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

Abstract

To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.

A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix

“…The second target protein for purification was an enzymatically biotinylated maltose binding protein (MBP-AviTag) mixed with a crude E. coli intracellular extract 39 . E. coli biotin ligase can enzymatically introduce a biotin to an AviTag 40 .…”

“…Based on the same principle, affinity matrices involving L-ABD(KC) fusions can also be prepared for different versions of streptavidin/avidin and their derivatives for affinity purification, capture and immunoprecipitation applications 41 . These include wild-type streptavidin and avidin 5 6 42 , SAVSBPM32 39 , Strep-Tactin 43 , traptavidin 3 and many other streptavidin/avidin muteins with different biotin binding affinities 4 5 44 . Besides being used in the traditional column format, an SAVSBPM18-L-ABD(KC) affinity matrix can also be easily prepared in the magnetic bead format with agarose coated magnetic beads for protein purification.…”

Enzymosomes: A Rising Effectual Tool for Targeted Drug Delivery System

“…This efficient communication between streptavidin and biotin containing lipid molecules is utilized in the indirect targeting procedures. Other drug discoveries like photoaffinity is used as additional mechanisms for molecular interactions and targets for the binding site via activation through light [91].…”

Engineering a disulfide-gated switch in streptavidin enables reversible binding without sacrificing binding affinity

“…Previous attempts to address these potential limitations have only been partially successful. One approach relies on engineering intermolecular disulfide bonds between biotinylated peptide tags or streptavidin binding peptides and streptavidin 19 , 20 . Although these intermolecular disulfide bonds successfully combine the high strength of covalent interactions and redox-dependent reversible binding, they can only be used for carefully engineered proteins and peptides, restricting their utility and increasing costs and complexity.…”