Grow cells in double rich medium*
at 20oC to OD=0.8
Induce (e.g. 0.01-0.1 mM IPTG)
Let grow overnight
Harvest, wash cells e.g. in 50 mM NaH2PO4, 100 mM NaCl, pH 8.0
Lyse cells e.g. by passing twice through a French Press (1500 psi)
Purify TEV protease by Ni-chromatography using standard protocol supplied by e.g. Qiagen
After elution with imidazole dialyze immediately against 50 mM Tris HCl pH=7.5 plus 1 mM EDTA, 5 mM DTT
Do not concentrate TEV protease too much (0.5 - 1 mg/ml)
Spin 10,000 rpm for 20 min
Take supernatant

Add glycerol for storage at -80oC. Freezing in the absence of glycerol will kill the activity.

* If TEV protease expression is IPTG-dependent do not use BactoTryptone in your medium since it contains lactose. Change BactoTryptone by NZ amine A (available from SIGMA)

Grow cells in double rich medium*
37oC to OD=0.8

Fully induce (e.g. 1 mM IPTG)
Let grow overnight
Harvest, wash cells e.g. in 50 mM NaH2PO4, 100 mM NaCl, pH 8.0
Lyse cells e.g. by passing twice through a French Press (1500 psi)
Harvest inclusion bodies by centrifugation (27,000xg, 20', 4oC)
Solubilize inclusion bodies by resuspending the pellet in 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris/HCl, pH 8.0
Remove insoluble aggregates by centrifugation (27,000xg, 10', room temperature
Purify TEV protease by Ni-chromatography using standard protocol
After elution with imidazole dialyze against 8 M urea in 50 mM Tris HCl pH=7.5 plus 1 mM EDTA, 5 mM DTT
Refold TEV protease by dilution

50 mM Tris HCl pH=7.5
1 mM EDTA
5 mM DTT
50% glycerol
0.1% Triton X100
Store at -80oC