PolQ HLD (Human) Purification Protocol

Used in our paper:¹

Plasmid gifted by the Richard Pomerantz Lab. Adapted by J. Schaub for PolQ cloned into the common lab pET19 vector.

Contents

1. Buffers
2. Protein pI’s
3. Procedure
4. Notes

1. Buffers

1.1 Lysis Buffer (100 mL)

• 50 mM HEPES pH 8.0: 5 mL (1M)
• 500 mM NaCl: 10 mL (5M)
• 5 mM BME: 34 uL (14.7M)
• 10 mM PMSF: 0.1 mL (200 mM)
• 10% glycerol: 20 mL (50% stock)
• 1% NP-40: 1 mL
• 1 tab/50 mL c0mplete
• H2O: 63.8 mL

1.2 Wash Buffer (250 mL)

• 50 mM HEPES pH 8.0: 12.5 mL (1M)
• 450 mM NaCl: 22.5 mL (5M)
• 5 mM BME: 85 uL (14.7M)
• 10 mM PMSF: 0.25 mL (200 mM)
• 0.005% IGEPAL: 12.5 uL (100%)
• 10% glycerol: 50 mL (50% stock)
• H2O: 147.25 mL

1.3 Wash 1M NaCl (25 mL)

• 50 mM HEPES pH 8.0: 1.25 mL (1M)
• 1 M NaCl: 5 mL (5M)
• 5 mM BME: 8.5 uL (14.7M)
• 10 mM PMSF: 0.025 mL (200 mM)
• 10% glycerol: 5 mL (50% stock)
• H2O: 13.72 mL

1.4 Wash 2M NaCl (25 mL)

• 50 mM HEPES pH 8.0: 1.25 mL (1M)
• 2 M NaCl: 10 mL (5M)
• 5 mM BME: 8.5 uL (14.7M)
• 10 mM PMSF: 0.025 mL (200 mM)
• 10% glycerol: 5 mL (50% stock)
• H2O: 8.72 mL

1.5 Elution Buffer (25 mL)

• 50 mM HEPES pH 8.0: 1.25 mL (1M)
• 450 mM NaCl: 2.25 mL (5M)
• 5 mM D-desthiobiotin: 28.6 mg
• 5 mM BME: 8.5 uL (14.7M)
• 0.005% Triton X-100: 1.25 uL (100%)
• 10% glycerol: 5 mL (50% stock)
• H2O: 15.25 mL

1.6 Dialysis Buffer (1L)

• 50 mM HEPES pH 8.0: 50 mL (1M)
• 500 mM NaCl: 100 mL (5M)
• 10% glycerol: 200 mL (50% stock)
• 5 mM DTT: 5 mL (1M)
• H2O: 645 mL

2. Protein pI’s

• PolQ FL: 6.92(N)/6.81(C) (~116 kDa)
• PolQ cleaved: 7.82(N)/7.58(C) (~100 kDa)
• Sumo: 6.11 (~16 kDa)
• SumoStar Pro (not SumoPro): 7.83

3. Procedure

3.1 Day 1

1. Transform pIF381 (PolQ-NSrtA) or pIF382 (PolQ-CSrtA) into BL21 cells. Plate on Carb plates.

3.2 Day 2

1. Select single colony and inoculate 100 mL of LB (+Carb). Grow at 37C O/N.
2. Grow Overnight at 37C.

3.3 Day 3

1. DInoculate 4x 1.5L flasks of TB with 15 mL of overnight LB culture. Grow at 37C until OD of ~0.6.
2. Induce cultures with 0.5 mM IPTG at 18C O/N.

3.4 Day 4

1. Spin cultures at 3500 rpm for 10m to pellet.
2. Suspend pellet in 5 mL Lysis Buffer per gram of cell pellet.
3. Homogenize and shear via sonication at 30% power for 15s ON/ 90s OFF (1:30 total).
4. Spin at 35,000 x g for 45 minutes at 4C.
5. Apply supernatant to 5 mL StrepTactin Column. Wash with Wash Buffer.
6. Wash with Wash 1M NaCl Buffer.
7. Wash with Wash 2M NaCl Buffer.
8. Elute with Elution Buffer in 2.5 – 5 mL fractions.
9. Run gel to determine fractions containing PolQ.
10. Pool fractions and add 1 tube of Sumo Protease. Dialyze against Dialysis Buffer O/N.

3.5 Day 5

1. Pool fractions and concentrate protein. Freeze in aliquots at -80C.

4. Notes

I see two common truncation products. Do not know how to remove them as of now. Pomerantz Lab purified protein did not have these truncation products. - Jeff 5.17.18

Footnotes

1. Schaub et al. (2022). Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends. NAR. ↩