A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization
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 Escherichi...
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| Veröffentlicht in: | Protein expression and purification 2008-02, Vol.57 (2), p.116-126 |
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| container_title | Protein expression and purification |
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| creator | Nielsen, Michael S. Petersen, Charlotte R. Munch, Astrid Vendelboe, Trine V. Boesen, Jane Harris, Pernille Christensen, Hans E.M. |
| description | 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 11mg/L culture and a specific activity of 0.60μmol/min/mg. The Km values were determined to Km,tryptophan=7.7±0.7μM, Km,BH4=324±10μM and Km,O2=39±2μM. Substrate inhibition by tryptophan was observed at concentrations above 15μM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-l-biopterin and a data set to 3Å resolution has been collected. |
| doi_str_mv | 10.1016/j.pep.2007.10.016 |
| format | Article |
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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 11mg/L culture and a specific activity of 0.60μmol/min/mg. The Km values were determined to Km,tryptophan=7.7±0.7μM, Km,BH4=324±10μM and Km,O2=39±2μM. Substrate inhibition by tryptophan was observed at concentrations above 15μM. 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Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-l-biopterin and a data set to 3Å resolution has been collected.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Biotechnology - methods</subject><subject>Catalytic Domain</subject><subject>Chickens - metabolism</subject><subject>Chromatography, Gel</subject><subject>Chromatography, Ion Exchange</subject><subject>Crystallization</subject><subject>Crystallography, X-Ray</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Escherichia coli</subject><subject>Expression</subject><subject>Gallus gallus</subject><subject>Humans</subject><subject>Kinetic</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>Purification</subject><subject>Sequence Alignment</subject><subject>Tryptophan hydroxylase</subject><subject>Tryptophan Hydroxylase - chemistry</subject><subject>Tryptophan Hydroxylase - isolation & purification</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUctu1TAUtBAVfX4AG-QVu1yO87ATsaqq8pAqsaFry3GOdX1JYmM70PAT_DJO75XYwcqe0cxYniHkNYMdA8bfHXYe_a4EEBnvMvOCXDDoeAGl6F5u95oXTVe25-QyxgMAYxyaV-SctdA0JesuyO9bGu3kR6Tpp6Mxoac-OI3DEpAaF6hfgjVWq2TdTJ2haY80IzWuyWo6uEnZZ17vrf6GM01h9cn5vZrpfh2Ce1pHFZEymmVqS5xoXGxS_XjM12GNOW20v56fuCZnRo0Rb07nFXn8cP_17lPx8OXj57vbh0JXbZkKPlRt1atWiFoh9KwHAKz1wHjHa6VY16lGcNX3kFFlkA2mNWg4lFWtupJXV-TtMTf_9vuCMcnJRo3jqGZ0S5Qit9kyUf9XmNuvm1JAFrKjUAcXY0AjfbCTCqtkILe55EHmuTaD2KjMZM-bU_jSTzj8dZz2yYL3RwHmLn5YDDJqi3PexwbUSQ7O_iP-D7a8qKs</recordid><startdate>200802</startdate><enddate>200802</enddate><creator>Nielsen, Michael S.</creator><creator>Petersen, Charlotte R.</creator><creator>Munch, Astrid</creator><creator>Vendelboe, Trine V.</creator><creator>Boesen, Jane</creator><creator>Harris, Pernille</creator><creator>Christensen, Hans E.M.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>200802</creationdate><title>A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization</title><author>Nielsen, Michael S. ; Petersen, Charlotte R. ; Munch, Astrid ; Vendelboe, Trine V. ; Boesen, Jane ; Harris, Pernille ; Christensen, Hans E.M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c382t-6d383ba8774ae0b1b000e4cd16964aa199a576abb04aa3fe1df8fef60234a9263</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Biotechnology - methods</topic><topic>Catalytic Domain</topic><topic>Chickens - metabolism</topic><topic>Chromatography, Gel</topic><topic>Chromatography, Ion Exchange</topic><topic>Crystallization</topic><topic>Crystallography, X-Ray</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Escherichia coli</topic><topic>Expression</topic><topic>Gallus gallus</topic><topic>Humans</topic><topic>Kinetic</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>Purification</topic><topic>Sequence Alignment</topic><topic>Tryptophan hydroxylase</topic><topic>Tryptophan Hydroxylase - chemistry</topic><topic>Tryptophan Hydroxylase - isolation & purification</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nielsen, Michael S.</creatorcontrib><creatorcontrib>Petersen, Charlotte R.</creatorcontrib><creatorcontrib>Munch, Astrid</creatorcontrib><creatorcontrib>Vendelboe, Trine V.</creatorcontrib><creatorcontrib>Boesen, Jane</creatorcontrib><creatorcontrib>Harris, Pernille</creatorcontrib><creatorcontrib>Christensen, Hans E.M.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nielsen, Michael S.</au><au>Petersen, Charlotte R.</au><au>Munch, Astrid</au><au>Vendelboe, Trine V.</au><au>Boesen, Jane</au><au>Harris, Pernille</au><au>Christensen, Hans E.M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2008-02</date><risdate>2008</risdate><volume>57</volume><issue>2</issue><spage>116</spage><epage>126</epage><pages>116-126</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><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 11mg/L culture and a specific activity of 0.60μmol/min/mg. The Km values were determined to Km,tryptophan=7.7±0.7μM, Km,BH4=324±10μM and Km,O2=39±2μM. Substrate inhibition by tryptophan was observed at concentrations above 15μM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-l-biopterin and a data set to 3Å resolution has been collected.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>18055219</pmid><doi>10.1016/j.pep.2007.10.016</doi><tpages>11</tpages></addata></record> |
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| subjects | Amino Acid Sequence Animals Biotechnology - methods Catalytic Domain Chickens - metabolism Chromatography, Gel Chromatography, Ion Exchange Crystallization Crystallography, X-Ray Electrophoresis, Polyacrylamide Gel Escherichia coli Expression Gallus gallus Humans Kinetic Kinetics Molecular Sequence Data Purification Sequence Alignment Tryptophan hydroxylase Tryptophan Hydroxylase - chemistry Tryptophan Hydroxylase - isolation & purification |
| title | 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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