title: Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris.
abstract: Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Bergen, Bergen, Norway. jeffrey.mckinney@student.uib.no
authors.forename: Jeffrey
authors.initials: J
authors.lastname: McKinney
authors.forename: Per M
authors.initials: PM
authors.lastname: Knappskog
authors.forename: Jacinto
authors.initials: J
authors.lastname: Pereira
authors.forename: Trude
authors.initials: T
authors.lastname: Ekern
authors.forename: Karen
authors.initials: K
authors.lastname: Toska
authors.forename: Baukje B
authors.initials: BB
authors.lastname: Kuitert
authors.forename: David
authors.initials: D
authors.lastname: Levine
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
authors.forename: Aurora
authors.initials: A
authors.lastname: Martinez
authors.forename: Jan
authors.initials: J
authors.lastname: Haavik
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D007501:Iron; D014365:Tryptophan Hydroxylase