High-level expression and deletion mutagenesis of human tryptophan hydroxylase.
Human tryptophan hydroxylase has been expressed as a soluble and active form in Escherichia coli by fusion with an affinity tag, maltose-binding protein. The fusion protein has been purified to near homogeneity by affinity chromatography on crosslinked amylose resin. The purified fusion protein has a specific activity of 86 nmol of 5-hydroxytryptophan per min per mg of fusion protein. A series of truncation mutants have also been made to explore the domain organization of tryptophan hydroxylase. All deletion mutants were subject to affinity purification and kinetic characterization. While removal of the N-terminal 164 amino acids completely inactivates the enzyme, deletion of the first 91 residues results in a 7-fold reduction in specific activity. From the C terminus, deletion of 36, 55, or 112 amino acids abolishes the activity, whereas deletion of 19 residues decreases the specific activity by approximately 11-fold. These results are consistent with a model for tryptophan hydroxylase in which the enzyme consists of an N-terminal regulatory domain, a catalytic core, and a small C-terminal region of uncertain but important function.
Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892.
X J
XJ
Yang
S
S
Kaufman
D018528:ATP-Binding Cassette Transporters; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014365:Tryptophan Hydroxylase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D014365:Tryptophan Hydroxylase