ESCORTing proteins directly from whole cell-lysate for single-molecule studies

• DOI: 10.1016/j.ab.2017.07.022
• Publication Date: 2017-07-26T17:48:04Z
• Authors: Shwetha Srinivasan; Jagadish P. Hazra; Gayathri S. Singa...; Debadutta Deb; Sabyasachi Rakshit
• Keywords: Cell Extracts; 0301 basic medicine; Cysteine Endopeptidases; Staphylococcus aureus; 0303 health sciences; 03 medical and health sciences; Bacterial Proteins; Surface Properties; 14. Life underwater; Aminoacyltransferases; Article; Recombinant Proteins; Single Molecule Imaging

We have developed a method for Enzymatic Sortase-assisted Covalent Orientation-specific Restraint Tethering (ESCORT) recombinant proteins onto surfaces directly from cell-lysate. With an improved surface passivation method, we obviate the cumbersome purification steps even for single molecule studies that demand high purity in the sample. We demonstrated high-specificity of the method, high-passivity of the surface and uncompromised functional integrity of anchored proteins using single molecule fluorescence and force-mapping. We anticipate that this method will substantially reduce the investment by way of time, money and energy in the area of single molecule studies.