**3.7 Testing for soluble protein expression in** *E. coli*

The following protocol is written for proteins expressed under the control of the lac, tac, or T7 promoters. The method as described is a generic protocol that can be expanded to test expression in different strains of *E. coli*, induction temperatures, concentrations of IPTG, or even in the presence of ligands or cofactors.

## *3.7.1 Protein expression*


**47**

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

5.Transfer the equivalent of 1 mL of cells at OD600 = 0.8 in a 1.5-mL microcentri-

6.Collect cells by centrifugation at 16,000× g on a tabletop centrifuge for at least 1 min. Carefully remove all of the supernatant. This is the uninduced sample.

8.Measure the OD600. Collect cells by centrifugation in two tubes containing the equivalent of 1 mL of cells at OD600 = 0.8 and remove the supernatant. These are the induced samples; one tube will be used to test for expression and the second for solubility. Store the cells at −20°C until ready to test for expression.

1.Take the tube of uninduced and one tube of induced cells and resuspend each in 100 μL of 1X SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample

4.Take 10 μL of each sample from the top of tube taking care not to disturb the

5.Analyze the results using SDS-PAGE following Section 3.9 (**Figure 2**), with

1.Resuspend the remaining induced cell pellet in 50 μL of lysis buffer containing

4.Carefully transfer all of the supernatant into a new microcentrifuge tube. Add

5.Resuspend the pellet in 100 μL of 1X SDS-PAGE buffer. This is the insoluble

<sup>6</sup> In order to have equal loading on an SDS-PAGE gel, the same amount of cells need to be harvested for gel analysis. To harvest the same number of cells each time, calculate the volume in mL needed of your culture

3.Spin down in a microcentrifuge at maximum speed for 10 min at 4°C.

2.Boil the samples for 10 min, then cool down to room temperature.

3.Centrifuge for 5 min at 16,000× g on a tabletop centrifuge at room

7.Add IPTG to a final concentration of 1.0 mM to the remaining culture.

Continue shaking at 250 rpm for 12–16 h at 18°C.

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

Store the cells at −20°C.

*3.7.2 Testing for expression*

buffer.

pellet.

temperature.

*3.7.3 Testing for solubility*

fraction.

western blotting if necessary.

protease inhibitors and 1 mg/mL of lysozyme.

2.Follow Section 3.8.1 for freeze-thawing to lyse the cells.

50 μL of 2X SDS-PAGE buffer. This is the soluble fraction.

6.Boil the samples for 10 min, then cool down to room temperature.

that would be the equivalent of 1 mL of OD600 = 0.8. E.g. X mL = 0.8/OD600 of your culture.

fuge tube.6

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

