Updated: 10/24/12
TEV Protease Purification
Expression and purification of TEV Protease
TEV protease is expressed as a fusion protein consisting of Maltose-binding protein and
TEV. TEV cleaves at the recognition sequence ENLYFQ(G/S) between the Gln and Gly/Ser
residues. Our construct contains a TEV L57V and S136G double mutant that increases its
intrinsic enzymatic activity.
Our version of TEV has been inserted into the pRK795 vector as follows:
N’MBP-ENLYFQG-PolyHis-TEV-PolyArgC’
TEV Protease (1-242)
MW = 27731.6 Da
ε280 (Cleaved product) = 32.22 cm-1mM-1
– STARTER CULTURE
1. Thaw 1 aliquot of electrically competent BL21 E.coli cells on ice.
2. Add 50 uL of sterile ddH2O to cells and mix gently with the pipet.
3. Prepare 5 mL of LB for overnight cultures and add Chloramphenicol and
Ampicilling to 1X concentration (5 uL of 1000X stocks).
4. From TEV-protease cell stock, take a stab w/ a pipette and inoculate 5 mL LB
overnights.
5. Place overnights in the warm room and let shake on shaker overnight at 37ºC.
Suggestions
 Prepare and autoclave 1L LB media per 5 mLs of overnight culture volume for
purification tomorrow.
[DAY 2] – GROWTH AND EXPRESSION – UNLABELED LB PREPS
1. In the morning, add antibiotics to sterile LB culture flask(s).
a. For 1L LB, add 1 mL 1000X Amp and 1 mL 1000X Chloramphenicol.
2. Remove overnight cultures from 37ºC warm room and verify that there is growth
present.
3. Add 5 mLs of overnight starter culture to each 1 L LB flask.
4. Let cell culture incubate on shaker at 37ºC until A600 reaches ~0.6-0.9.
5. Add dry IPTG to 1 mM to LB culture flask.
6. Let cells induce for ~4 hours @ 30ºC on a shaker or overnight at 16ºC.
7. Centrifuge cells at 5,000 rpm for 25 minutes in 1L centrifuge bottle.
8. Remove supernatant and resuspend cells in 10-15 mL Nickel Start Buffer.
9. Store cells in -80ºC freezer until ready for lysis.
Updated: 10/24/12
[DAY 3] – PURIFICATION OF TEV PROTEASE
1. Remove cells from -80ºC freezer and let cells thaw at room temperature.
2. Lyse resuspended cells via French Press twice.
a. NOTE: Do not add protease inhibitors for this prep!!! TEV protease is a
protease.
3. Add 1M MgCl2 to 10 mM to the pressed cells.
4. Add a small amount of DNase and RNase to pressed cells; mix by inversion and
let stand on ice until you are ready to French press.
5. Equilibrate two Nickel columns or one “jumbo” Nickel column while the DNase
and RNase digestion is proceeding.
a. If your Ni column(s) are currently stored in 20% EtOH, elute off EtOH w/
1-2 CV ddH2O.
b. Subsequently wash column(s) with 2-3 CV Ni2+-Start Buffer.
c. Column is now equilibrated.
6. Once DNA and RNA digestion is complete, spin the cell lysate at 13k rpm
(20000g) for 30 minutes in an SS-34 rotor.
7. Syringe filter (0.22 uM pore-size/low protein-binding) supernatant to remove
any residual material.
8. Save a small aliquot of the supernatant for SDS gels. Add the remaining
supernatant to the Ni resin while in a cold room (4ºC).
9. Run the Ni2+ Columns (half of total sample volume per Ni2+ column)
a. Allow supernatant to elute from columns and collect the flow through
(FT). Collect a sample for SDS-PAGE gels.
b. Wash with 2 CV (~15 ml/CV) 50 mM imidazole.
c. Wash 1 CV 100 mM imidazole
d. Elute with 1 CV 250 mM imidazole (the bulk of your protein should be
here)
e. Elute residual TEV with 1 CV 500 mM imidazole.
11. Run an SDS-PAGE on all collected samples for gel analysis to determine
effectiveness of purification.
a. Protein should be present primarily in 250 mM imidazole wash, with a
decent amount present in the 500 mM imidazole wash as well.
12. Dialyze elution fraction in 4L DTT Ni2+ Start Buffer (25 mM Tris-Hcl, 200 mM NaCl,
10 mM Imidazole, and 5 mM DTT, pH 7.6) overnight.
[DAY 4] – CONCENTRATION AND STORAGE
1. Determine concentration of TEV sample via A280.
a. TEV protease gives an absorbance of 1.19 at 1 mg/mL. (source)
2. Aliquot 500 uL samples into 1.5 mL microfuge tubes.
3. Flash freeze and store in -80C
Updated: 10/24/12