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

ACS Omega
Ellis DurnerHermann E Gaub

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 ex...Continue Reading

References

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Citations

Aug 30, 2019·Proceedings of the National Academy of Sciences of the United States of America·Achim LöfJan Lipfert
Mar 18, 2020·Nanoscale·Leonard C SchendelHermann E Gaub
Sep 27, 2019·Scientific Reports·Marie SynakewiczLaura S Itzhaki
Apr 2, 2018·The Journal of Chemical Physics·Fidan SumbulFelix Rico
Jun 9, 2020·Frontiers in Molecular Biosciences·Byeongseon YangMichael A Nash
Jan 4, 2019·Nanoscale·Katherine R ErlichHermann E Gaub
Dec 2, 2020·Nature Chemistry·Alvaro Alonso-CaballeroJulio M Fernandez
Jul 25, 2020·Biochemistry·Rohit H SubramanianF Akif Tezcan
Feb 15, 2020·Current Opinion in Structural Biology·Lukas F Milles, Hermann E Gaub
Aug 23, 2019·The Journal of Physical Chemistry Letters·Guodong YuanPeng Zheng
Sep 2, 2021·Biophysics Reviews·Jorge Alegre-Cebollada

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Methods Mentioned

BETA
atomic force microscopy
PCR
gel filtration
electrophoresis
AFM
affinity purification

Software Mentioned

Image Lab

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