Protein expression using a protein gel

A SDS-PAGE gel was used to determine the different sized proteins in 2 lysate samples with the goal of identifying expression of HRP in our construct. Protein samples were taken from yeast transformed with the plasmid pFL-HRP by lysing the cells and saving the lysate. When this yeast is grown in the presence of galactose, this yeast will express HRP. Any other media that does not contain galactose will inhibit the expression of HRP. To test expression of HRP, the yeast containing the plasmid pFL-HRP was grown in 1% galactose-containing media and the lysate from these colonies is labeled “pFL-HRP Gal 1%” in the image to the left. The second lysate sample was made from yeast containing the plasmid pFL-HRP as well. However, it was grown in YPD media that does not contain galactose. This sample is labeled “Negative Control” because this sample should not contain HRP in the lysate. The third sample is a positive control, which contains only pure HRP. After analyzing the gel, there is no definitive evidence that the pFL-HRP Gal 1% sample contains HRP.

The simplest way to test for the presence of functional HRP is to mix reagents necessary for a colorimetric into the sample of interest; in our case, the sample is full-cell lysate and the reagents include TMB and H2O2. To prepare samples, 50mL cultures of our transformed yeast were inoculated in both galactose and glucose media. When initial tests failed to demonstrate HRP production in the induced sample we investigated potential causes. Using store-bought samples of HRP, we tested the colorimetric reaction at different concentrations of lysate. Using constant final concentrations of HRP, TMB, and H2O2 we discovered that an increase in lysate concentration corresponds to an apparent decrease in the extent of reaction. Presumably, something in the lysate inhibits the reaction from occurring. Our advisors indicated that native yeast proteins like catalase or superoxide dismutase could potentially consume the added H2O2 before HRP has the opportunity to react. With this information we added copper ions, an inhibitor to these proteins, to the cell lysates at a final concentration of 50mM in the hopes of witnessing an HRP-based color change. Although evidence suggests the ions were successful in inhibiting the native proteins, they also reacted with the lysate to produce a precipitate and an unexpected color change.