RecBCD Purification

Used in our paper:¹

This 3-4 day protocol is to purify wtRecBCD from 3-6 liters of cell culture. The protocol is adapted from Wigley’s crystal structure paper: NATURE-V432-P187-2004. All RecBCD constructs were generated from plasmids provided by Jeff Gelles.

To save time, it is recommended that all columns be pre-washed in appropriate buffers and liquid cultures pre-warmed to 37oC before adding cells.

Contents

1. Materials
2. Buffers
3. Outline
   • Day 1 (long day)
   • Day 2 (long day, lots of waiting)
   • Day 3 (~3 hrs)
4. Detailed protocol
   • Day 0
   • Day 1
   • Day 2
   • Day 3

1. Materials

• Media
  • Liquid culture - 2YT (supplemented with 0.1 mM biotin for in vivo biotynilation; Sigma-Aldrich: Y2377-250G $27.30)
• Chemicals
  • Carbinicillin
  • Chloramphenicol
  • IPTG
  • DMSO - Make 10 mM stock
  • Biotin (10mM in DMSO, Sigma-Aldrich: Cat# B4501-1G $46.70)

2. Buffers

• 1M TrisCl pH 7.5 (filtered&sterile)
  • 1L – 121.14 g Tris base. Titrate with HCl to pH 7.5
• Buffer A (20 mM TrisCl pH 7.5, 0.1 mM DTT, 0.1mM EDTA)
  • 2L – 40 ml Tris (1M), 0.4 ml EDTA(0.5M), 0.2 ml DTT (1M)
• Buffer B (20 mM TrisCl pH 7.5, 0.1 mM DTT, 0.1mM EDTA, 1M NaCl)
  • 2L - 40 ml Tris (1M), 0.4 ml EDTA(0.5M), 0.2 ml DTT (1M), 117g NaCl
• Storage buffer (epicentre) 50% glycerol, 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton X-100.

3. Outline

3.1 Day 1 (long day)

• Grow cells, harvest, lyse, ammonium sulfate ppt (optional: collect ppt, dialysis overnight)
• Wash AKTA columns: 2xQ FF, Heparin, MonoQ, S300

3.2 Day 2 (long day, lots of waiting)

• Pass crude extract through Q FF column (last used: 1x5 ml Q FF from GE)
• Heparin column (last used: 1x5 ml Heparin from GE)
• Q column (last used: 1x1 ml Q)
• S300
• Dialysis into storage buffer overnight

3.3 Day 3 (~3 hrs)

• Aliquot, flash freeze and store

4. Detailed protocol

Note

It is critical to minimize the time RecBCD spends in high salt buffers, as the RecD unit is labile at high salt.[ref]

Danger

Be very careful about setting maximum pressure on AKTA. The MonoQ can take more pressure than superloop! (4MPa max on 10ml loop, 2MPa on 20ml and 150ml loops)

4.1 Day 0

1. Grow 50 ml overnight culture.
2. Prepare media: 31g 2YT into 1L.
3. Buffers – filtered and sterilized:
   • Buffer A: 20mM Tris pH 7.5; 0.1 mM EDTA; 0.1 mM DTT
   • Buffer B: 20mM Tris pH 7.5; 0.1 mM EDTA; 0.1 mM DTT; 1M NaCl
4. Other chemicals:
   • 3 ml of Carb and Cam stocks
   • 30 ml of 10mM biotin in DMSO (if growing biotinylated cells)
   • 3 ml IPTG
5. Make ~8 8% SDS-PAGE gels.

4.2 Day 1

1. Bacteria is grown in 1L cultures in 2YT supplemented with 0.1 mM biotin (for biotinylated RecBCD only) at 37˚C.
   1. At an OD650~0.6, bacteria is induced with 0.4 mM IPTG and grown for a further 4 hr.
   2. Bacteria may grow very slowly … prepare for a long wait.
2. Cells are harvested by centrifugation, (5K, 20 min) resuspended in resuspension buffer (50 mM TrisHCl pH 7.5/0.1 mM PMSF/10% sucrose) and frozen @ -80C.
3. The frozen cell suspension is slowly thawed and sonicated to lyse cells.
4. The suspension was ultracentrifuged for ~45 min, 36K RPM.
5. The lysate was then fractionated with 50% ammonium sulfate (good place to stop for the night) and spun at 20000g for 20 min to recover the precipitated protein.
   • Ammonium Sulfate Calculator: http://www.encorbio.com/protocols/AM-SO4.htm
6. Dialyze over night (or dilute with buffer A until conductance is equal or less than Buffer A + 100mM NaCl; a 4-5x dilution with Buffer A works).

4.3 Day 2

1. Collect dialyzed sample and ultracentrifuge at 35K for 30 min.
2. If growing > 3L of cells, run through 2x 5 mL Q FF columns in a 10-100% (Buffer A -> Buffer B) gradient.
3. Collect and pool fractions containing RecBCD.
4. Dilute with 5 volumes of Buffer A (no salt).
5. Run through a 5 ml heparin column in a 5%-100% gradient.
   • RecBCD will come out dilute, over many fractions.
6. Dilute with 3 volumes of buffer A and run through a 1 ml Q column in a 10%-100% gradient.
   • NOTE: This is done to concentrate RecBCD for gel filtration.
7. Run slowly on S300 column (~0.5 ml/min). Sample will elute at around the 45-65ml mark.
   • Make sure to run a gel of the eluted fractions. We typically see three overlapping peaks. The first (fastest) is very pure RecBCD. The second may have a slightly reduced RecD concentration. The third (slowest) peak is dirty with many contaminants. Typically we pool peaks one and two.
8. Dialyze into storage buffer overnight in cold room.

4.4 Day 3

1. Aliquot samples.
2. Flash freeze in liquid nitrogen and store in the -80C freezer.

Footnotes

1. Robison et al. (2014). Rapid prototyping of multichannel microfluidic devices for single-molecule DNA curtain imaging. Anal. Chem. ↩