Application of HaloTag Protein to Covalent Immobilization of Recombinant Proteins for Single Molecule Force Spectroscopy

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• 作者：Yukinori Taniguchi ; Masaru Kawakami
• 刊名：Langmuir
• 出版年：2010
• 出版时间：July 6, 2010
• 年：2010
• 卷：26
• 期：13
• 页码：10433-10436
• 全文大小：773K
• 年卷期：v.26,no.13(July 6, 2010)
• ISSN：1520-5827

文摘

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.