ESCORTing proteins directly from whole cell-lysate for single-molecule studies
| Autor: | Shwetha Srinivasan, Gayathri S. Singaraju, Debadutta Deb, Sabyasachi Rakshit, Jagadish P. Hazra |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Cell Extracts Staphylococcus aureus Surface Properties Protein-protein interactions Biophysics 010402 general chemistry 01 natural sciences Biochemistry Article law.invention Protein–protein interaction 03 medical and health sciences Surface modification Bacterial Proteins law Molecule Molecular Biology Pull-down assays Tethering Chemistry Cell Biology Single-molecule experiment Aminoacyltransferases Single Molecule Imaging Recombinant Proteins 0104 chemical sciences Cysteine Endopeptidases 030104 developmental biology Covalent bond Recombinant DNA |
| Zdroj: | Analytical Biochemistry |
| ISSN: | 1096-0309 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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