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Tryptophan hydroxylase (TPH) and dopamine β-hydroxylase (DβH) are two metal-containing monooxygenases that both function e.g. in the brain where they are involved in the biosynthesis of neurotransmitters. TPH catalyse the ratelimiting step in the biosynthesis of serotonin, namely the conversion of tryptophan to 5-OH-tryptophan, whereas DβH catalyse the conversion of dopamine into norepinephrine in the catecholamine neurotransmitter synthesis and thereby control the levels of both these neurotransmitters. With these functions both TPH and DβH are involved in a range of neurological disorders related to abnormal levels of the neurotransmitters serotonin, dopamine and norepinephrine and the regulation of tryptophan hydroxylase and dopamine β-hydroxylase. These include depression, anxiety disorders, obsessive compulsive disorder (OCD), schizophrenia, Parkinson's disease and attention deficit-hyperactive disorder (ADHD) among others. Since all these diseases are the cause of huge economical and personal costs it is very important to gain more knowledge of TPH and DβH since these two enzymes could be possible targets for medicine against the diseases mentioned above. TPH a three-domain, iron-containing enzyme which belongs to the aromatic amino acid hydroxylase (AAAH) family. It exist in two isoforms, TPH1 and TPH2, which are expressed in different tissues and have different properties. TPH is known as a very diffcult protein to work with especially due to instability and only truncated forms of TPH1 have been purified and crystallized. This project concern the human neuronal TPH or TPH2. In an attempt to overcome the problems with recombinant TPH two stability and solubility optimized variants of TPH2 are designed. Escherichia coli (E. coli) expression strains for these variants and full length human TPH2 are constructed and the expression levels analysed. Moreover purification of the three TPH2 variants are compared. As earlier reported in other studies, TPH2 expressed in this project was also unstable and only partly soluble, and these properties were not improved by the introduction of mutations. Since expression of TPH in E. coli systems seems not to be feasible, it was decided to try expression of TPH2 in an eukaryote expression system, namely Drosophila melanogaster (D. melanogaster ) S2 cells. Stable transfected S2 cell lines with human TPH2 with and without the secretion signal BiP were constructed, but also in this system TPH2 was problematic. No expression of TPH2 was detected from the S2/BiP-TPH2 cell line whereas TPH2 was found in the insoluble fraction when expressed from the S2/TPH2 cell line. Dopamine β-hydroxylase contains two copper ions in the active site and belongs to the family of ascorbate dependent type II Cu monooxygenases. Very little knowledge exist on DβH and most of it comes from investigations of related proteins. Attempts to express human DβH in bacterial systems have been done in the Metalloprotein Chemistry and Engineering Group, but at present no system expressing high amounts of soluble DβH have been found. On the other hand expression of DβH in D. melanogaster S2 was earlier reported and it was decided to use this system. Stable transfected cell lines for human DβH with and without the BiP secretion signal were constructed. From the S2/BiP-DβH cell line DβH was successfully expressed and secreted in spinner cultures. An improved purification procedure for glycosylated DβH was developed and up to 1.4 mg/l culture glycosylated tetrameric DβH and 1.0 mg/l glycosylated dimeric DβH were obtained. The oligomerisation states were determined by mass spectrometry analysis. Tetrameric DβH was deglycosylated and separated from the deglycosylation enzyme in another purification step. 0.2 mg/l culture deglycosylated DβH was obtained after this step and it was used for screening of crystallization conditions.
Original languageEnglish
Publication dateOct 2010
Place of publicationKgs. Lyngby, Denmark
PublisherTechnical University of Denmark (DTU)
Number of pages166
StatePublished

Keywords

  • expression, D. melanogaster, dopamine β-hydroxylase, E. coli, tryptophan hydroxylase, purification
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