Quantitative analysis by flow cytometry of green fluorescent protein-tagged human phenylalanine hydroxylase expressed in Dictyostelium

Abstract

We have developed a fluorescence assay system to monitor the protein levels of human phenylalanine hydroxylase (hPAH). Wild-type (WT) and three mutant hPAHs (I65T, L255V, and S349L) were expressed as green fluorescent protein (GFP)-tagged forms in a PAH knockout mutant (pah−) of Dictyostelium discoideum Ax2. The fluorescence-activated cell sorting (FACS) analysis showed that the GFP positive cells were the most frequent in WT but were rare in pah−, demonstrating the successful expression of GFP-tagged hPAHs in Dictyostelium. The fluorescence levels of mutants relative to WT were higher than expected from the protein amounts determined from the non-tagged forms, probably due to the presence of the N-terminal GFP. However, treatment of the cells with cumene hydroperoxide, which is known to accelerate protein degradation, decreased fluorescence levels, suggesting that protein stability changes in individual mutations can be monitored by FACS analysis. For an evaluation study, a putative pharmacological chaperone effect of yeast extract on S349L was examined by Western blot and FACS analysis. Both the protein amount and the fluorescence levels were increased by yeast extract, supporting that the FACS analysis could replace the time- and labor-consuming procedures such as the Western blot and cell culture. The fluorescence-based cell assay system may be valuable for the high-throughput screening of pharmacological chaperones for phenylketonuria mutations.

Keywords

• dictyostelium
• facs analysis
• pharmacological chaperone
• phenylalanine hydroxylase
• phenylketonuria