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.