title: Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme.
abstract: Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
authors.forename: A
authors.initials: A
authors.lastname: Martinez
authors.forename: P M
authors.initials: PM
authors.lastname: Knappskog
authors.forename: S
authors.initials: S
authors.lastname: Olafsdottir
authors.forename: A P
authors.initials: AP
authors.lastname: Døskeland
authors.forename: H G
authors.initials: HG
authors.lastname: Eiken
authors.forename: R M
authors.initials: RM
authors.lastname: Svebak
authors.forename: M
authors.initials: M
authors.lastname: Bozzini
authors.forename: J
authors.initials: J
authors.lastname: Apold
authors.forename: T
authors.initials: T
authors.lastname: Flatmark
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D000242:Cyclic AMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D000242:Cyclic AMP; D010651:Phenylalanine Hydroxylase; D011494:Protein Kinases; D010450:Endopeptidases