**2. Materials**

*Growing and Handling of Bacterial Cultures*

to smaller-scale centers and academic labs [3, 4, 13].

which is key in obtaining high-quality structural data.

protein. Meeting these requirements can be challenging, however, advancements in high-throughput technologies for recombinant expression of proteins have greatly advanced in the last decade or more, in large part due to efforts from large structural genomics and structural proteomics centers [1, 4, 10–12]. The lessons learned and technologies developed from these centers can allow for rapid assessment of different expression strategies, which can be transferred and scaled down

In addition to a demand for large quantities of highly pure protein, structural studies also often demand high solubility and stability of the protein in solution. To address this need, a high-throughput fluorescence-based thermal-shift assay, also known as differential scanning fluorimetry (DSF), has been implemented at the large structural genomics and structural proteomics centers [14]. DSF was originally developed as a high-throughput drug discovery assay to screen for small molecules that bind to and stabilize target proteins [15–17]. The DSF screen has been further adapted to optimize buffer conditions by varying the pH, buffer components, detergents, reducing agents and small molecules to screen for conditions that increase the stability and conformational homogeneity of a protein [14, 17–20],

We have established and optimized standard operating procedures for growing and handling bacterial cultures in a shared core laboratory to support Integrative Structural Biology and have used these in our own research [21–29]. The Integrative Structural Biology effort within the Biomolecular Research Center, a shared core facility, allows researchers at Boise State University and collaborating institutions to generate new knowledge about protein and RNA structure and function. We aim to understand how biomolecules assemble into stable structures and how structural dynamics can impact their function. Here we describe specific procedures for growing and handling bacterial cultures for overexpression and isolation of recombinant proteins, 15N/13C uniform labeling of recombinant proteins, protein isolation and purification, and analysis of protein solubility that are ideal for implementation in a shared research core laboratory that serves a multitude of diverse customers

**38**

**Figure 1.**

*A protein expression and purification workflow from plasmid to stable purified protein.*

All reagents listed in this chapter are commonly available from commercial vendors. A chemical hygiene plan including storage, shelf life, and safety of all chemicals should be in place at the institution.
