Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein.
Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 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. K'(d) values for B4F ranged from approximately 10(-11) to 10(-10) M, although K(d) 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.
Aslan
Filiz M
FM
Center for Advanced Biotechnology, Department of Chemistry, and Biomolecular Engineering Research Center, Boston University, Boston, MA 02215, USA.
Yu
Yong
Y
Mohr
Scott C
SC
Cantor
Charles R
CR
D000345:Affinity Labels / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D005452:Fluoresceins / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides; D017510:Protein Folding; D017422:Sequence Analysis, DNA; D019809:Streptavidin / Q000235:genetics* / Q000378:metabolism*
D000345:Affinity Labels; D005452:Fluoresceins; D009841:Oligonucleotides; C118194:biotin-4-fluorescein; D001710:Biotin; D019809:Streptavidin