DETECTION OF NONFUNCTIONAL OVEREXPRESSION GENE-PRODUCTS USING 2-DIMENSIONAL GEL-ELECTROPHORESIS WITH A NARROWED PH RANGE

A commonly used technique in the analysis of yeast protein function is the overexpression of cloned genes. In the case that overexpression d oes not lead to an altered phenotype a stable synthesis of the protein has to be demonstrated. Here an example is shown where overexpression of the yeast HYP2 genes, coding for a hypusine-containing protein, wa s alternatively analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) alone or by two-dimensional (2-D) gel elect rophoresis, using the narrowed pH range of 4.5-5.4. The results of the SDS-PAGE suggested a stable overproduction of HYP gene product, where as 2-D analysis revealed an accumulation of nonfunctional protein isof orms.