**3.12 Uniform 15N/13C labeling of recombinant proteins**

This protocol is for proteins expressed under the control of the lac, tac, or T7 promoters.

### *3.12.1 Day 1*

1.Transform 10 μL of competent BL21(DE3) cells (or derivatives) with 10 ng of plasmid DNA and plate cells on LB-agar containing the appropriate antibiotics (See Section 3.2).

#### *3.12.2 Day 2*

	- 5 mL 10X M9 medium.
	- 5 mL 10X ammonium chloride.
	- 0.75 mL 20% glucose.
	- 50 μL of each CaCl2, MgSO4, thiamine and biotin.
	- antibiotic at working concentration.
	- autoclaved water to 50 mL.

**51**

3.7.2.

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

3.Incubate cells in tilted tubes for a few hours at 37°C and 250 rpm in a shaking

5.Resuspend cell pellet in 50 mL unlabeled defined media, for a starting OD600 of ~0.03–0.08. Grow the culture overnight at 30°C in a shaking incubator.

1.Prepare 500 mL of 13C, 15N labeled defined medium as follows, in a 2 L baffled

• 0.5 g 15NH4Cl dissolved in 5 mL water and sterile filtered.

• 500 μL of each CaCl2, MgSO4, thiamine and biotin.

2.Prewarm the 500 mL of 13C, 15N labeled defined medium.

• antibiotic at working concentration.

• autoclaved water to 500 mL.

4000× g, 30°C) and discard supernatant.

starting OD600 of 0.03–0.08.

log growth (OD600 ~ 0.5–0.8).

Section 3.7).

• 1.5 g 13C glucose dissolved in 10 mL water and sterile filtered.

3.Centrifuge the overnight 50-mL unlabeled defined medium culture (5 min,

4.Resuspend the cell pellet in 500 mL of 13C, 15N labeled defined medium, for a

5.Grow culture at 37°C and 250 rpm in a shaking incubator until cells reach mid-

6.Once cells reach mid-log growth (OD600 ~ 0.5–0.8), measure the OD600. Calculate the corrected volume (in mL) to take for the sample aliquot equiva-

7.Transfer aliquot to a microcentrifuge tube, and spin it down at maximum speed for at least 1 min at room temperature. Remove the supernatant. This is

8.Induce protein expression by adding IPTG based on the optimal values of IPTG concentration, incubation time and incubation temperature (See

9.After the induced cells have grown for the proper length of time, dilute 200 μL of the culture 10-fold with 1X PBS and measure the OD600. To prepare an induced sample, take an aliquot containing the equivalent of 1 mL of cells at OD600 = 0.8 and immediately process it as described in Section

lent of 1 mL of cells at OD600 = 0.8 (See Section 3.7.1 for details).

an uninduced sample. Store the uninduced cells at −20°C.

4.Prewarm 50 mL of unlabeled defined medium to 30°C. While warming, centrifuge the LB culture (5 min, 4000× g, 30°C) and discard supernatant.

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

• 50 mL 10X M9 medium.

*3.12.3 Day 3*

flask:

incubator, until the culture is visibly turbid.

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


*3.12.3 Day 3*

*Growing and Handling of Bacterial Cultures*

priate antibiotic.

0.1 mM.

promoters.

*3.12.1 Day 1*

*3.12.2 Day 2*

(See Section 3.2).

in a 200 mL culture flask:

• 5 mL 10X M9 medium.

• 0.75 mL 20% glucose.

• 5 mL 10X ammonium chloride.

• antibiotic at working concentration.

• autoclaved water to 50 mL.

grown colonies (ca. 10–20).

• 50 μL of each CaCl2, MgSO4, thiamine and biotin.

**3.11 Large-scale expression of recombinant proteins**

1.Transform plasmid into an *E. coli* expression strain following Section 3.2.

2.Inoculate a liquid LB culture for an overnight growth following Section 3.4.

3.The next day, use the overnight culture to inoculate 1 L of LB with the appro-

4.Grow cultures at 37°C and 250 rpm shaking until the OD600 is ~0.6–0.8.

5.Induce expression of protein by adding IPTG to a final concentration of

6.Lower the temperature to 18°C and continue 250 rpm shaking for 12–16 h.

This protocol is for proteins expressed under the control of the lac, tac, or T7

1.Transform 10 μL of competent BL21(DE3) cells (or derivatives) with 10 ng of plasmid DNA and plate cells on LB-agar containing the appropriate antibiotics

1.Prepare 50 mL of unlabeled defined medium for overnight culture as follows,

2.Inoculate a 5 mL culture (LB with appropriate antibiotic) with several freshly

7.Follow Sections 3.7.1 and 3.7.2 to test for protein expression.

8.Harvest the cells by centrifugation at 6000× g.

9.Suspend cells in lysis buffer and store at −20°C.

**3.12 Uniform 15N/13C labeling of recombinant proteins**

**50**

	- 50 mL 10X M9 medium.
	- 0.5 g 15NH4Cl dissolved in 5 mL water and sterile filtered.
	- 1.5 g 13C glucose dissolved in 10 mL water and sterile filtered.
	- 500 μL of each CaCl2, MgSO4, thiamine and biotin.
	- antibiotic at working concentration.
	- autoclaved water to 500 mL.

10.Harvest the cells by centrifugation at 6000× g for 20–30 min at 4°C. Discard supernatant. Store the pellet at −20°C until ready for cell lysis.
