How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project

Bruce, D; Cardew, E; Freitag-Pohl, S; Pohl, E

Abstract

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To evaluate the potential biotechnical value of these proteins, the analysis of protein structures and functions is a central challenge in this program. The stability of protein sample is essential to provide meaningful assay results and increase the crystallizability of the targets. The thermal shift assay (TSA), a fluorescence-based technique, is established as a popular method for optimizing the conditions for protein stability in high-throughput. In TSAs, the employed fluorophores are extrinsic, environmentally-sensitive dyes. An alternative, similar technique is nano differential scanning fluorimetry (nanoDSF), which relies on protein native fluorescence. We present here a novel osmolyte screen, a 96-condition screen of organic additives designed to guide crystallization trials through preliminary TSA experiments. Together with previously-developed pH and salt screens, the set of three screens provides a comprehensive analysis of protein stability in a wide range of buffer systems and additives. The utility of the screens is demonstrated in the TSA and nanoDSF analysis of lysozyme and Protein X, a target protein of the Virus-X project.

Citation

Bruce, D., Cardew, E., Freitag-Pohl, S., & Pohl, E. (2019). How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project. Journal of Visualized Experiments, 144, Article e58666. https://doi.org/10.3791/58666

• Journal Article Type: Article
• Acceptance Date: Jul 30, 2018
• Online Publication Date: Feb 11, 2019
• Publication Date: Feb 11, 2019
• Deposit Date: Jul 30, 2018
• Publicly Available Date: Feb 12, 2019
• Journal: Journal of Visualized Experiments
• Electronic ISSN: 1940-087X
• Publisher: MyJove Corporation
• Peer Reviewed: Peer Reviewed
• Volume: 144
• Article Number: e58666
• DOI: https://doi.org/10.3791/58666
• Public URL: https://durham-repository.worktribe.com/output/1325264