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Materials

Chemicals

  • Lyticase [or Zymolase 100T (ICN, USA)]
  • Brij 58

Working buffer

  • 50 mM Tris-HCl pH 8.0
  • 10% sorbitol

Stock solutions

  • 100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol
  • 1 mg/ml leupeptin in water
  • 1 mg/ml pepstatin A in methanol
  • 100 mM spermidine (pH 7.8)
  • 100 mM EDTA
  • 1 M MgCl2.
  • 4 M KCl

Procedure

1. Resuspend the cells in an equal amount of chilled working buffer. Adjust the pH of the suspension to 8.2 with solid Tris base.
2. Freeze the suspension rapidly in liquid nitrogen in an ultracentrifuge tube. (Freezing and thawing is essential in this procedure).
3. Thaw the frozen suspension is thawed rapidly and place it on ice.
  • Add 10 µl PMSF (100 mM) per ml of celsuspension at this point.
4. Add the following solutions to the cell suspension: 4 M KCl to a concentration of 1 M, 100 mM spermidine to 5 mM, and lyticase to 2000 U/ml.
  • Instead of lyticase, Zymolase 100T could be used at a final concentration of 0.5 mg/ml.
5. Mix gently and incubate the suspension for 30 min on ice, with occasional swirling. Usually over 90% of the cells are converted to spheroplasts.
6. Add different protease inhibitors: 2 µl/ml EDTA (0.5 M), 1 µl/ml PMSF (100mM), 1 µl/ml leupeptin (1 mg/ml), and 1µl/ml pepstatin A (1 mg/ml) to the suspension.
7. Add Brij 58 to a concentration of 0.1% and heat shock the spheroplasts for 3 min at 37°C.
8. Remove cell debris by centrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).