Engineering monomeric streptavidin and its ligands with infinite affinity in binding but reversibility in interaction.
Natural tetrameric streptavidin captures and immobilizes biotinylated molecules with ultra-tight binding (K(d) approximately 10(-13) to 10(-14) M). In contrast, engineered monomeric streptavidin offers reversible binding (K(d) approximately 10(-7) M). To develop an ideal engineered streptavidin which possesses both the immobilization capability of the natural streptavidin and the reversible interaction reactivity of the monomeric streptavidin, a pair of engineered biomaterials was designed through molecular modeling. This system consists of two recombinant components: an engineered monomeric streptavidin M6, which has a cysteine residue (C118) near the biotin binding site, and a cysteine containing biotinylation tag. Interactions between M6 and the biotinylated peptide tag go through a two-stage process (capture and immobilization) to generate a covalently linked complex. Biotinylation is essential in the capture stage. Once the biotin moiety in the biotinylated tag is captured by M6, the biotinylated tag can fold back and rotate on the surface of the complex with the biotinylated lysine in the peptide tag as the axis until the formationof a disulfide bond. Consequently, cysteine residue in different positions flanking the biotin residue in the biotinylation tag can successfully form a disulfide bond with M6. Intermolecular disulfide bond formation between M6 and the tag containing protein offers the immobilization capability to M6. In the presence of reducing agent and biotin, bound ligands can be dissociated. This system has the potential to extend the biotin-streptavidin technology to develop reusable biosensor/protein chips and bioreactors.
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Sau-Ching
SC
Wu
Kenneth K-S
KK
Ng
Sui-Lam
SL
Wong
D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001665:Binding Sites / Q000502:physiology; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D003460:Crystallization; D018360:Crystallography, X-Ray; D008024:Ligands; D008969:Molecular Sequence Data; D015202:Protein Engineering; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D008024:Ligands; D001710:Biotin; D019809:Streptavidin