Stable High Affinity Streptavidin Protomers for Cell Biology Application

While the streptavidin-biotin interaction is the basis of many biotechnology and medical applications, the large size of streptavidin tetramer and the multivalency of the molecule present obstacles in some situations. The focus of the current study is thus to engineer smaller streptavidin variants that have sufficient stability and affinity to make the technology accessible to other applications, including in vivo cell imaging. To this end, we adopted a rational design approach to engineer a monomer and a “hairline” dimer mutant with improved stability and affinity. We tested monomer mutants containing one or more engineered disulfides to stabilize the biotin-bound conformation. The best mutant was then engineered further by introducing surface mutations to create an extended stabilizing salt bridge network. The T_m = 47 °C and K_d = 60 nM of the resulting monomer, which compare favorably with those of wt monomer (T_m = 32 °C and K_d = 130 nM), allow the molecule to be used in a cell biology experiment performed at 37 °C. It has long been suspected that “hairline” interaction across the dimer-dimer interface is important for high affinity biotin binding, but engineering such a dimer has been difficult due to limited interface between the subunits. To engineer a stable hairline dimer, we modeled the structure and introduced an interchain disulfide between two designed monomer subunits. We show that the purified dimer, which elutes as a single peak by gel filtration and has high monodispersity by dynamic light scattering, is more stable than the substituent monomer by ~ 6 °C and has an 8 fold higher affinity compared to wt monomer, making it an ideal molecule for applications that require a stable labeling reagent.

In addition to rational design of streptavidin protomers, we will present results from two other parallel works for improving the biophysical properties of streptavidin monomer. First, we have recently demonstrated that functional streptavidin monomer can be displayed on the yeast surface using biotin as a molecular chaperone. This allowed us to improve the stability of rationally designed molecule by directed evolution, which has not been possible until now due to the difficulties of folding streptavidin in yeast. Second, we used homology modeling of streptavidin and streptavidin-like molecules from other organisms to create a stable hybrid molecule that is roughly 20% of the mw of wt streptavidin. Toward improving the thermodynamic and binding properties of streptavidin monomer, we will present a comparative analysis of designed chimeras that reveal the challenges of engineering an optimized a monomer sequence.