title: 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.
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Dawson
authors.initials: D
authors.lastname: Fogen
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Sau-Ching
authors.initials: SC
authors.lastname: Wu
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Kenneth Kai-Sing
authors.initials: KK
authors.lastname: Ng
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Sui-Lam
authors.initials: SL
authors.lastname: Wong
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008024:Ligands; D058619:Maltose-Binding Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; C452700:streptavidin-binding peptide; D001710:Biotin; D019809:Streptavidin; D003545:Cysteine