TEV Purification

Purification of 7XHisTev

Expression

1. Grow TEV (pMHT238Δ, pMHTdelta238, self-cleavage to release 7XHis-TEV, http://dnasu.asu.edu/DNASU/POMOctNov09.jsp) in BL21 Rosetta 2 (DE3)pLysS in auto-induction medium or TB with 0.8% glycerol, 2 mM MgSO₄ and 0.375% aspartic acid

2. Induce with IPTG at a final concentration of 0.5 mM. 37 °C for for 3 h or 25 °C for 5 h.

Autoinduction: ~12 h at 37 °C or ~24 h at 25 °C4.

3. Spin down cells.5.

3. Resuspend cells in 50 mM phosphate, pH 7.5, containing 300 mM NaCl and 20% ethylene glycol6. Store cell pellet at −80 °C in 50 mL conical tubes.

Purification

Buffers:
Buffer A: 20 mM phosphate, pH 7.5, 500 mM NaCl and 0.3 mM <span style="background-color: navy; color: white;" />beta-ME.
Buffer B: 20 mM phosphate, pH 7.5, 350 mM NaCl, 500 mM imidazole and 0.3 mM <span style="background-color: navy; color: white;" />beta-ME.
Buffer C: 10 mM Tris, pH 7.5, 0.3 mM <span style="background-color: navy; color: white;" />beta-ME.
Buffer D: 10 mM Tris, pH 7.5, 1 M NaCl and 0.3 mM beta-ME.

1. Suspend cell paste in 50 mM phosphate, pH 7.5, containing 300 mM NaCl, 20% ethylene glycol, 1 mM EDTA and 0.3 mM beta-ME at a ratio of 6 mL of buffer per g of wet cell paste. (34 g cells + 200 ml buffer)

2. Sonicate cell suspension for 6 min at 4 °C.

3. Spin for 25 min at 95,000 × g.

4. Load to 2 X 5 ml Ni HisTrap HP column pre-equilibrated with buffer A at 3 mL/min.

5. Was columns with eight volumes of a mixture of 85% buffer A and 15% buffer B (7.5 mM imidazole) at 5 mL/min.

6. Elute TEV with 100% buffer B at 0.7 mL/min.

7. Eluate is loaded to the second purifier with two 5 mL SP Fast Flow columns arranged in series at 0.7 mL/min.

The columns were equilibrated with buffer C. The sample from the first purifier was injected into the mixing chamber mixed with 100% buffer C at 10 mL/min and loaded onto the columns of purification system 2 at a total flow rate of 10.7 mL/min. The resultant ~15-fold dilution of the sample prior to application to the cation exchange columns ensured that the ionic strength was low enough allow tight binding of the protease to the column.Upon completion of the IMAC elution, the flow through purifier system 1 was increased to 5 mL/min and directed to waste for column wash and reequilibration with buffer A prior to the injection of the next aliquot of lysate. The waste sample was collected so that possible losses of TEV protease could be determined.

8. flow through purifier system 2 was decreased to 5 mL/min and a six column volume gradient from 100% buffer C to a mixture of 40% buffer C and 60% buffer D was started.

Yield: ~400 mg per L of expression culture with >99% purity

Expression & Purification of His-TEV

1. Grow BL21-RIL cells containing pRK793 at 37 °C in LB containing 100 ug/ml ampicillin and 30 ug/ml chloramphenicol.

2. When the cells reach mid log phase (OD600 ~ 0.5), add IPTG to a final concentration of 1 mM and the temperature is reduced to 30 °C.

3. After 4 hrs of induction, collect the cells by centrifugation.

4. Dissolve the cell pellet in 10 ml of 50 mM PO4 (pH 8.0) + 100 mM NaCl + 10% glycerol + 25 mM imidazole (lysis buffer) per 1 gram of wet cell paste.

5. Lyse the cells using homogenizer, add 5% polyetheleneimine (adjusted to pH 7.9 with HCl) to a final concentration of 0.1%.

6. Mix by inversion and then immediately centrifuge at 15,000 x g for 30 minutes.

7.  Apply the supernatant to a Ni-NTA column equilibrated with lysis buffer, using ca. 2 ml of resin per gram of wet cell paste.

8.  Wash the column with 7 volumes of lysis buffer, and then elute the TEV protease with a linear gradient of lysis buffer to 50 mM PO4 (pH 8.0) + 100 mM NaCl + 10% glycerol + 200 mM Imidizole in 10 column volumes.

9.  Pool the appropriate fractions and then add EDTA and DTT to a final concentration of 1 mM each.

10. Concentrate the sample in a stirred cell using a YM10 membrane.

11.  Load the sample onto an S-100 (Sephacryl S-100 HR gel filtration) column equilibrated with 25 mM PO4 (pH 8.0) + 200 mM NaCl + 10% glycerol + 2mM EDTA + 10 mM DTT (sample volume = 3% of the column volume).
12. Pool the appropriate fractions.

