Durner Ellis, Ott Wolfgang, Nash Michael A, Gaub Hermann E
Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität, 80799 Munich, Germany.
Department of Chemistry, University of Basel, 4056 Basel, Switzerland.
ACS Omega. 2017 Jun 30;2(6):3064-3069. doi: 10.1021/acsomega.7b00478.
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 .
单分子力谱技术极大地受益于位点特异性的表面固定以及使用功能化悬臂进行特异性探测。在此，我们描述了一种简化的此类实验方法，即通过将机械稳定的受体共价连接到目标蛋白（POI）上，以提高捕获效率和特异性。该平台提高了通量，允许对拉伸几何结构进行精确控制，并允许使用相同的配体修饰悬臂对多种构建体进行探测。我们采用两种正交的酶促连接反应[分选酶和磷酸泛酰巯基乙胺基转移酶（Sfp）]将POI共价固定到聚乙二醇化表面，并随后将POI连接到蛋白质C末端的机械稳定的dockerin结构域，用作高强度的拉伸手柄。我们的配置允许POI的表达和折叠独立于用于特异性探测的机械稳定受体进行，并且仅需要两个短的末端肽序列（即ybbR标签和分选酶C标签）。我们成功地将该系统应用于使用体外转录和翻译反应表达的蛋白质，无需蛋白质纯化步骤，也应用于在……中表达的纯化蛋白质。
