TEV Protease Purification

Heat-shock transformation + Thaw Rosetta competent cells DE3) on ice. For transforming, use 50 l of competent cells. + Add 1 l plasmid TEV S219R to 50 l competent cells (entire ligation mix). Gently pipetting to homogenize the mixture and make sure that the pipette tip goes all the way down into the cells in order to add DNA to the cells. Keep on ice for 30 minutes. + Heat-shock cells at 42C for 30 seconds, then immediately put the tube on ice for 1-2 minutes. + After that, add 400 l LB medium (with no antibiotics added) to the tube. + Culture at 37C for 1 hour (shaking speed is at 110rpm). + Spread 200 l cells on to LB plates (with chloramphenicol and ampicillin added at appropriate concentration) then incubate overnight at 37C. (Too long time of incubation will allow the satellite colonies appear) 2. Starter culture cultivation for protein expression + To 10 ml of LB medium, add 10 l of ampicillin (Final concentration of ampicillin is) + Pick 1 colony from the plate and add to LB broth/ampicillin flask. + Incubate in a shaker at 37C (shaking speed is at 11 rpm) 3. Main culture cultivation 3.1 - LB medium preparation + Prepare two 5-litter-sized flasks. + For each flask, dissolve Tryptone, Yeast extract and NaCl (total mass is 50 g) Mili Q water to the final volume of 2 litters. + After that, flasks of LB medium are autoclaved followed by cooling medium before adding antibiotics. + To each flask, add 2 ml of ampicillin and store in appropriate

conditions. 3.2 - Culture of bacteria + Add the whole incubated starter culture to autoclaved LB medium with ampicillin added then incubate at 37C in the shaker for 3 hours at speed of 110 rpm. + Take 1 ml of culture solution and measure OD light absorbance at wavelength of 660 nm. Continue shaking until OD660 reaches 0.6 ~ 0.7. + Induce the culture to express protein by adding IPTG with appropriate concentration. + Keep culture shaking overnight at 20C at shaking speed of 110 rpm. 4. Harvesting cells + After one-night culture, harvest cells by centrifugation (using ROTAR 9.1) in 4C, 5.000 rpm for 15 minutes. + Carefully discard the supernatant then store the pellet at 80C until they can be further processed. 5. Sonication (Cell Lysis by Physical disruption) + Thaw frozen cell paste on ice and completely resuspend the pellet in 50 ml Binding buffer followed by adding 10 l of Protease inhibitor (to limit proteolysis and stabilize labile proteins) then transfer the whole solution into a new metal beaker. + For sonication, prepare ice with some NaCl to avoid ice meltability. + Place the beaker on ice and sonicate the suspension under conditions as follows: Power = 60%/In = 1.0 sec/Tmax = 7.0C/Time = 10 minutes + Divide the lysate into 2 new tubes then centrifuge at 40,000 x g for 30 minutes at 4C using ROTAR JA 25.5 After centrifugation, save the supernatant for purification (the desired protein is in soluble part). 6. Purification by Affinity Chromatography + Ni-NTA superflow column (2-ml sized column) is used for

purification. + Firstly, the column is equilibrated with 15 ml of Binding buffer. + Load the lysate into the column, followed by 25 ml of Binding buffer and 25 ml of Wash buffer respectively (save the flow through after loading sample and different buffers as 3 distinguished fractions). + Finally, the desired protein is eluted by 10 ml of Elution buffer. Compositions of Purification buffers:

Binding buffer:

1 M Tris-HCl NaCl 1 M Imidazole 100% Glycerol Mili Q water

5 ml 1.75 g 1 ml 10 ml 100 ml 5 ml 1.75 g 5 ml 10 ml 100 ml 5 ml 1.75 g 15 ml 10 ml 100 ml

Wash buffer:

1 M Tris-HCl NaCl 1 M Imidazole 100% Glycerol Mili Q water

Elution buffer:

1 M Tris-HCl NaCl 1 M Imidazole 100% Glycerol Mili Q water