Phenylketonuria (PKU) is an inherited genetic disease that causes mental retardation unless special dietary treatment is started in the newborn period of life. Mental retardation and birth defects also occur in the offspring of PKU women who are not under strict diet control before and during pregnancy. PKU diet therapy utilizes phenylalanine-free amino acid containing formulas and low protein diets that are poorly tolerated by most adolescent and adult patients due to poor odor and taste and poor heat stability of the formulas. We have hypothesized that dietary compliance of PKU adolescents and adults could be enhanced if a phenylalanine deficient protein supplement could be used in place of the amino acid mixtures. This project has previously identified a naturally occurring corn protein, gamma zein, whose low phenylalanine content and physical properties make it potentially suitable as a replacement. The gene encoding this protein has been modified to produce a series of twelve phenylalanine free mutants that are also more nutritionally appropriate for human consumption. The native gamma zein and mutant genes have been expressed in E. coli as thioredoxin linked fusion proteins that are detectable by standard staining and immunologic methods. Mutational removal of the N-terminal half of the native and three mutant proteins results in expression levels up to 50 percent of the cellular protein. This proposal seeks funding to evaluate the effects of the amino acid changes on several properties of the new proteins, including their ability to aggregate and form disulfide bonds. The results of these studies will be used to design a phenylalanine-free protein that retains solubility similar to the native protein and can be produced on a large scale to treat patients with PKU.
