Protein PurificationExtraction and Clarification
Preparation of cell lysates from yeast
Different methods are used for the preparation of cell lysates from yeast cells:
Autolysis. The oldest method is autolysis, A yeast suspension is mixed with toluene (or compounds such as ammonium hydroxide) and incubated at room temperature for 24-48 h. The toluene prevents bacterial growth and permeabilizes the yeast membrane. The latter releases a wide variety of hydrolases that attack the cell wall. This procedure is not recommended since it also releases many proteases that could damage the target protein.
Homogenization. The homogenizers can be used to disrupt yeast cells. 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 (at least 3) passes are required to achieve a reasonable degree of 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 more suited to lyze yeast cells since they can be operated at much higher pressures. At EMBL we have an Avestin Emulsiflex-C5 that adequately lyzes Pichia pastoris cells in 3-5 passage at 30,000 psi (200 MPa).
Glass bead vortexing. Probably the most widely used method is the disruption of yeast cells by agitation with glass beads (0.4-0.5 mm). The simplest method for agitating the glass beads is with the use of a vortex mixer. Several cycles of agitation (30-60 sec) must be interspersed with cycles of cooling on ice to avoid overheating of the cell suspension. Breakage is variable, but can be well over 50% (up to 95%). Above the method is described for small volumes (up to 15 ml) but it can be scaled up to many liters using specialized apparatus.
Ezymatic lysis. The enzymatic lysis of yeast cells is based on the digestion of the cell wall by a number of enzymes, of which zymolase and lyticase are the most widely used. The procedure yields spheroplasts that can be prepared and purified as an intermediary step or lysis can be carried out directly. The procedure can be used on any scale but for large scale preparations the enzymes can get expensive.
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.