Cell-free synthesis of cytochrome c oxidase, a multicomponent membrane protein.
Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.
Katayama
Yukie
Y
Picobiology Institute, Graduate School of Life Science, Center of Advanced Science and Technology, University of Hyogo, 3-1-1 Koto, Kamighori, Akoh, Hyogo, 678-1205, Japan.
Shimokata
Kunitoshi
K
Suematsu
Makoto
M
Ogura
Takashi
T
Tsukihara
Tomitake
T
Yoshikawa
Shinya
S
Shimada
Hideo
H
D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods*; D045304:Cytochromes c / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis* / Q000138:chemical synthesis; D004926:Escherichia coli / Q000235:genetics; D008722:Methods; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000235:genetics
D045304:Cytochromes c; D003576:Electron Transport Complex IV