Chapter 1: Introduction to Protein Expression

In this guide, we will explore the essential protocols for expressing isotopically labeled proteins in E. coli. These recipes are often hard to find, and I wanted to compile them for easy access.

Figure created by the author using Dall-E-2 and original illustrations.

Growth Medium Preparation (for 1 L)

To prepare the growth medium, gather the following components:

• 100 mL of 10x M9 medium
• 10 mL of 100x trace elements solution (or alternatives)
• 1 mL of 1M MgSO4
• 1 mL of 0.1M CaCl2
• 1 mL of biotin (1 mg/mL) - optional
• 1 mL of thiamin (1 mg/mL) - optional
• Antibiotic based on strain and plasmid
• 4 g of glucose (labeled with 13C) or 2-2.5 g
• 1 g of 15N-labeled ammonium chloride or 1.2 g of 15N-labeled ammonium sulfate
• Add distilled water to make up to 1 L

#### Preparation Steps

1. Combine all ingredients listed above except for the ammonium and glucose.
2. After autoclaving, introduce the glucose and ammonium by dissolving them in 10-20 mL of water and filtering them into the medium using a 0.22 µm filter.
3. This is the stage where you need to add the 15N and/or 13C-labeled compounds.

M9 Medium (10x for 1L)

• 60 g Na2HPO4 (0.42 mol, final concentration 0.42 M)
• 30 g KH2PO4 (0.22 mol, final concentration 0.22 M)
• 5 g NaCl (86 mmol, final concentration 86 mM)
• Distilled water to 1 L

Trace Elements Solution (100x for 1L)

• 5 g EDTA
• 0.83 g FeCl3 • 6 H2O
• 84 mg ZnCl2
• 13 mg CuCl2 • 2 H2O
• 10 mg CoCl2 • 6 H2O
• 10 mg H3BO3
• 1.6 mg MnCl2 • 6 H2O

#### Preparation Process

Dissolve the components gradually while adjusting the pH to 7.5. Gentle heating can assist in this process. If possible, sterilize the solution using a 0.22 µm filter instead of autoclaving.

Culture Growth Protocol

1. Grow bacteria overnight in 5-50 mL of LB medium (preferably from a previous culture).
2. Inoculate the saturated culture into 0.5 to 2 L of the prepared minimal medium.
3. For optimal labeling, wash the saturated culture gently with fresh medium to eliminate any unlabeled metabolites. This step is crucial for certain antibiotic resistances like ampicillin, as lactamase leakage can degrade the antibiotic.
4. When the optical density (OD600nm) reaches around 0.7 (this may require optimization), introduce your inducer (commonly 0.5-1 mM IPTG).
5. Allow the culture to induce protein expression for a minimum of 4-5 hours or up to overnight, adjusting temperature and other factors as needed.

Numerous parameters will require optimization. It may be wise to conduct preliminary runs with unlabeled substrates to refine your protocol before utilizing the more expensive labeled compounds.

Conclusion

I compiled this information here since I often find it challenging to locate these recipes. It turns out that content related to growth media has excellent search engine optimization (SEO), making it easier to find.

Chapter 2: Video Resources

In the video titled "Expression and purification of His-tagged proteins from E. coli," you will gain insights into the methods for expressing and purifying His-tagged proteins, which can enhance your understanding of protein expression techniques.

The second video, "QMUL Science Alive: Protein expression and purification," provides further information on protein expression and purification processes that complement the protocols discussed here.