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.

Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation

“…As expected 21, 22, the full‐length forms exists in an equilibrium of predominantly tetramers (~ 209 kDa) and some dimers (~ 104 kDa), and the catalytic core enzymes (ΔN102/ΔC24‐hPAH) were recovered as dimers (~ 70 kDa) (SEC data not shown). The protomers for the wt and mutant full‐length tetrameric forms revealed identical electrophoretic mobilities on SDS/PAGE (Fig.…”

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N‐Terminus of the Enzyme

“…Especially, Martinez et al (1995) expressed the recombinant human phenylalanine hydroxylase using the pMAL expression system and succeeded in purifying the enzyme as a tetrameric form. Therefore, we adopted the pMAL-c2 vector to express wild-type and N-terminusdeleted mutants of hTHl and to purify the fusion proteins.…”

Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

“…Wild-type and mutant forms of hPAH mutants were expressed as fusion proteins in E. coli with the maltosebinding protein (MBP) as a fusion partner [11]. Cells were grown at 37°C, but induction of hPAH by 1 mM isopropyl thio-β-D-galactoside was carried out at 28°C or 37°C.…”