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.
Highlights
Many biotechnologically-useful enzymes originate from viral sources, such as the tobacco etch virus (TEV) protease[1] and human rhinovirus type 3C (HRV 3C) protease[2]
The stability screens were designed for use in thermal shift assay (TSA) and nano differential scanning fluorimetry (nanoDSF) experiments to rapidly identify favorable conditions for a target protein (Figure 3, screen compositions are available in the supplementary information)
If an unsealed plate is used for a TSA experiment, there is a significant risk of solvent evaporating throughout the experiment, causing an increase in sample concentration and increasing the chance of premature protein aggregation
Summary
Many biotechnologically-useful enzymes originate from viral sources, such as the tobacco etch virus (TEV) protease[1] and human rhinovirus type 3C (HRV 3C) protease[2]. The stability screens were designed for use in TSA and nanoDSF experiments to rapidly identify favorable conditions for a target protein (Figure 3, screen compositions are available in the supplementary information). The screen can give a general indication of the affinity of a protein sample to the environments with high ionic strengths, but each subgroup of the compounds can provide information on the potential structure of a protein. Osmolytes are soluble compounds that affect the osmotic properties of their environment In nature, they can be used as "chemical chaperones", enforcing the folding of disordered proteins and stabilizing them, especially in stress conditions[20,21,22]. Each osmolyte is present in multiple concentrations based on its solubility and effective concentration ranges for comparison
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