Cloning, expression, purification and characterization of tryptophan hydroxylase variants

Publication: ResearchPh.D. thesis – Annual report year: 2010

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Cloning, expression, purification and characterization of tryptophan hydroxylase variants. / Boesen, Jane; Christensen, Hans Erik Mølager (Supervisor).

DTU Chemistry, 2010. 187 p.

Publication: ResearchPh.D. thesis – Annual report year: 2010

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@book{0ba5a77cfb92473081e9ccefac7368e2,
title = "Cloning, expression, purification and characterization of tryptophan hydroxylase variants",
publisher = "DTU Chemistry",
author = "Jane Boesen and Christensen, {Hans Erik Mølager}",
year = "2010",

}

RIS

TY - BOOK

T1 - Cloning, expression, purification and characterization of tryptophan hydroxylase variants

A1 - Boesen,Jane

AU - Boesen,Jane

A2 - Christensen,Hans Erik Mølager

ED - Christensen,Hans Erik Mølager

PB - DTU Chemistry

PY - 2010

Y1 - 2010

N2 - <p>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. <br/> This study concerns variants of both human TPH isoform 1 (<em>h</em>TPH1) and human TPH isoform 2 (<em>h </em>PH2). The main goal was to purify full-length <em>h</em>TPH1. Based on earlier results, <em>h</em>TPH1 was purified using detergent in the purification methods. After incubation of the <em>h</em>TPH1 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 <em>h</em>TPH1 and <em>h</em>TPH2 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. <br/> The regulatory domain of human TPH1 (r<em>h</em>TPH1), and two fusion proteins of r<em>h</em>TPH1 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-r<em>h</em>TPH1 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-r<em>h</em>TPH1 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. <br/> Characterization was performed on the three hTPH variants: The catalytic domain of both <em>h</em>TPH1 (c<em>h</em>TPH1) and <em>h</em>TPH2 (c<em>h</em>TPH2) as well as the catalytic and tetramerization domain of <em>h</em>TPH2 (cthTPH2). The kinetic parameters of chTPH1 was determined and compared with parameters of c<em>h</em>TPH2. Large differences were observed between the two isoforms and tryptophan inhibition was observed for c<em>h</em>TPH1 but not for c<em>h</em>TPH2. <br/> Mass spectrometric analysis of c<em>h</em>TPH1 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, c<em>h</em>TPH1 shows to be fully loaded with iron, whereas c<em>h</em>TPH2 showed to be heterogeneous with respect to Fe. c<em>h</em>TPH2 binds both tryptophan and tetrahydrobiopterin individually. Also characterization of ct<em>h</em>TPH2 by tandem mass spectrometry shows that the variant is a tetramer. <br/> Crystallization of c<em>h</em>TPH1 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 R<sub>free</sub> of 33.5 % and the overall structure is compared to the overall structure of the catalytic domain of <em>h</em>TPH1 co-crystallized with BH<sub>2</sub>. A structural change in the residues 125 to 130 is observed. This is the first structure of c<em>h</em>TPH1 without any substrates or inhibitors.</p>

AB - <p>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. <br/> This study concerns variants of both human TPH isoform 1 (<em>h</em>TPH1) and human TPH isoform 2 (<em>h </em>PH2). The main goal was to purify full-length <em>h</em>TPH1. Based on earlier results, <em>h</em>TPH1 was purified using detergent in the purification methods. After incubation of the <em>h</em>TPH1 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 <em>h</em>TPH1 and <em>h</em>TPH2 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. <br/> The regulatory domain of human TPH1 (r<em>h</em>TPH1), and two fusion proteins of r<em>h</em>TPH1 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-r<em>h</em>TPH1 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-r<em>h</em>TPH1 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. <br/> Characterization was performed on the three hTPH variants: The catalytic domain of both <em>h</em>TPH1 (c<em>h</em>TPH1) and <em>h</em>TPH2 (c<em>h</em>TPH2) as well as the catalytic and tetramerization domain of <em>h</em>TPH2 (cthTPH2). The kinetic parameters of chTPH1 was determined and compared with parameters of c<em>h</em>TPH2. Large differences were observed between the two isoforms and tryptophan inhibition was observed for c<em>h</em>TPH1 but not for c<em>h</em>TPH2. <br/> Mass spectrometric analysis of c<em>h</em>TPH1 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, c<em>h</em>TPH1 shows to be fully loaded with iron, whereas c<em>h</em>TPH2 showed to be heterogeneous with respect to Fe. c<em>h</em>TPH2 binds both tryptophan and tetrahydrobiopterin individually. Also characterization of ct<em>h</em>TPH2 by tandem mass spectrometry shows that the variant is a tetramer. <br/> Crystallization of c<em>h</em>TPH1 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 R<sub>free</sub> of 33.5 % and the overall structure is compared to the overall structure of the catalytic domain of <em>h</em>TPH1 co-crystallized with BH<sub>2</sub>. A structural change in the residues 125 to 130 is observed. This is the first structure of c<em>h</em>TPH1 without any substrates or inhibitors.</p>

BT - Cloning, expression, purification and characterization of tryptophan hydroxylase variants

ER -