To begin the transformation of Escherichia coli BL21 DE3 cells, thaw 50 microliter aliquot of competent cell suspension on ice. Add one microliter of DNA of pET 28a Sumo TRF2 plasmid to the competent cell suspension, and gently swirl the tube to mix the suspension. After transformation, spread 100 microliters of the cell suspension on a luria bertani, or LB, agar plate containing 50 micrograms per milliliter kanamycin.
Incubate the cells overnight at 37 degrees Celsius. The next day, pick a colony of the transformed cells, and culture it in five milliliters of LB medium, supplemented with 50 micrograms per milliliter kanamycin. Incubate for 18 hours at 37 degrees Celsius, with shaking at 220 RPM.
After incubation, induce the culture with one millimolar IPTG as a final concentration. Incubate at 20 degrees Celsius for 17 hours to promote protein expression. Load the crude protein extracts, along with the loading buffer, onto an SDS-PAGE gel.
Run the samples initially at 100 volts for 30 minutes, followed by 120 volts for 50 minutes in a tris-glycine buffer. Then, stain with Coomassie blue to visualize protein expression. For protein purification, centrifuge the crude protein extract at 38, 000 G for 40 minutes at four degrees Celsius, and filter the supernatant through a 0.22 micrometer syringe filter.
Load the filtered supernatant onto the pre-equilibrated nickel column in the binding buffer. Wash the column with binding buffer and elute the protein with the prepared imidazole gradient, collecting 12 fractions of one milliliter each. Add glycerol to a final concentration of 50%to the concentrated and buffer exchanged protein for storage.
The expression of TRF2 in Escherichia coli was successfully demonstrated, as shown by SDS-PAGE, where distinct bands corresponding to TRF2 appeared before and after induction.
