A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization
| Autor: | Jane Boesen, Trine V. Vendelboe, Pernille Harris, Astrid Munch, Hans Erik Mølager Christensen, Michael Ericsson Skovbo Nielsen, Charlotte Rode Petersen |
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| Rok vydání: | 2008 |
| Předmět: |
Molecular Sequence Data
Tryptophan Hydroxylase Crystallography X-Ray chemistry.chemical_compound Biosynthesis Catalytic Domain Animals Humans Amino Acid Sequence Polyacrylamide gel electrophoresis chemistry.chemical_classification Chromatography Chemistry Tryptophan Substrate (chemistry) Tryptophan hydroxylase Chromatography Ion Exchange Kinetics Enzyme Yield (chemistry) Chromatography Gel Electrophoresis Polyacrylamide Gel Serotonin Chickens Sequence Alignment Biotechnology |
| Zdroj: | Protein Expression and Purification. 57:116-126 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2007.10.016 |
| Popis: | 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 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected. |
| Databáze: | OpenAIRE |
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