Protein overexpression and SDS-PAGE

Ptac The tac promoter/operator (Ptac): Expression of Ptac is repressed by the LacI protein. The LacI^q allele is a promoter mutation that increases the intracellular concentration of LacI repressor, resulting in strong repression of Ptac. Addition of the inducer IPTG inactivates the IacI repressor

Allolactose inhibits the lac repressor’s DNA binding ability.

Protein overexpression Inoculate 2 ml of SOB or LB + Ampicillin (50 µg/ml) with a single recombinant E. coli colony. 2. Grow overnight at 37°C with shaking. 3. The next day, inoculate 50 ml of SOB or LB + Ampicillin (50 µg/ml) with 0.2 ml of the overnight culture. 4. Grow the culture at 37°C with shaking to an OD600 = 0.6 (the cells should be in mid-log phase). 5. Remove a 1 ml aliquot of cells prior to IPTG induction, centrifuge the sample in a microcentrifuge, aspirate the supernatant. Freeze at -20°C. This will be the time zero sample.

Bacterial growth curve

6. Add IPTG to a final concentration of 1 mM (0 6. Add IPTG to a final concentration of 1 mM (0.5 ml of 100 mM IPTG stock to 50 ml of culture) and grow at 37°C with shaking. Take samples at one hour intervals for 5 hours (or more). Centrifuge each sample and store both the supernatant and the pellet at +4°C. For long term storage (greater than 5 hours), store the samples at -20°C. 7. When all time points have been collected, resuspend each pellet in 100 µl of 20 mM phosphate buffer at neutral pH, and freeze in liquid nitrogen or methanol/dry ice (exercise caution when handling liquid nitrogen, it can cause severe burns if it comes in contact with the skin, wear appropriate protective equipment). Thaw the frozen lysate at 42°C. Repeat this freeze-thaw 2-3 additional times and pellet the insoluble protein in a refrigerated microcentrifuge for 10 minutes at maximum speed.

8. Remove the supernatant to a fresh labeled tube 8. Remove the supernatant to a fresh labeled tube. Resuspend the pellet in 100 µl of Laemmli Buffer. To 100 µl of supernatant sample, add an equal volume of 2X Laemmli Buffer. 9. Analyze 10-20 µl of both the supernatant and pellet samples on a 10% SDS polyacrylamide gel. Stain the gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the protein. Compare it to the negative control time course to distinguish the recombinant proteins from the background proteins

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis Theory of SDS-PAGE http://www.youtube.com/watch?v=IWZN_G_pC8U How to make SDS-PAGE http://www.youtube.com/watch?v=EDi_n_0NiF4 How to run a gel? http://www.youtube.com/watch?v=XUjLO-ek2C8

Protein loading (sample) buffer 4X stock for protein sample buffer 2.0 ml 1M Tris-HCl pH 6.8 0.8 g SDS 4.0 ml 100% glycerol 0.4 ml 14.7 M β-mercaptoethanol 1.0 ml 0.5 M EDTA 8 mg bromophenol Blue SDS contained in the sample buffer makes proteins negatively charged proportionally to their length. 2-mercaptoethanol/DTT breaks disulphide bonds.

Tetra-methyl-ethylene-di-amine 2.475 ml 1.575 ml 62.5 ul 5 ul (Ammonium persulfate) Tetra-methyl-ethylene-di-amine

Empty vector 0 0.5 1 2 3 4 hr after IPTG