120 L fermentor: Cell disruption and purification

Cell disruption

Select pressure according to information supplied by Constant Systems:   Switch on chiller (4°C) switch on disruptor run 1-2 fillings of water through at 10-20 KPSI (don't go beyond 40KPSI with the pressure adjustment, please) run your sample (foaming) run water through until machine is clean run 20% ethanol through to prevent growth switch off chiller and instrument please be aware that unless disassembled, this machine can not be used for Western blot samples (contamination possible) little pieces of plastic or tissue can block the jet. Easy to fix by taking off the chilled steel dome.

Example purification (Ni Sepharose FF)

120L BAI cells gave 1 Kg of wet cell pellet, frozen in LN2 smash frozen pellet into small pieces, be careful not to get pieces of plastic into suspension add 50 mM Tris pH 8 up to 2L, 20 EDTA-free PI tablets (Roche) stir until completely smooth run through cell disruptor at 20 KPSI, let foam settle for 10 min fill 12 type 19 rotor bottles and balance (because of foam) run two rotors at 19.000 rpm for 1 hour at 4°C decant supernatant and collect run supernatant at 20 ml/min over 50 ml Amersham Ni Sepharose FF follow your usual elution protocol, everything at 20 ml/min.

JL 3/6/2005