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Oligohis-tags : mechanisms of binding to Ni2+-NTA surfaces

Knecht, Steven and Ricklin, Daniel and Eberle, A. N. and Ernst Beat, . (2009) Oligohis-tags : mechanisms of binding to Ni2+-NTA surfaces. Journal of Molecular Recognition, Vol. 22. pp. 270-279.

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Official URL: http://edoc.unibas.ch/dok/A5260035

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Abstract

Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni(2+) immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C- or N-terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni(2+)-NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni(2+)-NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K(D)) of 14 +/- 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding.
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molekulare Pharmazie (Ernst)
03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel
UniBasel Contributors:Eberle, Alex N. and Ernst, Beat
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Heyden
ISSN:0952-3499
Note:Publication type according to Uni Basel Research Database: Journal article
Identification Number:
Last Modified:01 Feb 2013 08:42
Deposited On:22 Mar 2012 13:48

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