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

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Tryptophan hydroxylase (TPH) [EC] 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
Issue number2
Pages (from-to)116-126
Publication statusPublished - 2008

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