PARP1 Purification Protocol

Human PARP1 Expression & Purification 2019 - Detailed

Expression:

1. Transform in commercial Rosetta cells.
2. Inoculate two 100mL flasks (in case one doesn’t grow).
3. The next morning, evenly split one of the 100mL flasks among 6 1L flasks.
   1. Label the flasks 1-6 to avoid cross contamination between flasks.
   2. The media for the 6 1L flasks is made by hand pouring contents for LB media (i.e. yeast, NaCl, tryptone) and autoclaving on liquid 45. I stopped using LB capsules but you may if you’d like.
4. Grow media at 37ºC in an orbital floor shaker until OD reaches 0.4-0.6, and add 1mL 100mM ZnSO₄ to 0.1mM final concentration. Should take ~2.5 hours.
5. Continue growing until the OD reaches 0.8-1.0, should take another 30ish minutes. Induce with IPTG and take the flasks out onto a cart and roll into the cold room to sit while the shaker cools to 16ºC. Grow the cells overnight at 16ºC (>16 hours).
   1. Keep a chart of the ODs measured and at what time in your notebook.
   2. Take 1mL aliquots of uninduced cells (right before induction) and induced cells (before harvest) from each flask and prep them for the expression gel later. 
      1. Spin cells down in 1.5mL eppis at max speed at room temp for 5 minutes. 
      2. Discard media and resuspend in 200uL Bug Buster; vortex to help lysis.
      3.  Spin again and discard supernatant. Resuspend pellet in 100uL S200 buffer (or any buffer of your choice).
6. Harvest by spinning in JLA8.1 rotor at 6k RPM for 20 minutes.
7. Scrape pellets into labeled 50mL conicals and flash freeze in LN₂. Place in a rack in the -80ºC freezer.
8. Run a 4-12% Bis-Tris gel of all 6 uninduced/induced to see which flasks expressed and to what extent.

Buffers:

I make Ni and Hep buffers a day before to save time.

Nickel

Ni A Buffer (30mM Imidazole)

For  gfp mCherry use NO Imidazole

Ni B Buffer (1M Imidazole)

Heparin

Heparin 0 Salt Buffer

Heparin Low Salt Buffer A

Heparin High Salt Buffer B

S200

Keep buffers at 4ºC until ready for use. Use cold water from cold room when making them so that their pHs are more accurate.

Lysis: For 3 liters at a time

1. Take out 3 tubes of cells from the -80ºC freezer and place in ice.
2. In a graduated cylinder with 100mL Ni A Buffer, add:
   a. 2 ULTRA protease inhibitor tablets
   b. NP40 to 0.1% final concentration
   I use 10% NP40 and add 1mL
   c. PMSF to 1mM
   I have a 1M stock (0.174g in 1mL IPA), and add 100µL of that 
   d. Protease inhibitors:
   i. Leupeptin (0.5µg/mL final)
   I have a 5mM stock (0.00238g in 1mL H₂O), and add 21µL
   ii. Pepstatin A (0.7µg/mL final)
   I have a 1mM stock (0.00343g in 5mL EtOH), and add 102µL
   iii. Antipain (0.5µg/mL final)
   I have a 5mg/mL bottle (in H₂O), and add 10µL
   iv. Aprotinin (0.5µg/mL final)
   I have a 5mg/mL bottle (in H₂O), and add 10µL
   v. Benzadmidine (1mM final)
   I have a 1M stock (0.157g in 1mL H₂O), and add 100µL
3. Stretch parafilm over the mouth of the graduated cylinder and invert to mix until the tablets have completely dissolved.
4. Add buffer to each pellet’s tube up to the 20mL mark. Resuspend cells using a 25mL pipet and manual pipet pump.
5. Flash freeze in LN₂, put into a beaker of water to thaw (shake every now and then to help break frozen chunks), and combine all 3 tubes into a clean chilled beaker. Add another 20mL of buffer to one tube and shake to rinse the tube, then pour into the next tube and shake to rinse, and then pour into last tube to rinse and finally add to the beaker.
6. Stir on ice for 5 minutes. While it’s stirring, add MgCl₂ to 1.5mM, and 3µL benzonase per liter of cells.
   a. I fill a small container with ice and water and place the beaker in there to stir with a chunky stir bar over a stir plate. I usually use 135µL MgCl₂, assuming I have about 90mL of buffer + pellets, and 10µL benzonase.
7. Remove the stir bar. Sonicate for 8 minutes on recall 3 at 30% output. After first 4 minutes, remove beaker from container and pour out excess water. Place beaker back into remaining ice. Let the cells rest in ice for about 30 seconds before placing back into sonicator. Repeat this after the next 2 minutes. This ensures the cells do not heat up and keep as cool as possible during the sonication process.
8. Spin cells at 4ºC for 25 minutes in JA20 rotor at 18k RPM.
9. Filter using 0.45µ steriflips.

Purification:

Nickel

1. Equilibrate Ni column with Ni A buffer.
   1. Start by doing a pump wash through all the leads with water to clean them. In case the last user forgot, we don’t want their buffers in our prep.
   2. Start a flow of 0.5mL/min to attach the Ni column so there is no air between the column and tubing, only water.
   3. Next, do a pump wash with the buffers (Ni A in lead A1, Ni B in lead B1). 
   4. Equilibrate the column by running Ni A through the column at 2mL/min for 5 column volumes (CV). The column is now ready for our sample!
2. Make sure there are tubes and deep well blocks in the fraction collector.
3. Assemble and fill superloop with filtered sample. Attach superloop to AKTA and run program.
   1. May use Sofia’s programs for PARP1.
4. Pick fractions believed to contain PARP and run a 4-12% Bis-Tris gel to confirm.
5. Pool desired fractions. FL PARP1 requires a two-fold dilution to bring salt from 500mM down to 250mM (425mM for PARP2) using Hep 0 salt buffer.
   1. If protein precipitates, slowly add 4M NaCl until solution is clear again.

Heparin

1. Equilibrate Heparin column with Hep A buffer.
2. Refill tubes and deep well blocks in fraction collector.
3. Wash and reassemble superloop for next run. Fill superloop with diluted sample and attach to AKTA. Run program.
   1. At this step, the program is run over night since lysis, the Ni column, and running the Ni gel require a full day.
4. Run a gel to determine which fractions contain PARP.
5. Pool fractions and concentrate in a 50k concentrator to under 1mL.

S200

1. Equilibrate S200 column of your choice.
   1. I usually use the 120mL column (column #13), but if I’m tight on time then I use the 24mL column (#48).
2. Load concentrated sample into the AKTA using a 1mL syringe and 1mL loop.
3. Run program.
4. Run a gel to determine which fractions contain PARP.
5. Pool fractions and concentrate in a 50k concentrator to around 35µM. After every 10-15min spin, use a pipette to wash the walls of the membrane and disturb the concentration gradient.
   1. I’ll start by concentrating to less than 1 mL, and spec it while its still in the concentrator. I’ll then either continue to spin or dilute my sample, depending on its concentration. To determine concentration for PARP1 in µM: C=A280/120055*10⁶
6. Aliquot 10µL into PCR tubes and flash freeze in LN₂. Gather tubes into a labeled 50mL conical (45 PCR tubes fit per 50mL conical) and store in a rack in -80ºC freezer.