**Acknowledgements**

*Growing and Handling of Bacterial Cultures*

**Figure 3.**

**Figure 4.**

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**4. Conclusion**

**Figure 5.** *1*

We have described the workflow for protein expression and purification used in our shared core laboratory. These methods for growing and handling bacterial cultures work well for plasmid amplification, mini-expression screening, optimized larger-scale protein production, protein isolation and purification, and

*H, 15N 2D NMR spectrum of an RBP prepared using the methods described here.*

*SDS-PAGE gel of a typical Ni-NTA purification of an RBP (arrow indicates the recombinant RBP). (A) Samples appear in the following order: MW markers, Lysate, Supernatant, Pellet, Flowthrough, Wash #1: 50 mM Tris-Cl, 100 mM NaCl, pH 7.7; washes #2–4: 10 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. (B) MW markers, Elutions #1–9: 200 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. Some protein elutes from the column in the wash steps. All fractions are kept and can be pooled after SDS-PAGE analysis.*

*DSF analysis of an RBP in buffer (10 mM Tris-Cl) with different additives. (A) A graph of the fluorescence at 602 nm at increasing temperatures for the surveyed additive screen. The inflection point preceding the peak is the melting temperature. (B) A first derivative plot with a four-point smoothing applied helps to visualize the melting temperature, where the peak is the melting temperature. The legend provides a key for both A and B.*

This publication was made possible by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants P20GM109095 and P20GM103408. The authors wish to acknowledge Jackson Wall for careful reading and suggestions.
