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.
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Publication date: Available online 4 January 2018 Source: European Journal of Radiology Author(s): Peiyao Zhang, Jing Wang, Qin Xu, Zhen...
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Publication date: March 2017 Source: Free Radical Biology and Medicine, Volume 104 from #AlexandrosSfakianakis via Alexandros G.Sfak...
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Dtsch med Wochenschr DOI: 10.1055/s-0043-100054 Hintergrund und Fragestellung Ein etablierter Weg, die optimale Behandlung von Tumorpatien...
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Background Hyperthyroidism is associated with increased thrombotic risk. As contact system activation through formation of neutrophil extrac...
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Abstract Limited memory size is considered as a major bottleneck in data centers for intelligent urban computing. It is shown that there e...
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ecancer is supporting #BowelCancerAwarenessMonth https://t.co/opXxCAAxzE from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inore...
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Linked Article: Maintz et al. Br J Dermatol 2017; 176:481–487 . from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader h...
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Angelina Jolie’s openness about her diagnosis has undoubtedly raised public awareness of the importance of inherited cancers. Although Desai...
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