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Noncovalent, site-specific biotinylation of histidine-tagged proteins.
Institute of Biochemistry, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany.
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2007 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 22Article in journal (Refereed) Published
Abstract [en]

Site-specific conjugation of proteins to surfaces, spectroscopic probes, or other functional units is a key task for implementing biochemical assays. The streptavidin-biotin interaction has proven a highly versatile tool for detection, quantification, and functional analysis of proteins. We have developed an approach for site-specific reversible biotinylation of recombinant proteins through their histidine tag using biotin conjugated to the multivalent chelator trisnitrilotriacetic acid (BTtris-NTA). Stable binding of BTtris-NTA to His-tagged proteins was demonstrated, which is readily reversed by addition of imidazole, enabling versatile conjugation schemes in solution as well as at interfaces. Gel filtration experiments revealed that His-tagged proteins bind to streptavidin doped with BTtris-NTA in a 2:1 stoichiometry. Furthermore, an increased binding affinity toward His-tagged proteins was observed for BTtris-NTA linked to streptavidin compared to tris-NTA in solution and on surfaces. These results indicate an efficient cooperative interaction of two adjacent tris-NTA moieties with a single His-tag, yielding an extremely tight complex with a lifetime of several days. We demonstrate several applications of BTtris-NTA including multiplexed capturing of proteins to biosensor surfaces, cell surface labeling, and Western blot detection. The remarkable selectivity of the His-tag-specific biotinylation, as well as the highly stable, yet reversible complex provides the basis for numerous further applications for functional protein analysis.

Place, publisher, year, edition, pages
2007. Vol. 79, no 22
National Category
Cell and Molecular Biology
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URN: urn:nbn:se:umu:diva-116891DOI: 10.1021/ac0714922PubMedID: 17953454OAI: oai:DiVA.org:umu-116891DiVA: diva2:903295
Available from: 2016-02-15 Created: 2016-02-15 Last updated: 2016-02-15

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Al Furoukh, Natalie
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