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Phi29 Polymerase (pET19) Purification Procedure

Used in our papers:1234

Plasmid cloned from the previous phi29 into pET19. Adapted by J. Schaub for better purificaiton.

Contents

Section titled “Contents”
  1. Buffers
  2. Procedure
    1. Day 1
    2. Day 2
    3. Day 3
    4. Day 4

1. Buffers

Section titled “1. Buffers”

Lysis Buffer (250 mL)

  • 25 mM Tris pH 7.5 (1M): 6.25 mL
  • 500 mM NaCl (5M): 25 mL
  • 5% Glycerol (50% stock): 25 mL

Lysis Buffer (250 mL)

Lysis Buffer + High NaCl: (25 mL)

  • 25 mM Tris pH 7.5 (1M): 0.625 mL
  • 1M NaCl (5M): 5 mL
  • 5% Glycerol (50% stock): 2.5 mL

TwinStrep Elution Buffer: (25 mL)

  • 25 mM Tris pH 7.5 (1M): 0.625 mL
  • 500 mM NaCl (5M): 2.5 mL
  • 5% Glycerol (50% stock): 2.5 mL
  • 5 mM Desthiobiotin: 26.9 mg

Storage Buffer: (1L)

  • 10 mM Tris pH 7.5 (1M): 10 mL
  • 100 mM KCl: 7.45g
  • 1 mM DTT (1M): 1 mL
  • 0.1 mM EDTA (500 mM): 200 uL
  • 10% glycerol (50% stock): 200 mL

2. Procedure

Section titled “2. Procedure”

2.1 Day 1

Section titled “2.1 Day 1”
  1. pIF376 (WT) and pIF377 (7M). Transform in Rosetta pLysS (Carb + Cam). 37C O/N.

2.2 Day 2

Section titled “2.2 Day 2”
  1. Select single colony and inoculate 5-10 mL of LB media (+ Carb/Cam). Grow O/N.

2.3 Day 3

Section titled “2.3 Day 3”
  1. Add 5 mL of overnight culture to 1L of LB (+ Carb/Cam). Grow at 30C until OD600 of 0.6. Induce with 0.4 mM IPTG at 16C O/N.

2.4 Day 4

Section titled “2.4 Day 4”
  1. Spin culture at 4000 rpm for 10m. Go straight to purification.
  2. Suspend pellet in ~25 mL of Lysis Buffer. Homogenize with pipetting.
  3. Sonicate at 30% power for 15s ON and 90s OFF (90s total).
  4. Centrifuge pellet at 35k x g for 35 minutes.
  5. 5 mL StrepTactin Superflow Ni-NTA slurry washed with H2O and Lysis Buffer.
  6. Supernatant loaded and washed with Lysis Buffer until no detection via Bradford assay.
  7. Wash with high salt buffer. Re-equilibrate with Lysis Buffer.
  8. Elute resin with Elution buffer in 5 mL fractions.
  9. Dialyze in Dialysis Buffer with addition of SUMO Protease.
  10. Dialyze for 4 hours at 4C.
  11. Freeze in aliquots in L2. Store in -80C.

Footnotes

Section titled “Footnotes”

  1. Schaub et al. (2018). Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains. Langmuir. ↩

  2. Zhang et al. (2020). RADX condenses single-stranded DNA to antagonize RAD51 loading. Nucleic Acids Research. ↩

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

  4. Kozlov et al. (2022). How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein. J. Mol. Bio. ↩