**1. Introduction**

Shared research core facilities can provide support to campus-wide investigators by providing research infrastructure for the production and purification of recombinant proteins for a variety of research applications. We have designed a research support structure for investigators pursuing research in structural and functional studies that require high yields of pure proteins, particularly suited for structural studies including biomolecular nuclear magnetic resonance (NMR) and small angle X-ray scattering.

The *Escherichia coli* (*E. coli*) expression platform is commonly used for recombinant expression of proteins. The *E. coli* system has several advantages over yeast, insect cells, or mammalian cell expression systems: *E. coli* are relatively easy to handle, the doubling time is short, media are low-cost and there are abundantly established methods for protein expression [1–4]. The *E. coli* expression platform is also well-suited for stable isotope labeling of proteins for biological NMR studies [5–9]. Structural studies of proteins demand large quantities of high purity

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

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], which is key in obtaining high-quality structural data.

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

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1.Ice.

*Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated…*

and research laboratories. Here we outline a general workflow of essential steps in protein expression and purification that includes plasmid amplification, miniexpression screening, optimized larger-scale protein production, protein isolation and purification, and characterization of optimized experimental solution buffer

All reagents listed in this chapter are commonly available from commercial vendors. A chemical hygiene plan including storage, shelf life, and safety of all

2.Lysogeny broth (LB) medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L

3.Super optimal broth (SOB, a.k.a. Hanahan's Broth) medium for DH5α cells: 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 0.186 g/L KCl. Adjust the pH to 7.0 with NaOH. Sterilize by autoclaving and store at room temperature.

10.Competent Cell (CC) buffer: 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 15 mM CaCl2, 55 mM MnCl2, 250 mM KCl, pH 6.7. Dissolve all components except MnCl2 and adjust the pH to 6.7 with KOH. Then add the MnCl2 and filter sterilize the solution over a 0.22 μm filter.

**2.2 Transformation of cells for expression of desired plasmid**

2.LB or super optimal broth with catabolite repression (SOC).

NaCl. Sterilize by autoclaving and store at room temperature.

*DOI: http://dx.doi.org/10.5772/intechopen.81932*

chemicals should be in place at the institution.

**2.1 Preparation of chemically competent cells**

4.Culture tubes and flasks.

5.Incubator/shaker.

6.Centrifuge tubes.

7.Serological pipettes.

8.Repeating pipettor.

9.Dimethyl sulfoxide (DMSO).

3.Culture tubes and flasks.

4.Incubator/shaker.

5.Centrifuge tubes.

6.Serological pipettes.

conditions (**Figure 1**).

**2. Materials**

1.Ice.

**Figure 1.** *A protein expression and purification workflow from plasmid to stable purified protein.*

*Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated… DOI: http://dx.doi.org/10.5772/intechopen.81932*

and research laboratories. Here we outline a general workflow of essential steps in protein expression and purification that includes plasmid amplification, miniexpression screening, optimized larger-scale protein production, protein isolation and purification, and characterization of optimized experimental solution buffer conditions (**Figure 1**).
