Expression of Wild Type and Mutant Forms of Human Phenylalanine Hydroxylase in E. Coli

Abstract

Phenylketonuria (PKU) is an autosomal recessive disease caused by the absence or severely reduced enzymatic activity of the hepatic enzyme phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1, PAH). The loss of enzymatic activity found in PKU patients is a result of single base substitutions or small deletions in the PAH gene. Presently, more than 70 different mutations associated with the disease are known. PKU and non-PKU hyperphenylalaninemia (HPA) patients show extensive clinical heterogeneity. To understand the molecular mechanism of this heterogeneity, it is necessary to characterize the structural and functional properties of the different mutant PAH forms. Liver biopsies from PKU/HPA patients are usually not available, but in vitro expression in eukaryotic cells has been used for the characterization of certain mutant forms. However, it has been a problem to express stable forms of PAH in such in vitro systems¹, and therefore only a few mutant forms have been further characterized so far. This led us to clone and express the normal and several mutated forms of human PAH cDNA into the pET-plasmid system of E. coli (Novagen). Here we report the expression of both normal and seven disease associated forms of PAH, i.e. mutations in exon 7 and 8 of the PAH gene.