Purification of His-TEV
Information on the TEV protease construct can be found in the Sondek plasmid and
image databases; the ID is JS0215
All the work should be done as quickly as possible due to self-cleavage of the protease.
Use freshly transformed BL-21* cells for an overnight LB culture. Inoculate 4L of TB
containing 50ug/ml kanamycin with 10-15ml of ON per liter.
BL-21* (DE3) cells are grown at 37 degrees for 2-3 hours, and then the temperature is
reduced to 20 degrees. After 10-15hrs cells are collected by centrifugation, immediately
resuspended in N1 and lysed. The lysate is centrifuged at 40000 rpm for 30-45 min.
The supernatant is applied to two back-to-back 5 ml NiNTA columns equilibrated with
N1 buffer and washed extensively (at least 10 bv N1 followed by at least 10 bv of
5%N2). The TEV is eluted with 30% N2. First 0.7bv usually contains contaminants.
Collect eluate until absorbance (A280) drops below 50% of its maximum value.
Resuspend the protein to 1 mg/ml in N1. Add 5 mM DTT fresh from the 1M stock,
2mM EDTA from 0.5M stock and immediately snap-freeze the protein in orange tubes in
0.2 ml aliquots and store at –80 degrees. Run the gel only after freezing the
protein!
SUGESSTED USAGE: should not have to use more than 1/100 of TEV/YFFP for
complete proteolysis. e.g. ~ 1 tube TEV/ 20 mg of YFFP (YFFP = your favorite fusion
protein).
N1
20mM Hepes or Tris pH7.5
300mM NaCl
10% glycerol
10mM imidazole
N2 = N1+1M imidazole