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

Recombinant human phenylalanine hydroxylase (hPAH) was produced in hig h yields in Escherichia coli using the pET and pMAL expression vectors . in the pMAL system, hPAH was fused through the target sequences of t he 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 h igh specific activity even in crude extracts and was detected as a hom ogeneous band by Western-blot analysis using affinity-purified polyclo nal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and w as difficult to purify with a satisfactory yield. By contrast, when ex pressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affin ity 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 hydro xylapatite 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 ca talytic and physicochemical properties are essentially the same as tho se 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-depend ent protein kinase. The phosphorylated form of hPAH electrophoreticall y displayed an apparently higher molecular mass (similar to 51 kDa) th an the non-phosphorylated (similar to 50 kDa) form.