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

doi:10.1016/j.pep.2007.10.016

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

Department of Chemistry

Research output: Contribution to journal › Journal article › Research › peer-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 language	English
Journal	Protein Expression and Purification
Volume	57
Issue number	2
Pages (from-to)	116-126
ISSN	1046-5928
DOIs	https://doi.org/10.1016/j.pep.2007.10.016
Publication status	Published - 2008

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@article{7707ed6d072e417082126ad399136825,

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",

number = "2",

}

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

Research output: Contribution to journal › Journal article › Research › peer-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

C2 - 18055219

SN - 1046-5928

VL - 57

SP - 116

EP - 126

JO - Protein Expression and Purification

JF - Protein Expression and Purification

IS - 2

ER -

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

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