Abstract
A full-length human phenylalanine hydroxylase cDNA has been recombined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express phenylalanine hydroxylase immunoreactive protein and pterin-dependent conversion of phenylalanine to tyrosine. Recombinant human phenylalanine hydroxylase produced in E. coli has been partially purified, and biochemical studies have been performed comparing the activity and kinetics of the recombinant enzyme with native phenylalanine hydroxylase from human liver. The optimal reaction conditions, kinetic constants, and sensitivity to inhibition by aromatic amino acids are the same for recombinant phenylalanine hydroxylase and native phenylalanine hydroxylase. These data indicate that the recombinant human phenylalanine hydroxylase is an authentic and complete phenylalanine hydroxylase enzyme and that the characteristic aspects of phenylalanine hydroxylase enzymatic activity are determined by a single gene product and can be constituted in the absence of any specific accessory functions of the eukaryotic cell. The availability of recombinant human phenylalanine hydroxylase produced in E. coli will expedite physical and chemical characterization of human phenylalanine hydroxylase which has been hindered in the past by inavailability of the native enzyme for study.
Highlights
A full-length human phenylalanine hydroxylase cDNA has been recombinedwith a prokaryotic expression vector and introduced into Escherichiacoli
We report the cloning of the full length human phenylalanine hydroxylase cDNA into a prokaryotic expression vector, production of the recombinant human phenylalanine hydroxylase in Escherichia coli, partial purification of recombinant human phenylalanine hydroxylase, and comparison of the biochemical properties of this enzyme with native phenylalanine hydroxylase from human liver
This report describes the production of recombinant human phenylalanine hydroxylase in E. coli using a prokaryotic expression vector containing the phenylalanine hydroxylase cDNA
Summary
Recombinant DNA Clones-Phenylalanine hydroxylase coding sequences were derived from the full-length human phenylalanine hydroxylase cDNA phPAH247 (13). Plasmid phPAH247 was cut with EcoRI (IBI), which cuts the boundaries of the cDNA insert, and HpaII (Biolabs), which cuts twice within the 5’ untranslated region of the phenylalanine hydroxylase cDNA at positions 76 and 212 (Fig. 1).This 2236-base fragment contains the entire open reading frame and 3’ untranslated region of phenylalanine hydroxylase but retains only 12 bases 5’ to theAUG. This fragment was blunted with the Klenow (large fragment DNA polymerase).
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