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The Strep-tag system for one-step purication and high-afnity detection or capturing of proteins
Thomas GM Schmidt1 & Arne Skerra2
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2 natureprotocols
1IBA GmbH, Rudolf-Wissell-Strasse 28, 37079 Gttingen, Germany. 2Lehrstuhl fr Biologische Chemie, Technische Universitat Mnchen, 85350 Freising-Weihenstephan, Germany. Correspondence should be addressed to A.S. ([email protected]).
Published online 14 June 2007; doi:10.1038/nprot.2007.209
The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic afnity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purication of corresponding Strep-tag II fusion proteinsincluding their complexes with interacting partnersboth from bacterial and eukaryotic cell lysates using afnity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-afnity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.
INTRODUCTIONEfcient procedures for purication, detection and immobilization or separationpossibly in complex with cognate macromoleculesare of key importance in modern protein science. In particular for structural genomics and proteome research, the rapid isolation of a recombinant gene product, preferentially under standardized high-throughput conditions, and the characterization of its biochemical activity or identication of interaction partners are crucial. The Strep-tag II (ref. 1) is a small-afnity peptide, which, after fusion with the recombinant protein of interest, offers a practically useful solution to these tasks.
The Strep-tag was originally selected from a genetic random library2 as an eight-amino-acid peptide (WRHPQFGG) that specically binds to core streptavidin, a proteolytically truncated version of the natural bacterial protein3. Owing to its extraordinary afnity for the small group D-biotin, together with its high intrinsic stability and low nonspecic interaction, core streptavidinas soluble protein, in immobilized form, conjugated with enzymes or even as engineered versionsis in wide use as a powerful reagent for the detection as well as separation of...