Directed Evolution of Streptavidin Variants Using IVC

We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities (~10 −13 M), but the variants have off-rates almost 50 times slower and a half-life for dissociation of 24 hours at 25°C. The utility of streptavidin variants with altered specificities and kinetic properties was demonstrated by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents, and for evolving other extremely high affinity protein:ligand couples.