Producing GST-Cbx7 Fusion Proteins from Escherichia coli

This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside and they were purified by Glutathione Sepharose 4B. This protocol can be adapted for the purification of other proteins.


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Author manuscript been widely applied in various biochemical and structural studies (Harper and Speicher, 2011).

4.
BL21 competent cells (made in the laboratory)

A.
Expression of GST fusion protein

1.
Transform competent BL21 cells with pGEX-6P-1-GST plasmids that encode the Cbx7 variants by incubating plasmid with cells on ice for 10 min, then heat shock at 42 °C for 45 sec. The mixture was put on ice for 2 min, LB medium was added, and the mixture was incubated for 1 h at 37 °C while shaking at 250-300 rpm. After that, the cells were spread onto agar plate containing ampicillin.

2.
Transfer a single, isolated colony of transformed BL21 cells to 100 ml LB medium with 100 μg/ml ampicillin and incubate the inoculated culture overnight at 37 °C while shaking at 250-300 rpm.

3.
Transfer 50 ml of the overnight culture into 950 ml of warm, fresh LB medium with 100 μg/ml ampicillin.

5.
Induce the protein expression by adding IPTG to a final concentration of 1.0 mM (stock concentration: 100 mM, the powder was dissolved in MilliQ water and the solution was filtered before used) and incubating at 37 °C while shaking at 250-300 rpm for 5 h.

6.
Harvest cells by centrifugation at 4,000 × g for 20 min at 4 °C.

7.
Carefully decant the supernatant, leaving about 15-50 ml in the centrifuge bottle.

8.
Resuspend the cells and transfer to a 50 ml conical tube and centrifuge at 4,000 × g for 20 min at 4 °C.

1.
Resuspend the cell pellet in 25 ml of lysis buffer.

2.
Lyse cells by sonication at 4 °C using the standard probe with the following settings:

1.
To the mixture after sonication, add Triton X-100 to a final concentration of 1% and mix gently for 30 min at 4 °C with the mixer to increase the solubility of the protein.

2.
Centrifuge the mixture at 10,000 × g for 10 min at 4 °C.

3.
Wash 0.75 ml of Glutathione Sepharose beads with 3 × 10 ml of cold PBS, centrifuge at 500 × g for 3 min at 4 °C.

4.
Transfer the supernatant by pipetting from step C2 to pre-washed beads in a conical tube and rotate at 4 °C for 1 h.

5.
Centrifuge at 500 × g for 3 min at 4 °C and remove the supernatant.

6.
Wash the beads with 4 × 10 ml of wash buffer.

7.
Pour the beads into a Bio-Rad Chromatography Column and wash with 2 × 10 ml of cold PBS.

8.
Elute the fusion protein by incubating at room temperature for 10 min with 1 ml elution buffer. Repeat this step to get a total of three elutions.

10.
Run SDS-PAGE gel to determine the purity and identity of the fusion protein ( Figure 1).

Data analysis
The Pierce™ Coomassie (Bradford) Protein Assay Kit (Thermo Scientific) was performed to determine the concentration of the protein. SDS-PAGE gel was used to determine the identity of the proteins by their expected molar mass and also to check for contaminates (if yes, there will be other bands shown together with the protein band). Furthermore, GST protein was run with the SDS-PAGE gel as a control. The gel was stained with Coomassie Blue and is presented in Figure 6

1.
To monitor the OD, measure the absorbance at 600 nm. Estimate the amount of time by assuming the population of E. coli doubles every 20 min.

2.
The cell pellet from step C10 can be frozen at −80 °C for several months.

3.
For sonication: cell disruption is evidenced by partial clearing of the suspension. Avoid over sonication since it will heat the solution, leading to protein aggregation and denaturation.