TEV

Purification of His-TEV(S219V)-Arg

Materials

• Amp stock

• Chromophenocol stock

• 1 M IPTG stock

• Buffer A: 20mM Bicine, pH 8.0, 500mM NaCl, 20mM imidazole

• Buffer B: 50mM Tris, pH7.5, 20mM NaCl, 2mM EDTA, 5mM DTT, 30% glycerol, 0.1% Triton X-100

Expression

Bacterial Stock

1. Streak on the LB/amp+cm plate

2. Pick a single colony for liquid culture

3. Culture at 37oC overnight

4. Making glycerol stock by adding 1:1 50% glycerol

Large-scale liquid culture

1. Prepare 1 L LB medium+Amp+Cm

2. Throw the entire glycerol stock into the medium and grow at 37oC.

3. At OD600 reach 0.6, add IPTG (final conc. at 1 mM/L) and induce for 5 hours.

4. Harvested the cells by centrifuging at 5000x g for 15 mins.

5. Freeze the pellet at -80oC.

Protein purification

1. The cell pellet is thawed on ice and suspended in 50ml Buffer A + two Complete protease inhibitors.

2. French press

3. The sample is centrifuged at 15,000x g for 30 mins.

4. The supernatant is filtered (0.45 um) and then prepared for FPLC.

5. Wash the HisTrap column with 5CVs water.

6. Wash the column with 5CVs Buffer A.

7. Load sample and then wash with 20CVs Buffer A w/ 50mM imidazole

8. Elute in 0.5 CV with Buffet A w/500 mM.

9. Add EDTA (final conc. 1mM) and glycerol (final conc. at 30%).

10. Dialyze against 1L buffer B at 4oC overnight.