Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles.

ABSTRACT: Single-molecule force spectroscopy greatly benefits from site-specific surface immobilization and specific probing with a functionalized cantilever. Here, we describe a streamlined approach to such experiments by covalently attaching mechanically stable receptors onto proteins of interest (POI) to improve pickup efficiency and specificity. This platform provides improved throughput, allows precise control over the pulling geometry, and allows for multiple constructs to be probed with the same ligand-modified cantilever. We employ two orthogonal enzymatic ligation reactions [sortase and phosphopantetheinyl transferase (Sfp)] to covalently immobilize POI to a pegylated surface and to subsequently ligate the POI to a mechanically stable dockerin domain at the protein's C-terminus for use as a

SUBMITTER: Durner E 

PROVIDER: S-EPMC6044863 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature