| Streptavidin, a homotetrameric protein, binds biotin with extremely high affinity (Kd ∼ 10-14--10 -15 M) and has been widely used as an affinity reagent especially in medicine for both diagnostic and therapeutic applications. Its utility would be increased by engineering mutants with a wide spectrum of affinities and as single- rather than multiple-chain proteins. The latter feature is important for phage-display and chip technologies which cannot readily employ multi-subunit proteins. Taking into account the difference in calculated stability between the two subunit interfaces, we engineered reduced-size streptavidin mutants by destabilizing the dimer-dimer interface. The two-chain dimeric streptavidin mutants produced had biotin dissociation constants of about 10 -7 M. By a crossover-splicing procedure we converted one to a single-chain dimeric streptavidin (SCD), and introduced random mutations by error-prone PCR. Phagemid pCANTAB 5 E vectors bearing mutated genes were transformed into E.coli TG1 cells to form a bacterial library. These clones were expressed as fusion proteins on the 406-residue gene 3 protein (g3p) of M13 bacteriophage and panned with biotinylated beads to optimize for binding affinity and stability by phage display. Affinity-enriched phages were selected and sub-cloned into a pET system with T7 RNA polymerase to produce soluble proteins. Products from the original SCD gene and two candidate mutants for single-chain dimeric streptavidin were purified on a Superdex-75 10/300 GL column by Fast Performance Liquid Chromatography and then analyzed by MALDI-TOF mass spectrometry. The results showed that the dimers can tetramerize and exist in both dimeric and tetrameric forms. The two candidate mutant dimeric and tetrameric fractions were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization. Kd values ranged from 1--5 x 10-10 M. The binding affinity of these mutants to unmodified biotin---as determined by competition experiments with B4F---falls in the range of Kd = 10 -6--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. |
