The PURE system for the cell-free synthesis of membrane proteins.
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.
1] Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan. [2] Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
Yutetsu
Y
Kuruma
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
Takuya
T
Ueda
D002474:Cell-Free System; D008565:Membrane Proteins / Q000096:biosynthesis*; D014176:Protein Biosynthesis
D008565:Membrane Proteins