title: A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix.
abstract: SAVSBPM18 is an engineered streptavidin for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags. To develop a user-friendly approach for the preparation of the SAVSBPM18-based affinity matrices, a designer fusion protein containing SAVSBPM18 and a galactose binding domain was engineered. The galactose binding domain derived from the earthworm lectin EW29 was genetically modified to eliminate a proteolytic cleavage site located at the beginning of the domain. This domain was fused to the C-terminal end of SAVSBPM18. It allows the SAVSBPM18 fusions to bind reversibly to agarose and can serve as an affinity handle for purification of the fusion. Fluorescently labeled SAVSBPM18 fusions were found to be stably immobilized on Sepharose 6B-CL. The enhanced immobilization capability of the fusion to the agarose beads results from the avidity effect mediated by the tetrameric nature of SAVSBPM18. This approach allows the consolidation of purification and immobilization of SAVSBPM18 fusions to Sepharose 6B-CL in one step for affinity matrix preparation. The resulting affinity matrix has been successfully applied to purify both SBP tagged β-lactamase and biotinylated proteins. No significant reduction in binding capacity of the column was observed for at least six months.
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr., N.W. Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Sau-Ching
authors.initials: SC
authors.lastname: Wu
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr., N.W. Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Chris
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr., N.W. Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Dave
authors.initials: D
authors.lastname: Hansen
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr., N.W. Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Sui-Lam
authors.initials: SL
authors.lastname: Wong
mesh_terms: D000818:Animals; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D037161:Galectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D009835:Oligochaeta / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000302:isolation & purification
chemical_list: C542531:EW29 lectin, Lumbricus terrestris; D037161:Galectins; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D012710:Serum Albumin, Bovine; C047264:sepharose CL 6B; D012685:Sepharose; D019809:Streptavidin; D001618:beta-Lactamases