Elsevier

Analytical Biochemistry

Volume 357, Issue 2, 15 October 2006, Pages 289-298
Analytical Biochemistry

Thermofluor-based high-throughput stability optimization of proteins for structural studies

https://doi.org/10.1016/j.ab.2006.07.027Get rights and content

Abstract

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins’ probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.

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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