater than 2 mg/ml. Its kinetic parameters for interaction with biotin suggest excellent reversible biotin binding capability, which enables the mutein to be easily purified on the biotin-agarose matrix. Another mutein (D61A,W120K) was developed based on two mutations that have been shown to be effective in monomerizing avidin. This streptavidin mutein was oligomeric in nature. This illustrates the importance in selecting the appropriate residues and approaches for effective monomerization of streptavidin.(Strept)avidin with reversible biotin binding capability can extend the applications of the biotin-(strept)avidin technology. These molecules can be applied for affinity purification of biotinylated biomolecules, screening of ultratight binders binding to biotinylated biomolecules displayed on the phage display system, and development of reusable biosensor chips, protein/ antibody microarrays, and enzyme bioreactors (1, 2). (Strept)avidin is a homotetrameric molecule with a biotin binding site in each subunit (3). The three-dimensional structure of (strept)avidin (4, 5) suggests that a complete biotin binding pocket in each subunit requires the contribution of a tryptophan residue from an adjacent subunit. Site-directed mutagenesis studies also demonstrate the importance of this residue for tight biotin binding and subunit communications (6 -8). Therefore, development of monomeric (strept)avidin can be an attractive approach to engineer (strept)avidin with reversible biotin binding capability.The engineering of (strept)avidin to its monomeric form is technically challenging. In the case of avidin, the first generation of engineered monomeric avidin can exist in the monomeric state only in the absence of biotin (9). This problem has been solved by the recent development of the second generation of monomeric avidin (10), which carries two mut...

The 38-residue SBP-Tag binds to streptavidin more tightly (K d ' 2.5-4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12-14) and a C-terminal HPQ sequence (residues 31-33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17-28) adopts a regular -helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1-10 and 35-38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11-34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of -lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tagstreptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.