A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Michael Skovbo Windahl, Charlotte R. Petersen, Astrid Munch, Trine Vammen Vendelboe, Jane Boesen, Pernille Harris, Hans Erik Mølager Christensen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 μmol/min/mg. The Km values were determined to Km,tryptophan = 7.7 ± 0.7 μM, Km,BH4=324±10 μM and Km,O2=39±2 μM. Substrate inhibition by tryptophan was observed at concentrations above 15 μM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-l-biopterin and a data set to 3 Å resolution has been collected.
Original languageEnglish
JournalProtein Expression and Purification
Volume57
Issue number2
Pages (from-to)116-126
ISSN1046-5928
DOIs
Publication statusPublished - 2008

Cite this

Windahl, Michael Skovbo ; Petersen, Charlotte R. ; Munch, Astrid ; Vendelboe, Trine Vammen ; Boesen, Jane ; Harris, Pernille ; Christensen, Hans Erik Mølager. / A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization. In: Protein Expression and Purification. 2008 ; Vol. 57, No. 2. pp. 116-126.
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title = "A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization",
abstract = "Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 μmol/min/mg. The Km values were determined to Km,tryptophan = 7.7 ± 0.7 μM, Km,BH4=324±10 μM and Km,O2=39±2 μM. Substrate inhibition by tryptophan was observed at concentrations above 15 μM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-l-biopterin and a data set to 3 {\AA} resolution has been collected.",
author = "Windahl, {Michael Skovbo} and Petersen, {Charlotte R.} and Astrid Munch and Vendelboe, {Trine Vammen} and Jane Boesen and Pernille Harris and Christensen, {Hans Erik M{\o}lager}",
year = "2008",
doi = "10.1016/j.pep.2007.10.016",
language = "English",
volume = "57",
pages = "116--126",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press",
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A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization. / Windahl, Michael Skovbo; Petersen, Charlotte R.; Munch, Astrid; Vendelboe, Trine Vammen; Boesen, Jane; Harris, Pernille; Christensen, Hans Erik Mølager.

In: Protein Expression and Purification, Vol. 57, No. 2, 2008, p. 116-126.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

AU - Windahl, Michael Skovbo

AU - Petersen, Charlotte R.

AU - Munch, Astrid

AU - Vendelboe, Trine Vammen

AU - Boesen, Jane

AU - Harris, Pernille

AU - Christensen, Hans Erik Mølager

PY - 2008

Y1 - 2008

N2 - Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 μmol/min/mg. The Km values were determined to Km,tryptophan = 7.7 ± 0.7 μM, Km,BH4=324±10 μM and Km,O2=39±2 μM. Substrate inhibition by tryptophan was observed at concentrations above 15 μM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-l-biopterin and a data set to 3 Å resolution has been collected.

AB - Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 μmol/min/mg. The Km values were determined to Km,tryptophan = 7.7 ± 0.7 μM, Km,BH4=324±10 μM and Km,O2=39±2 μM. Substrate inhibition by tryptophan was observed at concentrations above 15 μM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-l-biopterin and a data set to 3 Å resolution has been collected.

U2 - 10.1016/j.pep.2007.10.016

DO - 10.1016/j.pep.2007.10.016

M3 - Journal article

VL - 57

SP - 116

EP - 126

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

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ER -