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Post-translational Sortase-mediated Attachment of High-strength Force Spectroscopy Handles

Durner, Ellis and Ott, Wolfgang and Nash, Michael A. and Gaub, Hermann E.. (2017) Post-translational Sortase-mediated Attachment of High-strength Force Spectroscopy Handles. ACS Omega, 2 (6). pp. 3064-3069.

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Official URL: http://edoc.unibas.ch/58783/

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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 high-strength pulling handle. Our configuration permits expression and folding of the POI to proceed independently from the mechanically stable receptor used for specific probing and requires only two short terminal peptide sequences (i.e., ybbR-tag and sortase C-tag). We applied this system successfully to proteins expressed using in vitro transcription and translation reactions without a protein purification step and to purified proteins expressed in Escherichia coli.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie
05 Faculty of Science > Departement Chemie > Chemie > Synthetic Systems (Nash)
UniBasel Contributors:Nash, Michael
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:American Chemical Society
ISSN:2470-1343
e-ISSN:2470-1343
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
Identification Number:
Last Modified:15 Jan 2018 09:48
Deposited On:15 Jan 2018 09:48

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