Expression and Purification of His-TEV

PURPOSE

To overexpress and purify the His-TEV protease from E. coli bacteria.

OVEREXPRESSION

Materials

1 x glycerol stock of HisTEV protease in BL21(DE3)-RIL cells (AddGene 8827 pRK793)
1 x LB + amp. + chloramp. agar plate
1 x 100 mL LB + amp. + chloramp. in 500 mL erlenmeyer flask
1 x 1,500 mL LB + amp. + chloramp. in 2.8 L fernbach flask.
Final ampicillin concentration (selects for pRK793): 100 μg/mL
Final chloramphenicol concentration (selects for pRIL): 34 μg/mL

Protocol

Day 1

• Streak out HisTEV protease glycerol stock onto LB+amp+chloramp plate
• Grow overnight at 37 °C

Day 2

• Sometime in the afternoon, inoculate culture of 100 mL LB+amp+chloramp with a single colony from the plate.
• Grow overnight at 37 °C, shaking at 225 rpm

Day 3

• In the morning, inoculate 1.5 L LB+amp+chloramp culture with entire contents of 100 mL overnight culture.
• Place 37 °C, shaking at 225 rpm.
• Check the OD600 of the culture periodically using the Eppendorf spectrophotometer in the lab.  A typical growth curve is shown here.
• When the OD600 reaches 1.0, add 1.5 mL of 1 M IPTG.
• Place the culture at 15 °C, shaking at 225 rpm overnight.

Day 4

• In the morning, split the contents of the flask evenly between two 1 L centrifuge bottles.
• Spin at 3,500 rpm for 15 minutes in the preparative centrifuge.
• Pour off the supernatant, and then place the bottles on their sides, pellet-side down, at -20°C for ~1/2 hr.
• Pop the pellets out of the bottles with a spatula and place them in a 50 mL conical tube.
• Measure the total mass of the pellets.
• Continue to purification.  If necessary, these pellets can then be frozen for a later prep.  Write “TEV Protease”, your name, the date, and the pellet mass on the side of the tube and place at -20°C.  Use them within ~1 month.

PURIFICATION

Materials

• B-Per Bacterial Protein Extraction Solution
• Lysozyme (50 units/mL)
• DNaseI (2500 units/mL)
• IMAC Buffer (see below)
• Elution buffer (see below)

IMAC buffer (Final volume:  2L)

Filter 500 mL of IMAC buffer at 0.22 μm for the FPLC.   Set aside remaining 1.5 L for overnight dialysis at the end of the purification.

Elution buffer (Final volume: 250 mL)

Filter at 0.22 μm for FPLC.

Protocol

Day 1

• Lyse cells with 3.5 mL IMAC buffer/g pellet with lysozyme at 8 μL/g and with DNase I at 8 μL/g.  Vortex periodically until the pellet is thoroughly resuspended/dissolved.  This can take ~30 minutes.
• Centrifuge at 15,000 x g for 60 minutes.
• In the meantime, equilibrate a stack made from 2 of the 5 mL HisTrap columns with IMAC buffer (see Affinity FPLC protocol)
• Collect the supernatant from the centrifuge run.  Make sure you are there immediately after the run finishes so the pellet remains intact.  If there is visible junk floating in the supernatant, syringe filter @ 0.45 μm prior to placing on the FPLC.
• Do a conventional affinity column FPLC run (@ 1 mL/min: load using superloop, 25 mL wash, 50 mL step elution in elution buffer).
• Assess fraction purity using an SDS-PAGE gel.
• Pool the pure fractions and dialyze overnight against the 1.5 L unfiltered IMAC buffer you set aside earlier.

Day 2

• If precipitate is visible in the dialysis bag, spin down samples at 15,000 x g for 10 minutes.  Keep supernatant.
• Check concentration of protein by A280. Dilute with dialysis buffer so A280 is approximately 1.
• Flash freeze dropwise in liquid nitrogen – store at -80°C and thaw as needed.