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.
Highlights
Streptavidin-biotin technology has been widely used in many in vitro and in vivo applications including the capture and immobilization of biotinylated biomolecules, cell imaging, drug delivery, radioimmunotherapy, the generation of artificial cellulosomes and the building of PLOS ONE | DOI:10.1371/journal.pone.0139137 September 25, 2015Affinity-Driven Thiol Coupling of Streptavidin and Its Binding Tag analysis, decision to publish, or preparation of the manuscript
A86 of streptavidin and A18 of the streptavidin-binding peptide tag (SBP-tag) were chosen for mutagenesis to cysteine residues (Fig 1, panels B and C) as they are the best candidates for three reasons
To limit the loss of binding interactions caused by mutations, the residues in the SBP-tag selected for mutagenesis should not have doi:10.1371/journal.pone.0139137.g001
Summary
Streptavidin-biotin technology has been widely used in many in vitro and in vivo applications including the capture and immobilization of biotinylated biomolecules, cell imaging, drug delivery, radioimmunotherapy, the generation of artificial cellulosomes and the building of PLOS ONE | DOI:10.1371/journal.pone.0139137 September 25, 2015. It would be ideal to have a designer streptavidin-SBP tag system which allows the engineered SBP-tag to have infinite affinity to streptavidin via covalent bond formation With this feature, the peptide-tagged biomolecules could be immobilized to streptavidin with a bond strength that is even stronger than the traditional streptavidin-biotin interaction. Breakage of the covalent bond between streptavidin and SBP tag would be desirable This would allow reversible interactions between these molecules. Under optimized wash conditions, the new system does not show protein leakage in the wash fractions even when an affinity column is overloaded with the tagged protein, a problem previously observed in the SAVSBPM18-SBP system [13] This newly developed system further advances the streptavidin-SBP technology to have the flexibility for both immobilization and reversible binding applications. This method can be applied to other systems for analyzing the composition of disulfide-bonded protein complexes
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