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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 (Engelska)Ingår i: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, nr 22Artikel i tidskrift (Refereegranskat) 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.

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2007. Vol. 79, nr 22
Nationell ämneskategori
Cell- och molekylärbiologi
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URN: urn:nbn:se:umu:diva-116891DOI: 10.1021/ac0714922PubMedID: 17953454OAI: oai:DiVA.org:umu-116891DiVA, id: diva2:903295
Tillgänglig från: 2016-02-15 Skapad: 2016-02-15 Senast uppdaterad: 2018-06-07

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