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 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.
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Yukinori
Y
Taniguchi
Masaru
M
Kawakami
D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods*; D008962:Models, Theoretical; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D006867:Hydrolases; C046137:haloalkane dehalogenase