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Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris
We developed a method for efficient chromosome tagging in Pichia pastoris , using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followe...
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| Published in: | Journal of structural and functional genomics 2014-12, Vol.15 (4), p.191-199 |
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| Main Authors: | , , , , , , , , |
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| Language: | English |
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| container_end_page | 199 |
| container_issue | 4 |
| container_start_page | 191 |
| container_title | Journal of structural and functional genomics |
| container_volume | 15 |
| creator | Higo, Toshiaki Suka, Noriyuki Ehara, Haruhiko Wakamori, Masatoshi Sato, Shin Maeda, Hideaki Sekine, Shun-ichi Umehara, Takashi Yokoyama, Shigeyuki |
| description | We developed a method for efficient chromosome tagging in
Pichia pastoris
, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from
P. pastoris
. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes. |
| doi_str_mv | 10.1007/s10969-014-9190-1 |
| format | article |
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Pichia pastoris
, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from
P. pastoris
. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.</description><identifier>ISSN: 1345-711X</identifier><identifier>EISSN: 1570-0267</identifier><identifier>DOI: 10.1007/s10969-014-9190-1</identifier><identifier>PMID: 25398586</identifier><language>eng</language><publisher>Dordrecht: Springer Netherlands</publisher><subject>Biochemistry ; Bioinformatics ; Biology ; Biomedical and Life Sciences ; Chromatography, Affinity - methods ; Crystallization ; Forestry Management ; Fungal Proteins - biosynthesis ; Fungal Proteins - genetics ; Fungal Proteins - isolation & purification ; Histidine - biosynthesis ; Histidine - genetics ; Histidine - isolation & purification ; Human Genetics ; Life Sciences ; Methods ; Microbial Genetics and Genomics ; Multiprotein Complexes - biosynthesis ; Multiprotein Complexes - genetics ; Multiprotein Complexes - isolation & purification ; Pichia - chemistry ; Pichia - genetics ; Pichia - metabolism ; Pichia pastoris ; Plant Genetics and Genomics ; Proteins ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; RNA polymerase ; Yeast</subject><ispartof>Journal of structural and functional genomics, 2014-12, Vol.15 (4), p.191-199</ispartof><rights>The Author(s) 2014</rights><rights>Springer Science+Business Media Dordrecht 2014</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-p2151-d9fe3e95174c1588bc082832350daec396bba6d70d605299babd91c9c50bc2ef3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.proquest.com/docview/1626119284?pq-origsite=primo$$EHTML$$P50$$Gproquest$$H</linktohtml><link.rule.ids>230,314,780,784,885,11688,27924,27925,36060,36061,44363</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25398586$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Higo, Toshiaki</creatorcontrib><creatorcontrib>Suka, Noriyuki</creatorcontrib><creatorcontrib>Ehara, Haruhiko</creatorcontrib><creatorcontrib>Wakamori, Masatoshi</creatorcontrib><creatorcontrib>Sato, Shin</creatorcontrib><creatorcontrib>Maeda, Hideaki</creatorcontrib><creatorcontrib>Sekine, Shun-ichi</creatorcontrib><creatorcontrib>Umehara, Takashi</creatorcontrib><creatorcontrib>Yokoyama, Shigeyuki</creatorcontrib><title>Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris</title><title>Journal of structural and functional genomics</title><addtitle>J Struct Funct Genomics</addtitle><addtitle>J Struct Funct Genomics</addtitle><description>We developed a method for efficient chromosome tagging in
Pichia pastoris
, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from
P. pastoris
. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.</description><subject>Biochemistry</subject><subject>Bioinformatics</subject><subject>Biology</subject><subject>Biomedical and Life Sciences</subject><subject>Chromatography, Affinity - methods</subject><subject>Crystallization</subject><subject>Forestry Management</subject><subject>Fungal Proteins - biosynthesis</subject><subject>Fungal Proteins - genetics</subject><subject>Fungal Proteins - isolation & purification</subject><subject>Histidine - biosynthesis</subject><subject>Histidine - genetics</subject><subject>Histidine - isolation & purification</subject><subject>Human Genetics</subject><subject>Life Sciences</subject><subject>Methods</subject><subject>Microbial Genetics and Genomics</subject><subject>Multiprotein