PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays
| Autor: | James W. Saville, Franck Duong Van Hoa, Lucy Troman |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
General Chemical Engineering
protein-protein interactions receptors membrane proteins transporters Plasma protein binding channels biosensor General Biochemistry Genetics and Molecular Biology Protein–protein interaction lipids biotin-streptavidin 03 medical and health sciences 0302 clinical medicine nanodiscs Protein purification Biotinylation 030304 developmental biology 0303 health sciences General Immunology and Microbiology Chemistry General Neuroscience Membrane Proteins Membranes Artificial Ligand (biochemistry) Kinetics Interferometry Membrane Solubility Membrane protein Colicin membrane mimetics Biophysics Streptavidin colicin 030217 neurology & neurosurgery Protein Binding |
| Zdroj: | Saville, J, Troman, L A & Duong Van Hoa, F 2019, ' PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays ', Journal of Visualized Experiments, vol. 153, e60661 . https://doi.org/10.3791/60661 |
| ISSN: | 1940-087X |
| DOI: | 10.3791/60661-v |
| Popis: | Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a “one-size-fits-all” approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one day in a detergent-free environment. |
| Databáze: | OpenAIRE |
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