The yeast Saccharomyces cerevisiae is one of the premier organisms for genetic and biochemical studies. However, the isolation of novel factors from yeast or the overproduction of proteins in yeast by overexpression of cloned genes can be seriously complicated by proteolysis during extract preparation and protein purification. Suppression of proteolysis can be achieved by using protease-deficient strains of yeast and by including various protease inhibitors during cell breakage and extract fractionation. Whole cell extracts are prepared by blending yeast cells with glass beads. Procedures are described which maximize the yield of nuclear proteins while minimizing proteolytic degradation.
|Number of pages||6|
|Journal||Methods in Molecular and Cellular Biology|
|State||Published - Dec 1 1994|