TEV Protease Prep- Lang Lab Protocol

March 4th 2022

Protocol prepared by: Evan Langille

1. Inoculate pRK793 overnight in 10 mL LB broth supplemented with 100 ug/mL Ampicillin

at 37C.
2. Inoculate 500 mL 2xYT in a 1L baffle flask with 1/100 dilution of the overnight culture.
(eg. 5mL /500 mL broth).
3. Incubate at 37 with shaking at 250 rpm for about 90 minutes, then start taking 700 uL
aliquots for OD measurement.
4. When the OD reaches 0.5, remove the flasks from the incubator and reduce the
incubator temp to 30C. Add IPTG to a final conc of 0.3 mM. (eg. 1.5 mL/ 500 mL culture)
5. Grow the cells for four hours after induction, at 30C.
6. Harvest the cells by transferring to pre-weighed 250 mL centrifuge bottles. Spin in the
floor centrifuge that you have pre-cooled to 4C. Transfer the rotor from the cold room
to the chilled centrifuge. Spin at 4000xg for 20 mins to pellet cells.
7. Decant the supernatant by pouring, and weight the wet cell paste to estimate
resuspension buffer volume.
8. Resuspend in HisTrap loading buffer A (20 mM sodium phosphate, 0.5 M NaCl, 5 mM
imidazole, pH 7.4 ) to 5 mL of buffer A for every gram of wet cell paste. Mix by vortexing
or pipetting up and down. Transfer to 50 mL centrifuge tubes and freeze overnight at -
20C. Stable at this step for up to 4 days.
9. Thaw the frozen tubes in a 37C incubator just until the solution starts to melt, about 30
mins. Transfer to room temp for another 30 mins, then take to cold room, and vortex to
suspend cells.
10. Add 5 uL/ mL of 1M PMSF (in 20% EtOH) and 2.5 uL/mL of 1M Benzamidine to each
tube.
For 50 mL- 250 uL PMSF 0.2M and 125 uL Benzamidine.
11. Sonicate in 75W bath sonicator at 4C for 2 rounds of 5-minute sonication.
12. Press the cells through the large French press with a single pass, the lysate should look
less yellow, and opaquer white/frothy and partially clarified. Add 5 uL of benzonase
nuclease to the start of the eluate.
13. Transfer lysate to 50 mL ultracentrifuge tubes (weigh and balance them) and add 5 uL
benzonase nuclease to each tube (if you haven’t already). Invert several times and
incubate 5 mins at 4C.
14. Pellet cell debris by spinning at 20,000 x G for 20 minutes.
15. Using a serological pipette, transfer supernatant to new 50 mL tube on ice. Ensure you
do not transfer the raft floating on the solution or disturb the pellet. The consistency
should be much thinner than when you loaded the tubes, and the solution should be a
clear straw colour. If not, spin longer.
16. Equilibrate the His Trap column with >20 CV of Buffer A at 2 mL/min.
17. Load the entire sample volume (50 mL for 1L starting culture) at the same rate.
18. Wash with 20 CV buffer A to remove cellular debris.
 19. Elute using step-gradients at 25, 50 , 75 and 100% B prepared in 50 mL syringes. Elute
using 12 CV of each (2 mL/min; 6 fractions; 12 mL).
20. Prepare samples for SDS-PAGE by adding 10 uL fractions to 5 uL 3x SDS Loading buffer.
21. Heat for 5 mins at 95C, followed by cooling to 10C.
22. Run using precast TG gels at 225V for 30 mins.
23. Stain using standard CBB protocol.
24. Combine fractions that are pure. Concentrate the fractions in an amicon-15 centrifugal
filter device. Exchange the buffer with:

Storage Buffer
50 mM Tris-HCl 
250 mM NaCl 
1 mM DTT 
1 mM EDTA 
50% Glycerol 
pH 7.5 @ 25°C

Setting up Reactions with homemade TEV protease:

Use 1 unit (OD or ug/mL etc. ) to cleave 100 units of protein. 4C overnight or 20C for a few
hours works well. Check by SDS-PAGE. Use reaction buffer below:

1X TEV Protease Reaction Buffer 

50 mM Tris-HCl 
0.5 mM EDTA 
1 mM DTT 
(pH 7.5 @ 25°C)