2.5.1. Preparation of cell free extracts.
Frozen cell paste (25-35 g) was thawed and suspended in 50 mM sodium
phosphate buffer (pH 7.0). The cell suspension was disrupted by
sonication (12 W, 6 x 15 pulses with 15 sec intervals) in 2 mL Eppendorf
tubes containing 1.5 mL of cell suspension, supplemented with lysozyme
(250 µg/mL,) in a sonicator at 4 °C. Unbroken cells and cell debris were
removed by centrifugation at 30000 x g for 30 min at 4 °C. The pellets
were re-suspended in 50 mM sodium phosphate buffer (pH 7.0) and made up
to appropriate volume and subjected to ammonium sulphate precipitation.
Solid ammonium sulphate was added and the proteins precipitated at 40-60
% saturation was collected by centrifugation, dialyzed and used for
assay.