ENGINEERED CHIMERIC STREPTAVIDIN TETRAMERS AS NOVEL TOOLS FOR BIOSEPARATIONS AND DRUG-DELIVERY

We report the construction of chimeric streptavidin tetramers that are composed of subunits of both wild-type (WT) streptavidin and genetica lly-engineered streptavidin variants designed for enhanced bioseparati on and drug delivery performance, Subunit mixing is accomplished by gu anidine thiocyanate-induced denaturation of an equimolar mixture of WT streptavidin and the respective site-directed mutant, followed by ren aturation and reassociation of mixed tetramers, In the first example, we demonstrate the mixing of WT subunits with an Asn49Cys (N49C) mutan t, The WT/N49C tetramers can be used for site-specific and stoichiomet ric attachment of therapeutics/imaging agents or targeting proteins th rough the genetically-engineered thiol while retaining unhindered acce ss to biotin-binding at the WT subunits, Second, we demonstrate that t he His127Cys mutation (H127C) results in a streptavidin mutant that fo rms a disulfide-linked dimer under non-reducing conditions, Mixing of H127C and WT streptavidin subunits results in chimeric tetramers where both the stoichiometry (WT:H127C::1:1) and subunit architecture is co ntrolled by the unique disulfide bridge engineered into H127C. In the third example, WT subunits were mixed with the subunits of a site-dire cted mutant, Trp120Ala (W120A), which displays a biotin dissociation c onstant that is enhanced by more than 10(4) compared to WT streptavidi n, The W120A biotin-binding affinity is sufficiently high (K-a approxi mate to 10(7) M(-1)) to immobilize the mutant on a biotin-agarose affi nity chromatography column, but the engineered off-rate allows for fac ile elution with excess biotin at physiological pH, whereas WT strepta vidin is irreversibly immobilized on the column, We demonstrate that t he purified WT/W120A chimeric tetramers combine the advantages of both subunits, allowing for irreversible immobilization of biotinylated ta rgets at the WT subunit, while retaining the reversible separation cap abilities of the W120A subunits via biotin-agarose affinity chromatogr aphy.