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
Hauptverfasser:	Taniguchi, Yukinori, Kawakami, Masaru
Format:	Artikel
Sprache:	eng
Schlagworte:	Chemistry Exact sciences and technology General and physical chemistry Hydrolases - chemistry Hydrolases - genetics Hydrolases - metabolism Microscopy, Atomic Force - methods Models, Theoretical Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism
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creator	Taniguchi, Yukinori Kawakami, Masaru
description	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.
doi_str_mv	10.1021/la101658a
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subjects	Chemistry Exact sciences and technology General and physical chemistry Hydrolases - chemistry Hydrolases - genetics Hydrolases - metabolism Microscopy, Atomic Force - methods Models, Theoretical Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism
title	Application of HaloTag Protein to Covalent Immobilization of Recombinant Proteins for Single Molecule Force Spectroscopy
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