Common plasmid vectors for protein expression in E. Coli

• GST-fusion protein
• His-tagged protein
• MBP-fusion protein

Inducible promoter

Protein expression is frequently induced by the addition of IPTG (isopropyl-D-thiogalactopyranoside) that induces the lac-promoter on the vectors.

Scale up culture volume

1. For large scale expression (500 ml), 5-50 ml overnight culture is used to inoculate 500 ml LB medium + appropriate antibiotics.

2. Cells are grown at 30 °C or 37 °C with shaking (225-300 rmp) until mid-log phase (OD₆₀₀ ~ 0.5-1).

3. IPTG is added to the final concentration of 0.1-1 mM. Cells are grown at 30 °C or 37 °C with shaking (225-300 rmp) for an additional 2-4 hours.

4. Cells are then pelleted by centrifugation at 4 °C. Resuspend the pellet in an appropriate buffer, and freeze immediately until disruption.

Disruption of bacterial cells

1. Cells can be disrupted by repeated freeze and thaw (3 cycles). Freeze in liquid nitrogen or dry-ice/ethanol bath, thaw quickly in 37 °C water bath.

2. Lysozyme: add lysolyme to 0.5-1 mg/ml. Incubate at room temperature for 20-30 minutes.

3. Sonication: sonicate for 5-15 seconds and cool on ice for 30-60 seconds. Repeat 4-6 times or until the concentration of proteins in the lysate don't increase anymore.

4. Protein may be insoluble. If insoluble, add 0.25% Tween 20, 0.1 mM EGTD, to extract the proteins from the membrane fraction.

5. If still insoluble, extraction must be made under denaturing conditions (e.g., 1% SDS or 6 M guanidine or Urea).

Clear insoluble material

The lysate should be centrifuged at high speed to clear insoluble material. Transfer the clear supernatant to a fresh tube. Proceed to purification steps.