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The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins

Nature Protocols volume 2, pages 1528–1535 (2007)Cite this article

Abstract

The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins—including their complexes with interacting partners—both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity 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.

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Figure 1: Schematic illustration of the Strep-tag purification cycle.
Figure 2: Purification of rhtTGase from a mammalian cell lysate.
Figure 3: Immobilization of rhtTGase carrying the Strep-tag II via StrepMAB-Immo as detected in a Biacore measurement.

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Acknowledgements

A.S. wishes to thank the DFG und CiPSM for financial support and Ina Theobald, TU Munich, for providing Biacore data. T.S. wishes to thank Neil Smyth, University of Southampton, for kindly providing HEK 293 cells transfected with an expression plasmid for rhtTGase, Thomas I. Koblizek, IBA, for experimental data and critically reading the manuscript, and Kristian Stanar, IBA, for technical assistance.

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Authors and Affiliations

  1. IBA GmbH, Rudolf-Wissell-Strasse 28, Göttingen, 37079, Germany

    Thomas GM Schmidt

  2. Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, 85350, Germany

    Arne Skerra

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Correspondence to Arne Skerra.

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Competing interests

T.S. is an employee (COO) of IBA, a company that commercializes the Strep-tag technology.

A.S. is a consultant to IBA and inventor on patents relating to the Strep-tag technology.

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Schmidt, T., Skerra, A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2, 1528–1535 (2007). https://doi.org/10.1038/nprot.2007.209

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