Complexes - biosynthesis</subject><subject>Multiprotein Complexes - genetics</subject><subject>Multiprotein Complexes - isolation & purification</subject><subject>Pichia - chemistry</subject><subject>Pichia - genetics</subject><subject>Pichia - metabolism</subject><subject>Pichia pastoris</subject><subject>Plant Genetics and Genomics</subject><subject>Proteins</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>RNA polymerase</subject><subject>Yeast</subject><issn>1345-711X</issn><issn>1570-0267</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>M0C</sourceid><recordid>eNqFks9qFTEUxgdRbK0-gBsJuHETzZ9JMtkIpbVVuKALBXchk5y5kzKTjMnc0q58DB_IFzOXW6W6cZWE8-PLd875muY5Ja8pIepNoURLjQltsaaaYPqgOaZCEUyYVA_rnbcCK0q_HjVPSrkihErW6cfNERNcd6KTx833c7iGKS0zxBWlAVk0wo0dQ1mDDxEw__kDXWxOL_Fqo4cZ2WEIMay3aNnlMARn15AimmEdk0dDygiiT1uIaVfQktMKISKX5mWCGyioPj4FNwaLFlvWlEN52jwa7FTg2d150ny5ePf57D3efLz8cHa6wQujgmKvB-CgBVWto6Lrekc61nHGBfEWHNey7630inhJBNO6t73X1GknSO8YDPykeXvQXXb9DN7VdrOdzJLDbPOtSTaYvysxjGabrk3LuNK0rQKv7gRy-raDspo5FAfTZCPUZg2V1QuXvPL_R5kiSmkmK_ryH_Qq7XKsk9hTklLNuv3fL-6b_-P69xorwA5AqaW4hXxPhph9VswhK6ZmxeyzYij_BRrysiU</recordid><startdate>201412</startdate><enddate>201412</enddate><creator>Higo, Toshiaki</creator><creator>Suka, Noriyuki</creator><creator>Ehara, Haruhiko</creator><creator>Wakamori, Masatoshi</creator><creator>Sato, Shin</creator><creator>Maeda, Hideaki</creator><creator>Sekine, Shun-ichi</creator><creator>Umehara, Takashi</creator><creator>Yokoyama, Shigeyuki</creator><general>Springer Netherlands</general><general>Springer Nature B.V</general><scope>C6C</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>3V.</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PYYUZ</scope><scope>Q9U</scope><scope>RC3</scope><scope>7X8</scope><scope>7QO</scope><scope>5PM</scope></search><sort><creationdate>201412</creationdate><title>Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris</title><author>Higo, Toshiaki ; Suka, Noriyuki ; Ehara, Haruhiko ; Wakamori, Masatoshi ; Sato, Shin ; Maeda, Hideaki ; Sekine, Shun-ichi ; Umehara, Takashi ; Yokoyama, Shigeyuki</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p2151-d9fe3e95174c1588bc082832350daec396bba6d70d605299babd91c9c50bc2ef3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Biochemistry</topic><topic>Bioinformatics</topic><topic>Biology</topic><topic>Biomedical and Life Sciences</topic><topic>Chromatography, Affinity - 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Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of structural and functional genomics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Higo, Toshiaki</au><au>Suka, Noriyuki</au><au>Ehara, Haruhiko</au><au>Wakamori, Masatoshi</au><au>Sato, Shin</au><au>Maeda, Hideaki</au><au>Sekine, Shun-ichi</au><au>Umehara, Takashi</au><au>Yokoyama, Shigeyuki</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris</atitle><jtitle>Journal of structural and functional genomics</jtitle><stitle>J Struct Funct Genomics</stitle><addtitle>J Struct Funct Genomics</addtitle><date>2014-12</date><risdate>2014</risdate><volume>15</volume><issue>4</issue><spage>191</spage><epage>199</epage><pages>191-199</pages><issn>1345-711X</issn><eissn>1570-0267</eissn><abstract>We developed a method for efficient chromosome tagging in
Pichia pastoris
, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from
P. pastoris
. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.</abstract><cop>Dordrecht</cop><pub>Springer Netherlands</pub><pmid>25398586</pmid><doi>10.1007/s10969-014-9190-1</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Journal of structural and functional genomics, 2014-12, Vol.15 (4), p.191-199 |
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| language | eng |
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| source | ABI/INFORM Global |
| subjects | Biochemistry Bioinformatics Biology Biomedical and Life Sciences Chromatography, Affinity - methods Crystallization Forestry Management Fungal Proteins - biosynthesis Fungal Proteins - genetics Fungal Proteins - isolation & purification Histidine - biosynthesis Histidine - genetics Histidine - isolation & purification Human Genetics Life Sciences Methods Microbial Genetics and Genomics Multiprotein Complexes - biosynthesis Multiprotein Complexes - genetics Multiprotein Complexes - isolation & purification Pichia - chemistry Pichia - genetics Pichia - metabolism Pichia pastoris Plant Genetics and Genomics Proteins Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification RNA polymerase Yeast |
| title | Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris |
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