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.
KeywordsE. coli cell-free system Heme A Paracoccus denitrificans
Unable to display preview. Download preview PDF.
- Isaacs NS (1995) In: Longman AW (ed) Physical organic chemistry, 2nd ed. Harlow Essex, pp 235–286Google Scholar
- Raitio M, Jalli T, Saraste M (1987) Embo J 6:2825–2833Google Scholar
- Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Coldspring Harbor Laboratory, New YorkGoogle Scholar
- Unger BP, Gunsalus IC, Sligar SG (1986) J Biol Chem 261:1158–1163Google Scholar
- Xie K, Dalbey RE (2008) Nat Rev Microbiol 6:234–244Google Scholar