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

Yutetsu Kuruma, Takuya Ueda

研究成果: Article査読

53 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)1328-1344
ページ数17
ジャーナルNature protocols
10
9
DOI
出版ステータスPublished - 2015 10 1
外部発表はい

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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