HEAT-INDUCED PRECIPITATION OF CELL HOMOGENATES - AN INVESTIGATION OF THE RECOVERY OF THERMOSTABLE PROTEINS

The use of heat treatment as a means of partially purifying thermostable proteins from more labile proteins has been studied. This system, where a thermostable T. aquaticus β-galactosidase was added to and recovered from a yeast cell homogenate, is a useful model for the recovery of intracellular thermostable mesophilic proteins or thermophilic proteins cloned and expressed in mesophilic hosts. Components of the yeast cell homogenate whose removal may be facilitated by heat treatment include insoluble debris and soluble macromolecules. While the former can be partially removed using continuous industrial centrifuges, the remaining cell debris has a deleterious effect on the performance of subsequent protein purification steps. Incubation at high temperatures (70°C to 90°C) yielded protein aggregates that were readily removed along with the majority of the cell debris components by low speed centrifugation. Almost complete recovery of β-galactosidase activity was achieved at these high temperatures, leading to a purification factor with respect to total cell protein of 35- to 50-fold. The application of purification by heat treatment in pilot and industrial-scale processing is discussed. © 1990.