Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein

Streptavidin, a homotetrameric protein with extremely tight biotin binding (Kd ≤ 10-14 M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. Kd′ values for B4F ranged from ≈10-11 to 10-10 M, although Kd values for biotin ranged from 10-6 to 10-5 M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin–biotin for composite separation or analysis steps.