Cloning, expression, purification and characterization of tryptophan hydroxylase variants

Jane Boesen

Research output: Book/ReportPh.D. thesis

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Tryptophan hydroxylase (TPH) catalyzes the first and rate-limiting step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). Serotonin is involved in many physiological functions, such as appetite and sleep rhythm, as well as a wide range of psychiatric disorders such as depression and obsessive-compulsive disorder (OCD). Characterization of TPH and elucidation of the enzymes regulation and catalytic mechanism is therefore vital to our understanding of the serotonin balance.
This study concerns variants of both human TPH isoform 1 (hTPH1) and human TPH isoform 2 (h PH2). The main goal was to purify full-length hTPH1. Based on earlier results, hTPH1 was purified using detergent in the purification methods. After incubation of the hTPH1 sample with 0.1 % of n-dodecyl-β-D-maltopyranoside (DDM) the protein binds to the anion exchange column and elutes over a large area in the anion exchange, indicating that the protein still exists in different oligomer forms. This was also observed in the gel filtration. Variants of both hTPH1 and hTPH2 containing the regulatory domain or parts of it were constructed and tested for expression in Escherichia coli as well as solubility. It was observed that changes in the amino acid sequence of the regulatory domain by point mutations or truncations in the N-terminal had a huge impact on the solubility of the protein and caused the protein to be insoluble.
The regulatory domain of human TPH1 (rhTPH1), and two fusion proteins of rhTPH1 fused to the green fluorescent protein (GFP) in the C-terminal and the glutathione S-transferase (GST) in the N-terminal, respectively, were expressed in a soluble form. The purification trials of the variants containing the regulatory domain showed that a high salt concentration was necessary to stabilize the variant. The GST-rhTPH1 variant could be purified using affinity chromatography followed by gel filtration with high purity and a yield of 40 mg/l culture. The purified GST-rhTPH1 exists as a dimer in solution due to dimerization of GST. The GST could be cleaved successfully from the fusion protein using Factor Xa and rhTPH1 was successfully purified from GST after cleavage.
Characterization was performed on the three hTPH variants: The catalytic domain of both hTPH1 (chTPH1) and hTPH2 (chTPH2) as well as the catalytic and tetramerization domain of hTPH2 (cthTPH2). The kinetic parameters of chTPH1 was determined and compared with parameters of chTPH2. Large differences were observed between the two isoforms and tryptophan inhibition was observed for chTPH1 but not for chTPH2.
Mass spectrometric analysis of chTPH1 shows that the sample contains two species: chTPH1 and another species, which could not be seen during purification or electrophoresis of the chTPH1 sample. Additionally, chTPH1 shows to be fully loaded with iron, whereas chTPH2 showed to be heterogeneous with respect to Fe. chTPH2 binds both tryptophan and tetrahydrobiopterin individually. Also characterization of cthTPH2 by tandem mass spectrometry shows that the variant is a tetramer.
Crystallization of chTPH1 was achieved both without substrate and with bound substrate (tryptophan and pterin) but resulted in very small crystals. A data set of the variant without bound substrate was collected to 4 Å and the structure was solved by molecular replacement. The structure was refined to an Rfree of 33.5 % and the overall structure is compared to the overall structure of the catalytic domain of hTPH1 co-crystallized with BH2. A structural change in the residues 125 to 130 is observed. This is the first structure of chTPH1 without any substrates or inhibitors.

Original languageEnglish
PublisherDTU Chemistry
Number of pages187
Publication statusPublished - 2010


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