Thermofluor-based high-throughput stability optimization of proteins for structural studies
Section snippets
Sample preparation
All of the proteins used in the thermal shift assay are expressed from genes from E. coli (Table 1). The molecular weights of these proteins range from 19 to 79 kDa. Recombinant proteins were expressed with either a six-residue His fusion tag both N-terminally and C-terminally, a C-terminal His fusion tag (STHHHHHH-C) and an N-terminal Flag fusion tag (N-MDYKDDDDKGSTSLYKKAGSTELYIQG), or only an N-terminal His fusion tag (N-MHHHHHHGSTSLYKKAGFEDRT) using a PT73.3HisGW, PT73.3FlagGW, or modified
Initial protein screen
An initial experiment was performed with the fluorescent dye Sypro Orange and 25 proteins (Fig. 1 and Table 1). Sypro Orange has low quantum yield in aqueous solution but is highly fluorescent in nonpolar environments with low dielectric constants such as hydrophobic sites in proteins. When a protein starts to unfold or melt, the dye binds to exposed hydrophobic parts of the protein, resulting in a significant increase in fluorescence emission. The fluorescence intensity reaches a maximum and
Discussion
In current structural genomics initiatives focused on bacterial proteins, only some 3–15% of the genes entering the pipeline end up as structures. At the same time, more than 50% of proteins are expressed in a soluble form. Many proteins can be purified and yield lead crystals, but the optimization of these crystals often is difficult or too time-consuming, and new proteins are entered into the pipeline to keep productivity instead of solving the structures of the difficult proteins. Many
Acknowledgments
We thank the Swedish Research Council, the Wallenberg Consortium North, NIH GM074899, and the EU framework V Network SPINE for financial support. We thank Benita Engvall, Marie Hedren, and Martin Andersson for the initial cloning of the proteins used in this study. We also thank Alexei Savchenko for providing some of the clones and Tove Sjögren (AstraZeneca) for help with preparing ligands.
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