2005年
Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli
Protein Expression and Purification
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- 巻
- 44
- 号
- 2
- 開始ページ
- 130
- 終了ページ
- 135
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.pep.2005.06.001
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 angstrom resolution with synchrotron radiation. (c) 2005 Elsevier Inc. All rights reserved.
- リンク情報
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- DOI
- https://doi.org/10.1016/j.pep.2005.06.001
- J-GLOBAL
- https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200902275505314256
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000233462500006&DestApp=WOS_CPL
- URL
- http://www.scopus.com/inward/record.url?eid=2-s2.0-27644583963&partnerID=MN8TOARS
- ID情報
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- DOI : 10.1016/j.pep.2005.06.001
- ISSN : 1046-5928
- J-Global ID : 200902275505314256
- ORCIDのPut Code : 58433882
- SCOPUS ID : 27644583963
- Web of Science ID : WOS:000233462500006