Editing TEV Purification (section)

== Purification of HisMBP-TEV<br> ==

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. <br>

[[Category:Protocols]]