Protein PurificationExtraction and Clarification
Preparation of cell lysates from E.coli
Different methods are used for the preparation of cell lysates from E. coli cells:
Sonication. Sonication is the most popular technique for lysing small quantities of cells (1-6 L of cell culture). Cells are lysed by liquid shear and cavitation. DNA is also sheared during sonication, so it is not necessary to add DNase to the cell suspension. The main problem is controlling the temperature. This is addressed by keeping the suspension on ice and using a number of short pulses (5-10 sec) with pauses (10-30 sec) to re-establish a low temperature. For cell quantities larger than 50 g the method is of limited value because of the difficulty in maintaining low temperatures and the long sonication times needed to reach adequate lysis.
Homogenization. Homogenizers are the most common devices to lyse bacteria. The presses lyse cells by pressurizing the cell suspension and suddenly releasing the pressure. This creates a liquid shear capable of lysing cells. Typical operating pressures for the older type of homogenizers, the French press and Manton-Gaulin homogenizer, are 6000-10,000 psi. Multiple (2-3) passes are generally required to achieve adequate lysis. The high operating pressures, however, result in a rise in operating temperatures. Therefore, pressure cells are cooled (4°C) prior to use. In addition to temperature control, care should be taken to avoid inactivating proteins by foaming.
Modern homogenizers are often continuous and can be operated at higher pressures. At EMBL we have an Avestin Emulsiflex-C5 that efficiently lyzes E. coli cells in one passage at 15,000 psi (100 MPa).
Enzymatic lysis using lysosyme. Enzymatic lysis is based on the digestion of the peptidoglycan layer of the bacterial cell wall by lysosyme. Gram-negative bacteria, however, have an outer membrane that is external to the cell wall and needs to be permeabilized to expose the peptidoglycan layer. Tris, often used as a buffer in lysis methods, effectively permeabilizes outer membranes. This effect can be enhanced by the addition of EDTA (1 mM). EDTA chelates the magnesium ions that stabilize membranes. During cell lysis often a lot of DNA is liberated and it becomes necessary to add DNase (1 mg/ml) to reduce the viscosity of the preparation. Enzymatic cell lysis can be carried out on any scale but for large-scale preparations the lysosyme and DNase can get expensive. To increase the level of cell lysis the solution can be sonicated (see above).
Freezing and grinding. An alternative lysis method is to freeze the cells directly in liquid nitrogen and ground the frozen cells to a powder using a mortar and pestle that are chilled with liquid nitrogen. The powder can be stored indefinitely at -80°C and the cell lysate can be prepared by adding the powder to 5 volumes of buffer.