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.

An Overview of Antibody Conjugated Polymeric Nanoparticles for Breast Cancer Therapy

“…Thus, other forms of avidin, like streptavidin or neutravidin, are preferred [132]. Contrary to avidin, streptavidin is not a glycoprotein [133,134] and its pI is much lower, which prevents interactions with sugar receptors [135]. One of the most elegant strategies applied consisted in engineering monomeric avidin/streptavidin to monovalent biotin binding [136] and nanosized poly-avidins named Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) [137].…”

A bio-coupling approach using a dextran-binding domain to immobilize an engineered streptavidin to Sephadex for easy preparation of affinity matrix

“…In this study, DBD is applied as an immobilization domain to anchor capturing proteins to chromatographic matrices such as Sephadex to generate the affinity matrices. It is interesting to monitor the retention ability of the bound DBD fusions in their monomeric and tetrameric states to Sephadex using fluorescent microscopy 4,22 . A His-Tagged version of SC-L-DBD with a cysteine in the C-terminal region was constructed (Fig.…”

“…The His-Tag enables purification of His-SC-L-DBD(Cys) by IMAC affinity chromatography 23 . Presence of a unique cysteine allows the fluorescent labelling of this protein through thiol coupling 4 . Pure M18-L-ST·His-SC-L-DBD(Cys) complexes with four DBD domains per tetrameric M18 (Fig.…”

“…A recently reported molecular biological approach has greatly simplified this process 4 . Molecular fusion of the capturing molecule to an agarose-binding domain (ABD) combines the purification and coupling processes into one step.…”

A suitable and effective stepwise oxidative refolding procedure for highly‐cationic tetrameric avidin in nucleic acid free conditions

“…5 Based on these technological backgrounds, various streptavidin derivatives have been developed for biotechnological application. [6][7][8][9] Many procedures have been reported for the production of recombinant cationic avidin. The first report in 1998 demonstrated refolding from bacterial inclusion bodies, 10 but subsequent studies employed secretion expression systems.…”