{"title":"在 Pichia pastoris 中开发内源蛋白复合物的六脒-3×FLAG-串联亲和纯化方法。","authors":"Toshiaki Higo, Noriyuki Suka, Haruhiko Ehara, Masatoshi Wakamori, Shin Sato, Hideaki Maeda, Shun-ichi Sekine, Takashi Umehara, Shigeyuki Yokoyama","doi":"10.1007/s10969-014-9190-1","DOIUrl":null,"url":null,"abstract":"<p><p>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. </p>","PeriodicalId":73957,"journal":{"name":"Journal of structural and functional genomics","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2014-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237914/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris.\",\"authors\":\"Toshiaki Higo, Noriyuki Suka, Haruhiko Ehara, Masatoshi Wakamori, Shin Sato, Hideaki Maeda, Shun-ichi Sekine, Takashi Umehara, Shigeyuki Yokoyama\",\"doi\":\"10.1007/s10969-014-9190-1\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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. </p>\",\"PeriodicalId\":73957,\"journal\":{\"name\":\"Journal of structural and functional genomics\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2014-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237914/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of structural and functional genomics\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1007/s10969-014-9190-1\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2014/11/15 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of structural and functional genomics","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/s10969-014-9190-1","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2014/11/15 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
摘要
我们利用一种有用的串联亲和纯化（TAP）标签，开发了一种在 Pichia pastoris 中高效标记染色体的方法。这种 TAP 标签在这里被命名为 THF 标签，它包含一个凝血酶蛋白酶裂解位点，用于去除 TAP 标签，以及一个六组苷序列（6× His）和三份 FLAG 序列（3× FLAG），用于亲和纯化。利用这种方法，我们成功地从 P. pastoris 中纯化出了 THF 标记的 RNA 聚合酶 I、II 和 III。我们还利用这种方法大规模纯化了标记的 RNA 聚合酶 II，并将其结晶化，进行了初步的 X 射线晶体学分析。本文介绍的方法将广泛用于快速、大规模制备结晶级真核生物多亚基蛋白复合物。
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) 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.
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