Protocol for T7 Polymerase Expression and Purification(thanks to Emily Goers and Kate Green)
1. Use toothpick to scrape frozen cell stock. Place toothpick into 100mL LB + amp (75μg/mL) supplemented with 2% glucose. Incubate on incubator/shaker O/N at 37oC at ~200 RPM.
2. Into 1L of LB (75 μg/mL Amp.) add 50mL of overnight culture. Do this twice (so you have  1L sol’ns). Grow to an O.D. of 0.5 at 600nM (2-5 hrs)
3. Remove 200μL of sample (spin down on table top centrifuge and keep pellet) and save in freezer for later gel (‘uninduced’ sample).
4. Induce protein expression with 0.1mM IPTG for 3 hours at 37oC, 200RPM.
o Remove 200uL of sample and save at 4oC for later gel (‘induced’ sample).
5. Pellet cells at 6000 RPM. You can now freeze at –80 for overnight to months or continue on. Resuspend each (2) cell pellet in 50mL lysis/wash buffer. Rotate on cold rack for 5 – 20 mins to help loosen and break up pellet from container.
o Lysis/Wash Buffer
• 300mM NaCl, 25mM Tris, pH 8.0, 5% glycerol
6. Transfer 50mL (x2) mixture to pre-chilled 100mL beakers.
7. Sonicate cells at power setting 7, 6X, 15 sec. each time.* Be sure to ice sample in between sonications to keep sample at 4oC. Go to page 3.
o Remove 200uL of sample and pellet down at 11,000 RPM, 2-5mins. Keep both supernatent and pellet (to see how much of the protein is soluble). Save sample in freezer for later gel.
8. Pre-clear lysate by spinning at 14,000 RPM for 15 mins in 50mL Nalgene tubes. Balance containers before centrifuging at high rotation speeds.
9. Apply supernatent to pre-equilibrated metal affinity beads and rotate for 30mins, 4oC.
o Remove 50μL of sample and save at 4oC for later gel (‘bead’ sample).
Preparation of TALON Metal Affinity Resin
• Place appropriate amount of bead solution in 50mL Corning tube (5mL of beads (more of slurry) per 1L of E.Coli culture stock).
• Wash beads with excess amount of lysis/wash buffer.
• Centrifuge at 500-1000RPM for ~2 min.
• Discard supernatant.
• Do this 3-5 times.
10. Pellet down the metal affinity beads (which contain the T7 Polymerase) at 1000 RPM. Discard supernatant.
11. Wash beads with bound T7 polymerse 5X with (~50mL) lysis/wash buffer.
12. Elute T7 Pol. off of the beads with 15mL of elution buffer. Rotate for 15 mins. at 4oC.
o Elution buffer:
• 300mM NaCl, 50mM Tris, pH 7.0, 5% glycerol and 200mM Imidizole
13. Dialyze into 1L of storage buffer O/N at 4oC. After dialysis protein solution may look orange-brown. o Storage buffer:
• 100mM NaCl, 50mM Tris, pH 8.0, 10mM DTT and 50% glycerol
14. Next Day: Check purity of all samples taken throughout the purification by running them on a 10% SDS-PAGE gel.
• Loading Dye is bromophenyl blue
• Need protein ladder for MWM (molecular weight marker).
• Staining sol’n: 40% ethanol, 10% acetic acid, 1g Coomassie blue in 1L. Destaining sol’n: same as staining sol’n but w/ no Coomassie.
The MW of T7 Pol = 99 g/mol.
The Extinction Coeff. = 1.4 x105
Check concentration at 280nm
15. Store enzyme at ~ 6mg/mL for use, but this may vary, so make sure to check by trying out different concentrations for transcription. Most likely you will have to dilute the enzyme significantly to get to 6mg/mL. Make sure to dilute in storage buffer.
- Sonication Instructions
Be sure to be trained by qualified lab member before operating sonicator.
Also, bring a 100-mL ‘rescue beaker’ on ice in case of accident.
➢ Set sonicator to zero (it should be there already).
➢ Turn on, let warm up ~1 min.
➢ Check to make sure probe is screwed on tightly. Wash tip w/ dI H2O.
➢ Tune sonicator:
o Press ‘Tune’ button.
o Turn intensity knob to level 3.
o Turn ‘tune’ knob until bar graph on display is below 20%. The bar graph should NEVER be above 50%!
o Turn intensity knob slowly to desired level, watching that the bar does not exceed 50%.
o Once at desired level, use the tune knob to move the bar on the bar graph as low as it will go (usually ~%5).
o Press ‘tune’ again to turn off the tuning mode.
➢ Place earmuffs on to protect from loud noise. Do not operate sonicator without the appropriate safety gear. Period.
➢ In general, the width of the probe is the depth at which the probe should be placed in the sol’n. However, with E.Coli sol’ns this usually results in excessive foaming, splattering and loss of protein. To sonicate the E.Coli sol’n, hold the beaker under the probe. Immerse the probe until you can feel it tap the bottom of the beaker. Lower the beaker so the probe is ~1/2” to 1” above the bottom of the beaker. Take care not to touch the beaker to ANY part of the sonicator during sonication – at best, the beaker will crack at the point of contact (which is usually the bottom of the beaker), at worst it will break the beaker. ➢ Press “Start” button. A timer will begin in the corner of the digital screen. At 15 sec., hit “Start” again and sonication will stop.
➢ Place beaker on ice for about 1 min. In the meantime, check the sonication probe for excessive heat. (If excessive heat is observed, STOP experiment and report to Dan Graham).
➢ Repeat sonication until appropriate # of sonications are completed. E.Coli sol’n should be watery (not viscous) at the end of sonication.
➢ Bring intensity knob back down to zero.
➢ Turn off sonicator. Clean off probe.
➢ Sign Logbook.
➢ Return to pg 1, step 7.