13. Concentrate the protease to ca. 1 mg/ml and flash freeze with liquid nitrogen.
14. Store at –80 °C.
The final yield should be approximately 10 mg of protein per gram of wet cell paste (ca. 30 mg/liter)

Expression & Purification of His-TEV(S219V)-Arg

1. Grow BL21-RIL cells containing pRK793  at 37 °C in L-broth containing 100 ug/ml ampicillin and 30 ug/ml chloramphenicol.
2. When the cells reach mid log phase (OD600 ~ 0.5), IPTG is added to a final concentration of 1 mM and the temperature is reduced to 30 °C.
3. After 4 hrs of induction, the cells are collected by centrifugation.
4. Dissolve the cell pellet in 10 ml of 50 mM PO4 (pH 8.0) + 100 mM NaCl + 10% glycerol + 25 mM imidazole (lysis buffer) per 1 gram of wet cell paste.
5. Lyse the cells.
6. Add 5% polyetheleneimine (adjusted to pH 7.9 with HCl) to a final concentration of 0.1%.
7. Mix by inversion and then immediately centrifuge at 15,000 x g for 30 minutes.
8. Apply the supernatant to a Ni-NTA column equilibrated with lysis buffer, using ca. 2 ml of resin per gram of wet cell paste.
9. Wash the column with 7 volumes of lysis buffer, and then elute the TEV protease with a linear gradient of lysis buffer to 50 mM PO4 (pH 8.0) + 100 mM NaCl + 10% glycerol + 200 mM Imidizole in 10 column volumes.
10. Pool the appropriate fractions and then add EDTA and DTT to a final concentration of 1 mM each.
11. Concentrate the sample in a stirred cell using a YM10 membrane.
12. Load the sample onto anS-100 column equilibrated with 25 mM PO4 (pH 8.0) + 200 mM NaCl + 10% glycerol + 2mM EDTA + 10 mM DTT (sample volume = 3% of the column volume).
13. Pool the appropriate fractions.
14. Concentrate the protease to ca. 1 mg/ml and flash freeze with liquid nitrogen.
Store at –80 °C.
The final yield should be approximately 10 mg of protein per gram of wet cell paste (ca. 30 mg/liter)

Purification of HisMBP-TEV

TEV protease recognizes the epitope E-Xaa-Xaa-Y -Xaa-Q-(G/S) and cuts C terminal to Q. The most efficient substrate sequence is ENLYFQS.

TEV Protease Digestion

1. Current stock made 5/30/09 in -80C in tubes labeled with red "T." Concentration is 1.2 mg/mL. It contains a C terminal His tag and is 27kDa. Storage buffer is 10mM TrisHCl pH8, 0.1M NaCl, 50% glycerol, 1mM EDTA, 1.5mM 2-ME

2. Use 1:100 (w/w) in 25mM Tris-HCl pH 8.0, 50mM NaCl at 4C overnight. EDTA up to 1mM and DTT up to 1mM are tolerated by TEV. For more information on TEV reaction conditions, read http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf

TEV Protease Purification

1. Glycerol stocks for E. coli expression are in the -80C. Grow in LB + Amp + Cam + 2g/L glucose. Tev is expressed as an MBP-TEV fusion separated by a TEV Site. TEV contains the S219V mutation for improved activity and reduced autoinactivation.

2. Grow to OD600~0.6-0.8, induce overnight at 30C with 1mM IPTG

3. Freeze pellets overnight in -20C and resuspend in 2xPBS+protease inhibitor cocktail.

4. Sonicate and clarify with centrifugation for 20 min at 35000g

5. Prepare a mixture of 2 Talon:1amylose resin, use ~4mL total resin for 500mL culture.

6. Batch absorb at 4C for 40 minutes, Wash resin on column with 2xPBS, Elute with 20mM TrisHCl pH 8.0, 150mM NaCl, 10% glycerol, 1mM 2-ME, 200mM imidazole.

7. Flow through 1mL amylose resin to remove residual MBP. Wash with elution buffer above with 1.2M NaCl. Collect flow through and 10mL of wash

8. Concentrate with Centricon spin concentrators (10kDa MWCO) for loading onto FPLC. Some precipitate will form. Remove precipitate with Spin-X tube.

9. Run on Superdex 200 using 20mM TrisHCl pH8, 0.2M NaCl, 10% glycerol, 2mM EDTA, 3mM 2-ME, at 0.2mL/min. Due to glycerol, lower flow rates (0.2-0.25mL/min) are required to prevent high-pressure errors on the FPLC. Remember to account for the longer equilibration time. For collection, 2 min per tube is useful to reduce number of tubes.

10. Check fractions on SDS-PAGE and pool fractions containing TEV (should elute around 14-16mL)

11. Add glycerol to 50%, aliquot into individual tubes, flash freeze in LN2, store in -80C.

12. Quantify TEV concentration by quantitative coomassie blue.