Physical and chemical cell disruption for the recovery of intracellular proteins.
There are many ways to disrupt microorganisms and plant and animal tissue. Selecting the best cell disruption method depends on the factors listed in Table 6. The kind or type of cells is an important consideration. For example, some disruption methods which work well for animal tissue do not work at all for microorganisms. A guideline for the suitabiity of a given disruption method for some cell types is given in Table 7. The ratings in this table are not incontestable and, as mentioned earlier, combinations of methods can sometimes produce satisfactory results whereas one method alone fails. The disruptibility of cells can be influenced by their growth and storage history. For microorganisms, cells in log phase growth tend to produce thinner cell walls which are more easy to disrupt. This and other conditions which can influence microbial cell disruptiability are listed in Table 8. The cell disruption method selected will depend on its capability to process samples of a certain size or to be able to process multiple samples in a reasonable period of time. Other considerations are the availability, cost, and general utility of the disruption equipment. Thus, in a research environment the purchase of an expensive cell disrupter which processes a wide variety of cell types may be more easy to justify than a specialized disrupter. And if the long-term goal is to scale up, the choice of disruption methods narrow considerably. Indeed, several of the most successful laboratory cell disruption methods have no possibility of being scaled up. Despite possible scale-up difficulties, in the case of many bioactive recombinant products expressed at high levels in microorganisms, this concern may be irrelevant. Few of these products are likely to be manufactured in really large amounts and current laboratory scale or pilot plant scale production equipment may be entirely adequate. For instance, active human TNF (tissue necrosis factor) can be expressed in Pichia pastoris yeast at levels of 100 g/kg of yeast (dry weight). At this level of expression, only a few kilograms of r-DNA yeast needs be disrupted to meet the worldwide demand for this research material. Finally, the operating and energy requirements which affect the economics of the disruption process (batch versus continuous, disruption yield, cell fragment size, effect of added enzymes on downstream separation, etc.) are important considerations in the selection of production equipment.
Hopkins
T R
TR
Phillips Petroleum Company, Bartlesville, Oklahoma.
D000818:Animals; D001419:Bacteria; D002458:Cell Fractionation / Q000379:methods*; D006867:Hydrolases; D007202:Indicators and Reagents; D010944:Plants; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014465:Ultrasonics
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D006867:Hydrolases