Purification of recombinant Taq 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 Taq. 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 - His14-TEV-Taq polymerase, MW 97.8 kd Expression in DH5a (with z1 pSB161, KanR), plain DH5a is ok. BL21 do not work according to Steffen Frey/Görlich lab. z1: contains LacI, reduces uninduced expression. pSB161 contains genes for rare Arg-tRNA

We also have an older version of Taq expression available that is enriched by thermal denaturation only. The plasmid is in the cDNA-misc box. Please find here the protocol.

    Expression

  • Transform E. coli strain, plate on selective plates and inocculate a culture (300 ug/ml Ampicillin, 25 ug/ml Kanamycin). 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 100 ml of a fresh overnight culture. The resulting OD600 should be 0.3. shake at 37C until OD reaches 0.8. record grow curve, take samples hourly.
  • take a 1 ml sample (0h control). Spin at 13k for 1 min. Discard the clear supernatant. Dissolve the pellet in 80 ul Lämmli buffer. Load 5 ul for SDS-PAGE.
  • Add 500 ul of 1 M IPTG (final conc. 0.5 mM). Shake for further 6 h. Take sample hourly for growth curve and analysis with SDS-PAGE. Use 100 ul Lämmli for 1 OD unit.
  • After 6 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 TrisHCl pH 7.5, 50 mM glucose, 1 mM EDTA). Use a 25 ml pipette for suspension. The volume depends on density. Use 1 ml for each 200 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 20 mM (stock is 14.3 M)
  • Add lysozyme to 4 mg/ml, incubate at RT for 15 min
  • take 5 ul (corresponds to 1 OD) mix with 95 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 75C
  • Add one volume of boiling extraction buffer 2 (10 mM Tris pH8, 50 mM KCl, 1 mM EDTA, 2 mM imidazol, 0.5% Tween20, 0.5% NP-40)
  • Incubate in water bath at 75C for 30 min
  • Cool on ice
  • spin in ultra centrifuge for 15 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 Tris pH8, 100 mM NaCL, 0.1 mM EDTA, 10 mM imidazol. wash the column with about 10 volumes of 50 mM Tris pH8, 100 mM NaCl, 0.1 mM EDTA, 10 mM imidazol, 5 mM DTT wash the column with 50 mM Tris pH8, 100 mM NaCl, 0.1 mM EDTA, 10 mM imidazol, 5 mM DTT, 25% Glycerol
  • Elute the protein with 50 mM Tris pH8, 100 mM NaCl, 0.1 mM EDTA, 300 mM imidazol, 5 mM DTT, 25% Glycerol
  • pool the peak fractions

    • Storage and quality control

  • buffer exchange with NAP-10 columns with 50 mM Tris pH8, 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 25% Glycerol
  • measure the concentration: 1 OD280 corresponds to 0.88 mg/ml
  • slowly (drop wise) add and mix with 1 volume of storage buffer (50 mM Tris-HCl pH 8, 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 75% glycerol, 2% TritonX-100)
  • store at -20C in aliquots.
  • perform activity assays in comparision to a known polymerase sample.