“Plug-and-Go” Strategy To Manipulate Streptavidin Valencies

Abstract

The streptavidin-biotin set is one of the most widely utilized conjugation pairs in biotechnological applications. The tetravalent nature of streptavidin and its homologues, however, tends to result in such undesirable complications as cross-linking or ill-defined stoichiometry. Here, we describe a mutagenesis-free strategy to manipulate the valencies of wild-type streptavidin that only requires commercially available reagents. The basic idea is simple: one obtains the desired streptavidin valency by blocking off unwanted binding sites using ancillary biotin ("plug"); this way, the extraordinary fM-biotin-binding affinity is fully retained for the remaining sites in streptavidin. In the present implementation, the ancillary biotin is attached to an auxiliary separation handle, negatively charged DNA or His-tagged protein, via a photochemically or enzymatically cleavable linker. Mixing streptavidin with the ancillary biotin construct produces a distribution of streptavidin valencies. The subsequent chromatographic separation readily isolates the construct of desired streptavidin valency, and the auxiliary handles are easily removed afterward ("go"). We demonstrate how this "plug-and-go" strategy allows a precise control for the compositions of streptavidin-biotin conjugates at the single-molecule level. This low-entry-barrier protocol could further expand the application scope of the streptavidin technology.

Streptavidin interfacing as a general strategy to localize fluorescent membrane tension probes in cells

“…The schematic structures of all streptavidin complexes including 19 show the molar ratios of the components used. In reality, the multivalency of streptavidin complicates the situation 26–28. Without cooperativity effects, the complex stoichiometries reflecting the substrate ratio should dominate clearly 27.…”

“…1c and 2). Streptavidin–biotin interfacing is one of the best explored methods in biotechnology 24–28. The multivalency of the streptavidin tetramer provides unique versatility; examples extend from the combination of cellular uptake with fluorescent labeling, molecular recognition, self-assembly and catalysis24 to the construction of ordered multicomponent architectures on solid surfaces (Fig.…”

Single-Molecule Fluorescence Methods to Study Plant Hormone Signal Transduction Pathways

“…To resolve this problem, modified streptavidin with only two binding sites can be used. This modification is performed using anion-exchange columns that prepare multivalent streptavidin (Sun et al, 2014 ). Streptavidin with two dsDNA can be used for linkage because of the prior blocking of the two biotin-binding sites.…”

“…If protein dimerization or stoichiometry is under investigation, the proteins can be fused with StrepII-tag or Avi-tag for direct linkage without the biotinylated His-tag antibody. Furthermore, bivalent streptavidin (Sun et al, 2014 ) were prepared to eliminate the possibility of additional binding sites on regular streptavidins by using the following procedure: the mixtures of streptavidin and biotinylated DNA were incubated for 0.5 h at room temperature at a molar ratio of 1:2. Streptavidins with different valences were separated through Mono-Q anion-exchange chromatography under alkaline conditions (optimal pH 8–9; Figure 3 ).…”

Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

“…When using biotinylated DNA as a binding ligand, the majority of streptavidin complexes have two to three DNA fragments per streptavidin even when the DNA:streptavidin ratio is five [ 46 ]. With the recent development of cis - and trans -divalent streptavidin [ 47 , 48 ], steric hindrance is shown to be a factor accounting for the observed negative cooperativity for binding two biotinylated molecules to the cis -sites in streptavidin. In the SAVSBPM32-SBP(A18C) system described above, each bound SBP-tag occupies the two biotin binding pockets located on the same side of streptavidin ( Fig 8 ).…”