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A03 KURUMA, Yutetsu |Proposed Research Projects (2014-2015)

Paper | Original Paper

2015

*Yutetsu Kuruma and Takuya Ueda,
The PURE system for the cell-free synthesis of membrane proteins,
Nature Protocols 10, 1328-1344 (2015).

[Summary] 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 SecYEG translocon or ATP synthase. Here, we describe a protocol for the cell-free synthesis of membrane proteins using the PURE system, and subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane, and complex formation in the membrane. Additionally, 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 one day. This protocol can also avoid the risks in membrane protein purification and in reconstruction to lipid membrane, compared to alternative methods using living cells.

2014

Hideaki Matsubayashi, Yutetsu Kuruma, and Takuya Ueda,
Cell-Free Synthesis of SecYEG Translocon as the Fundamental Protein Transport Machinery,
Origins of Life and Evolution of Biospheres 44, 331-334 (2014).

Hideaki Matsubayashi, Yutetsu Kuruma, and Takuya Ueda,
In vitro synthesis of the E. coli Sec Translocon from DNA,
Angewandte Chemie International Edition 53, 7535-7538 (2014).

*Yoshihiro Shimizu, Yutetsu Kuruma, Takashi Kanamori, Takuya Ueda ,
The PURE system for protein production,
Methods in Molecular Biology 1118, 275-284 (2014).