論文

査読有り
2015年9月

The PURE system for the cell-free synthesis of membrane proteins

NATURE PROTOCOLS
  • Yutetsu Kuruma
  • ,
  • Takuya Ueda

10
9
開始ページ
1328
終了ページ
1344
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1038/nprot.2015.082
出版者・発行元
NATURE PUBLISHING GROUP

Cell-free gene expression systems are biotechnological tools for the in vitro production of proteins of interest. The addition of membrane vesicles (liposomes) enables the production of membrane proteins, including those in large-molecular-weight complexes, such as the SecYEG translocon or ATP synthase. Here we describe a protocol for the cell-free synthesis of membrane proteins using the protein synthesis using recombinant elements (PURE) system, and for subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane and complex formation in the membrane. In addition, measurements of ATP synthase activity are used as an example to demonstrate the functional nature of the cell-free synthesized proteins. This protocol allows the rapid production and the detailed analysis of membrane proteins, and the complete process from template DNA preparation to activity measurement can be accomplished within 1 d. In contrast to alternative methods using living cells, this protocol can also help to prevent the difficulties in membrane protein purification and the risks of protein aggregation during reconstitution into lipid membranes.

Web of Science ® 被引用回数 : 53

リンク情報
DOI
https://doi.org/10.1038/nprot.2015.082
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/26270393
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000360493200006&DestApp=WOS_CPL

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