Cookies

We use cookies to ensure that we give you the best experience on our website. By continuing to browse this repository, you give consent for essential cookies to be used. You can read more about our Privacy and Cookie Policy.


Durham Research Online
You are in:

How to stabilize protein: stability screens for thermal shift assays and nano differential scanning fluorimetry in the Virus-X Project.

Bruce, D and Cardew, E and Freitag-Pohl, S and Pohl, E (2019) 'How to stabilize protein: stability screens for thermal shift assays and nano differential scanning fluorimetry in the Virus-X Project.', JoVE - journal of visualized experiments., 144 . e58666.

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.

Item Type:Article
Full text:(VoR) Version of Record
Available under License - Creative Commons Attribution.
Download PDF
(1661Kb)
Status:Peer-reviewed
Publisher Web site:https://doi.org/10.3791/58666
Publisher statement:Copyright © 2019 Creative Commons Attribution 3.0 License.
Date accepted:30 July 2018
Date deposited:12 February 2019
Date of first online publication:11 February 2019
Date first made open access:No date available

Save or Share this output

Export:
Export
Look up in GoogleScholar