Detection of nonfunctional overexpression gene products using two-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 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.
Wöhl
T
T
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Klier
H
H
Lottspeich
F
F
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000031:analogs & derivatives; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; C100028:hypusine; D008239:Lysine