Back to Search
Start Over
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.
- Source :
-
The Biochemical journal [Biochem J] 1995 Mar 01; Vol. 306 ( Pt 2), pp. 589-97. - Publication Year :
- 1995
-
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.
- Subjects :
- Amino Acid Sequence
Blotting, Western
Carrier Proteins genetics
Chemical Phenomena
Chemistry, Physical
Cyclic AMP pharmacology
Electrophoresis, Polyacrylamide Gel
Genetic Vectors
Humans
Kinetics
Maltose-Binding Proteins
Molecular Sequence Data
Molecular Weight
Phenylalanine Hydroxylase chemistry
Phenylalanine Hydroxylase metabolism
Phosphorylation
Protein Kinases metabolism
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins isolation & purification
Recombinant Fusion Proteins metabolism
ATP-Binding Cassette Transporters
Endopeptidases metabolism
Escherichia coli genetics
Escherichia coli Proteins
Gene Expression
Monosaccharide Transport Proteins
Phenylalanine Hydroxylase genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0264-6021
- Volume :
- 306 ( Pt 2)
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 7887915
- Full Text :
- https://doi.org/10.1042/bj3060589