High-efficient production and biophysical characterisation of nicastrin and its interaction with APPC100
Nicastrin, the largest member among the four components of the γ-secretase complex, has been identified to be the substrate recognizer for the proteolytic activity of the complex. Here we report that full-length human nicastrin (hNCT) can be obtained by heterologous expression in E. coli . Milligram...
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| Veröffentlicht in: | Scientific reports 2017-03, Vol.7 (1), p.44297-44297, Article 44297 |
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| creator | Yu, Kun Yang, Ge Labahn, Jörg |
| description | Nicastrin, the largest member among the four components of the γ-secretase complex, has been identified to be the substrate recognizer for the proteolytic activity of the complex. Here we report that full-length human nicastrin (hNCT) can be obtained by heterologous expression in
E. coli
. Milligram quantities of the target protein are purified in a two-step purification protocol using affinity chromatography followed by SEC. The FOS-choline 14 purified tetrameric hNCT exhibits a proper folding with 31% α-helix and 23% β-sheet content. Thermal stability studies reveal stable secondary and tertiary structure of the detergent purified hNCT. A physical interaction between nicastrin and the γ-secretase substrate APPC100 confirmed the functionality of hNCT as a substrate recognizer. |
| doi_str_mv | 10.1038/srep44297 |
| format | Article |
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E. coli
. Milligram quantities of the target protein are purified in a two-step purification protocol using affinity chromatography followed by SEC. The FOS-choline 14 purified tetrameric hNCT exhibits a proper folding with 31% α-helix and 23% β-sheet content. Thermal stability studies reveal stable secondary and tertiary structure of the detergent purified hNCT. A physical interaction between nicastrin and the γ-secretase substrate APPC100 confirmed the functionality of hNCT as a substrate recognizer.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/srep44297</identifier><identifier>PMID: 28276527</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/1647/527/873 ; 631/45/612/1237 ; 82/58 ; 82/80 ; 82/83 ; Affinity chromatography ; Alzheimer's disease ; Amino acids ; Choline ; Chromatography ; E coli ; Humanities and Social Sciences ; Molecular weight ; multidisciplinary ; Nicastrin ; Peptides ; Protein purification ; Protein structure ; Proteins ; Proteolysis ; Science ; Secretase ; Spectrum analysis ; Tertiary structure ; Thermal stability</subject><ispartof>Scientific reports, 2017-03, Vol.7 (1), p.44297-44297, Article 44297</ispartof><rights>The Author(s) 2017</rights><rights>Copyright Nature Publishing Group Mar 2017</rights><rights>Copyright © 2017, The Author(s) 2017 The Author(s)</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c438t-1f1d0adb61b9a86a541cd146d5265ac267ad4ea5b0fb9e917a05decb459ff74a3</citedby><cites>FETCH-LOGICAL-c438t-1f1d0adb61b9a86a541cd146d5265ac267ad4ea5b0fb9e917a05decb459ff74a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343570/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343570/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,728,781,785,865,886,27929,27930,41125,42194,51581,53796,53798</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28276527$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yu, Kun</creatorcontrib><creatorcontrib>Yang, Ge</creatorcontrib><creatorcontrib>Labahn, Jörg</creatorcontrib><title>High-efficient production and biophysical characterisation of nicastrin and its interaction with APPC100</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><addtitle>Sci Rep</addtitle><description>Nicastrin, the largest member among the four components of the γ-secretase complex, has been identified to be the substrate recognizer for the proteolytic activity of the complex. Here we report that full-length human nicastrin (hNCT) can be obtained by heterologous expression in
E. coli
. Milligram quantities of the target protein are purified in a two-step purification protocol using affinity chromatography followed by SEC. The FOS-choline 14 purified tetrameric hNCT exhibits a proper folding with 31% α-helix and 23% β-sheet content. Thermal stability studies reveal stable secondary and tertiary structure of the detergent purified hNCT. A physical interaction between nicastrin and the γ-secretase substrate APPC100 confirmed the functionality of hNCT as a substrate recognizer.</description><subject>631/1647/527/873</subject><subject>631/45/612/1237</subject><subject>82/58</subject><subject>82/80</subject><subject>82/83</subject><subject>Affinity chromatography</subject><subject>Alzheimer's disease</subject><subject>Amino acids</subject><subject>Choline</subject><subject>Chromatography</subject><subject>E coli</subject><subject>Humanities and Social Sciences</subject><subject>Molecular weight</subject><subject>multidisciplinary</subject><subject>Nicastrin</subject><subject>Peptides</subject><subject>Protein purification</subject><subject>Protein structure</subject><subject>Proteins</subject><subject>Proteolysis</subject><subject>Science</subject><subject>Secretase</subject><subject>Spectrum analysis</subject><subject>Tertiary structure</subject><subject>Thermal stability</subject><issn>2045-2322</issn><issn>2045-2322</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNplkU1rFTEUhgdRbGm78A_IgBsVpuZ7JhuhXKoVCnah63AmH3dS5iZjMlPpv2_q1MvVZpPA-_DkHN6qeoPROUa0-5STnRgjsn1RHRPEeEMoIS8P3kfVWc63qBxOJMPydXVEOtIKTtrjarjy26GxznntbZjrKUWz6NnHUEMwde_jNNxnr2Gs9QAJ9GyTz_AHiK4OJclz8ivt51z7UAhYDb_9PNQXNzcbjNBp9crBmO3Z031S_fxy-WNz1Vx___ptc3HdaEa7ucEOGwSmF7iX0AngDGuDmTCcCA6aiBYMs8B75HppJW4BcWN1z7h0rmVAT6rPq3da-p01uiyVYFRT8jtI9yqCV_8mwQ9qG-8Up4zyFhXB-ydBir8Wm2e181nbcYRg45IV7lrBpEBSFPTdf-htXFIo6yksEaWCSokL9WGldIq5tOX2w2CkHitU-woL-_Zw-j35t7ACfFyBXKKwtengy2e2B9sUp3Y</recordid><startdate>20170309</startdate><enddate>20170309</enddate><creator>Yu, Kun</creator><creator>Yang, Ge</creator><creator>Labahn, Jörg</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>C6C</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20170309</creationdate><title>High-efficient production and biophysical characterisation of nicastrin and its interaction with APPC100</title><author>Yu, Kun ; 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Here we report that full-length human nicastrin (hNCT) can be obtained by heterologous expression in
E. coli
. Milligram quantities of the target protein are purified in a two-step purification protocol using affinity chromatography followed by SEC. The FOS-choline 14 purified tetrameric hNCT exhibits a proper folding with 31% α-helix and 23% β-sheet content. Thermal stability studies reveal stable secondary and tertiary structure of the detergent purified hNCT. A physical interaction between nicastrin and the γ-secretase substrate APPC100 confirmed the functionality of hNCT as a substrate recognizer.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>28276527</pmid><doi>10.1038/srep44297</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 631/1647/527/873 631/45/612/1237 82/58 82/80 82/83 Affinity chromatography Alzheimer's disease Amino acids Choline Chromatography E coli Humanities and Social Sciences Molecular weight multidisciplinary Nicastrin Peptides Protein purification Protein structure Proteins Proteolysis Science Secretase Spectrum analysis Tertiary structure Thermal stability |
| title | High-efficient production and biophysical characterisation of nicastrin and its interaction with APPC100 |
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