Detection of nonfunctional overexpression gene products using two-dimensional gel electrophoresis with a narrowed pH range.

Abstract

A commonly used technique in the analysis of yeast protein function is the overexpression of cloned genes. In the case that overexpression does 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, was alternatively analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) alone or by two-dimensional (2-D) gel electrophoresis, using the narrowed pH range of 4.5-5.4. The results of the SDS-PAGE suggested a stable overproduction of HYP gene product, whereas 2-D analysis revealed an accumulation of nonfunctional protein isoforms.