A Self-Assembled Monolayer for the Binding and Study of Histidine-Tagged Proteins by Surface Plasmon Resonance

This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag:  a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a g...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1996-02, Vol.68 (3), p.490-497
Main Authors: Sigal, George B, Bamdad, Cynthia, Barberis, Alcide, Strominger, Jack, Whitesides, George M
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Chemistry
Fundamental and applied biological sciences. Psychology
Histidine - chemistry
Immobilization of enzymes and other molecules
Immobilization techniques
Indicators and Reagents
Kinetics
Membranes, Artificial
Methods. Procedures. Technologies
Protein Binding
Proteins
Proteins - chemistry
Proteins - isolation & purification
Surface Properties
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Summary:This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag:  a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a gold surface of a mixture of two alkanethiols:  one thiol that terminated with a nitrilotriacetic acid (NTA) group, a group that forms a tetravalent chelate with Ni(II), and a second thiol that terminated with a tri(ethylene glycol) group, a group that resists protein adsorption. His-tagged proteins bound to the SAM by interaction of the histidines with the two vacant sites on Ni(II) ions chelated to the surface NTA groups. Studies with model proteins showed the binding was specific for His-tagged proteins and required the presence of Ni(II) on the surface. Immobilized His-tagged proteins were kinetically stable in buffered saline at pH 7.2 but could be desorbed by treatment with 200 mM imidazole. Surface plasmon resonance studies for two model systems showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than proteins immobilized in a thin dextran gel layer by covalent coupling.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac9504023