Purification of recombinant Pfu polymerase

The expression plasmid was obtained from Steffen Frey in Görlich's laboratory. It has been succesfully used in student courses and yields more than 100 k units of Pfu (from Pyrococcus furiosus). Purification is performed by themal denaturation of all the other proteins and in addition by affinity chromatography with a His tag at the N-terminus.

Construct in pQE - His10-TEV-Taq polymerase, MW 92.7 kd Expression in BL21DE3 Star Rosetta (ChloramphenicolR). Rosetta (ChlorampR) carries genes for rare tRNA

    Expression

  • Transform E. coli strain, plate on selective plates and inocculate a culture (300 ug/ml Ampicillin, 34 ug/ml chloramphenicol). Grow overnight.
  • inocculate prewarmed (37C) 1l 2YT, 2% glucose, 40 mM K2HPO4, 300 ug/ml Amp, 50 ug/ml Kan in a 5 l flask with about 30 ml of a fresh overnight culture. The resulting OD600 should be 0.1. shake at 37C. record grow curve.
  • take a 1 ml sample (0h control). Spin at 13k for 1 min. Discard the clear supernatant. Dissolve the pellet in 50 ul Lämmli buffer. Load 5 ul for SDS-PAGE.
  • Add 200 ul of 1 M IPTG (final conc. 0.2 mM). Shake for another 3 h. Take sample hourly for growth curve and analysis with SDS-PAGE. Use 100 ul Lämmli for 1 OD unit.
  • After 3 h add 10 ml of 100mM PMSF (final conc 1 mM) and 2.5 ml of 100 mM ortho Phenanthrolin (final conc 250 uM). Spin at 4C at 4-5k for 10 min.
  • Suspend the pellet on ice in Extraction buffer (50 mM Na-phosphate pH 7.5, 200 mM NaCl, 1 mM imidazol). Use a 25 ml pipette for suspension. The volume depends on density. Use 1 ml for each 100 OD units.
  • Transfer the suspension into 50 ml falcon tubes. Freeze in liquid nitrogen.

    • Lysis

  • Thaw the suspension in a water bath.
  • Add ß-mercaptoethanol to 10 mM (stock is 14.3 M)
  • Lyse by sonification: 3 x 2 min, maximal output, 40% duty cycle on ice, in a precooled metal container
  • take 5 ul (corresponds to 1 OD) mix with 45 ul Lämmli, load 5 ul on SDS-PAGE
  • spin 15 min at more than 4.5 k at 4C.
  • carefully transfer the supernatant to a 50 ml falcon tube. Take a 5 ul sample (0.5 OD) mix with 45 ul Lämmli, load 5 ul on SDS-PAGE
  • Alternatively, the cells may be lysed by the microfluidizer in Ficner's lab

    • Purification

  • Prepare a waterbath at 85C
  • Incubate in water bath at 85C for 15 min
  • Cool on ice
  • spin in ultra centrifuge for 15-30 min at 38k, take a sample of 5 ul (0.5 OD), add 45 ul of Lämmli, load 5 ul on SDS-PAGE

    • Affinity chromatography

  • Apply the clear supernatant on a Ni-column. Equilibrate with 50 mM Na-phosphate pH7.5, 500 mM NaCl, 10 mM imidazol.
  • wash the column with about 10-20 volumes of 50 mM Na-phosphate pH7.5, 500 mM NaCl, 10 mM imidazol, 5 mM DTT
  • Elute the protein with 50 mM Na-phosphate pH7.5, 500 mM NaCl, 5 mM DTT 300 mM imidazol,
  • pool the peak fractions, take samples for SDS-PAGE

    • Storage and quality control

  • buffer exchange with NAP-10 columns with 40 mM TrisHCl pH7.5, 200 mM KCl, 2 mM DTT, 0.2 mM EDTA
  • measure the concentration in a 1:20 dilution with storage buffer: 1 OD280 corresponds to 0.75 mg/ml
  • add and mix with 1 volume glycerol (final 50%),NP40 to finally 0.5% and Tween20 to 0.5%.
  • store at -20C in aliquots.
  • perform activity assays in comparision to a known polymerase sample.