Application of HaloTag Protein to Covalent Immobilization of Recombinant Proteins for Single Molecule Force Spectroscopy
We have developed the HaloTag system for the covalent immobilization of polyproteins onto a mica substrate for single molecule force spectroscopy using the atomic force microscope. A recombinant fusion polyprotein of titin I27 with HaloTag7 protein was produced, and the covalent and site-specific at...
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Veröffentlicht in: | Langmuir 2010-07, Vol.26 (13), p.10433-10436 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | We have developed the HaloTag system for the covalent immobilization of polyproteins onto a mica substrate for single molecule force spectroscopy using the atomic force microscope. A recombinant fusion polyprotein of titin I27 with HaloTag7 protein was produced, and the covalent and site-specific attachment on a HaloTag-ligand-modified mica surface was confirmed by force−extension measurements. Two mechanical unfolding intermediates of HaloTag7 protein were found by contour length analysis. This tethering method allows site-specific covalent immobilization of a protein that complements the standard method utilizing thiol−gold interaction, thus facilitating force−extension measurements for cysteine-containing proteins. |
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ISSN: | 0743-7463 1520-5827 |
DOI: | 10.1021/la101658a |