Co-Expression of human DNA primase p49-p58 subunits

Adapted from W.C. Copeland, Protein Exp. & Purif 9, (1997) 1-9 and Schneider, et. al., JBC 273:34 (1998) 21608-21615.
Obtained from Fanning lab and revised by LSM, 7/23/01
Obtained cell stock of BL21(DE3) cells cotransformed with pET21a-Hp49 (ampicillin resistance) and pACYC184-Hp58 (low copy, chloramphenicol resistance) from Fanning lab. Despite what the plasmid names might suggest, Hp49 is expressed native, while Hp58 is expressed w/an NT his tag (see Copeland ref).
Antibiotic concentrations used: [AMP] = 100 mg/mL; [CAP] = 34 mg/mL
  1. Streaked cells from cell stock on a LB/AMP/CAP plate. Incubated O/N at 37°C.
  2. In the late morning, pick a single colony and add to 2 mL of LB w/AMP and CAP in a culture tube. Shake at 37°C.
  3. As late in the afternoon as possible, inoculate 100 mL 2xYT with AMP and CAP and 10 mL of LB culture. Grow O/N at 37°C. I used a 1:10,000 dilution to avoid having an oversaturated culture after ~15hr O/N incubation.
  4. Inoculate 1 L 2xYT with 100 mL O/N culture + fresh antibiotics. Use baffled 2800 mL flasks for proper aeration. Shake vigorously at 37°C.
  5. After ~1½ hours, add IPTG to a final concentration of 1 mM. I have induced at OD600 as high as 1.2 with good results. Shift cultures to 20-22°C and grow an additional 3 hours with shaking. Note: You don't have to shake (the Fanning lab simply transfers their flasks to a RT H2O bath), but I found that it does improve expression levels.
  6. Harvest by centrifugation, 5000 rpm, 10 min., 4°C. Freeze pellets at -80°C.
Purification of p49-p58 human primase subunits
The most important thing in doing this purification is to make sure that EVERYTHING is kept cold. Prepare all buffers ahead of time and pre-chill them. Pre-chill rotor, centrifuge tubes, falcon tubes, etc. It's best if you can do all of the spin downs in refridgerated centrifuges. If you can't do this, then work quickly and keep all buffers ice cold.
 
Lysis buffer:
Wash buffer:
Elution buffer:
  • 250 mM imidazole
  • 40 mM Tris pH 8.0
  • 3mM BME
    1. Thaw frozen cell pellet at RT till semi-soft.
    2. While pellet is thawing, take ~60 mL lysis buffer (amount based on cell pellet obtained from 4L growth) and add protease inhibitors. Use ROCHE EDTA-free cocktail tablets-1 tablet/10mL lysis buffer (It's important that all your solutions are EDTA-free because EDTA is incompatable w/Ni-NTA resin). Rotate ~5 min 4ºC to dissolve tablets.
    3. Add lysis buffer containing protease inhibitors to cell pellet. Pipet up and down to resuspend cells thoroughly. Add 3 mg/mL lysozyme. Swirl to mix.
    4. Keep solution on ice and wait until it becomes viscous, ~1/2 hour. (Cells lyse, genomic DNA released -> solution becomes snotty.) You can check viscosity by drawing up some solution in a pasteur pipet.
    5. Sonicate solution on ice: 2' sonicate, 2' rest, repeat. Used microtip at highest power. Don't sonicate continuously for very long cause it heats up the solution. Check w/a Pasteur pipet to make sure that the genomic DNA is broken up (solution should dribble out freely, not stick to the tube). Continue sonicating until you achieve this.
    6. Centrifuge 17K, 15', 4ºC to pellet cell debris.
    7. Meanwhile, wash QIAGEN Ni-NTA agarose beads. Use at least 3 mL packed beads (amount based on a 4L induction). Solution is a packaged as a 50% slurry. An easy way to obtain desired amount is to cut off the very tip of a blue,1 mL pipet tip w/a razor blade and then use it measured out 6 mL of slurry. The resin pellets best in 15 mL Falcon tubes. Centrifuge at 1000 rpm, 1' to pellet beads. Pipet or aspirate off storage buffer. Wash beads 2x with 10mL cold lysis buffer.
    8. Pipet off supernatant of cell lysate into 2x 50 mL Falcon tubes, add beads and rotate solution at 4ºC for 1-1½ hr.
    9. Transfer solution to 15 mL Falcon tubes. Centrifuge at 1000 rpm, 1' to pellet beads. Pipet or aspirate off supernatant.
    10. Add ice cold wash buffer to the beads (I used ~12 mL/ 15 mL falcon tube of beads). Invert to resuspend beads. Centrifuge at 1000 rpm 1' to pellet beads. Pipet or aspirate off supernatant. Repeat at least 4 times.
    11. After final wash, resuspend beads in some wash buffer and pour slurry into a plastic disposable BIORAD column in cold room. Try to get every last bead. Equilibrate & pack column with more wash buffer. If beads are grayish, it's a good sign cause it means you've got a lot of protein retained on them.
    12. Elute protein with ice cold elution buffer (~8 mL). Collect 1.0 mL fractions in 1.5 mL epp tubes. The majority of the protein should elute in the first ~3 mL. After elution, beads should be a light blue color.
    13. Run SDS-PAGE on fractions to make sure you got everything off of column and to check purity.
    14. Pool fractions containing protein. Typical yields are ~0.5-0.8 mg/L culture.
    The protein is relatively pure at this stage, but for biophysical studies, further purification can be achieved by heparin affinity chromatography:
    Buffer A:
    Buffer B:
    1. To lower the salt conc. of pooled fractions from Ni-NTA purification, dialyze O/N against Buffer A, 4ºC.
    2. Take dialysate and spin down to remove any particulate matter. Take supernatant and load onto a Pharmacia Hi-trap heparin column equilibrated in Buffer A. I found it easiest to load the sample onto the column by hand using a syringe and used the FPLC only for gradient salt elution.
    3. Wash column with 5 column volumes of Buffer A by hand using a syringe.
    4. Hook column up to FPLC. Gradient salt elution from 0%-100% Buffer B was accomplished over 20 column volumes. Protein elutes at ~0.4M NaCl as a broad peak w/a side shoulder.
    5. Run SDS-PAGE on fractions to look for protein and to check purity. I found that even though the elution peak was a funny shape, all fractions contained protein. I think the weird shape is due to differential heparin binding properties of the 2 subunits.
    6. Pool fractions containing protein. I found that the heparin column got rid of some higher and lower MW impurities, but there were always 2 lower MW bands that I couldn't get rid of. I think they may be degradation products since I've seen similar bands present in published gels of the complex.