Abstract
Recently cell-free translation systems became a laboratory research tool to obtain an objective protein. However, a general method for in vitro protein synthesis and multi-subunit complex formation on lipid membrane has not been established. Here, we describe the procedure for the production of subcomplexes of FoF1-ATP synthase using a reconstructed cell-free translation system. As for the membrane part Fo (a 1 b 2 c (10–15)), cosynthesis of c-subunit and UncI proteins, which are integral membrane proteins, and further c 11-ring formation in lipid bilayer are performed by use of a liposomes-containing cell-free system. Moreover, as for the cytoplasm part F1 (α3β3γδε), subcomplex formations are successfully achieved by mixing the translation products.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shimizu, Y., Kuruma, Y., Ying, B. W., Umekage, S., and Ueda T. (2006) Cell-free translation systems for protein engineering. FEBS J. 273, 4133–4140.
Ozaki, Y., Suzuki, T., Kuruma, Y., Ueda, T., and Yoshida, M. (2008) UncI protein can mediate ring-assembly of c-subunits of FoF1-ATP synthase in vitro. Biochem. Biophys. Res. Commun. 367, 663–666.
Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. (2001) Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755.
Suzuki, T., Ozaki, Y., Sone, N., Feniouk, B. A., and Yoshida, M. (2007) The product of uncI gene in F1Fo-ATP synthase operon plays a chaperone-like role to assist c-ring assembly. Proc. Natl. Acad. Sci. U.S.A. 104, 20776–20781.
Suzuki, T., Ueno, H., Mitome, N., Suzuki, J., Yoshida, M. (2002) Fo of ATP synthase is a rotary proton channel: Obligatory coupling of proton translocation with rotation of c-subunit ring. J. Biol. Chem. 277, 13281–13285.
Acknowledgments
The authors thank Dr. Yoko Ozaki for the works on the c-ring formation and Mr. Kurumada for the works on in vitro production of F1 subunits. The authors also thank Prof. Masasuke Yoshida (Tokyo Institute of Technology, Japan) who kindly provided us the FoF1 samples.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kuruma, Y., Suzuki, T., Ueda, T. (2010). Production of Multi-Subunit Complexes on Liposome Through an E. coli Cell-Free Expression System. In: Endo, Y., Takai, K., Ueda, T. (eds) Cell-Free Protein Production. Methods in Molecular Biology, vol 607. Humana Press. https://doi.org/10.1007/978-1-60327-331-2_14
Download citation
DOI: https://doi.org/10.1007/978-1-60327-331-2_14
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-330-5
Online ISBN: 978-1-60327-331-2
eBook Packages: Springer Protocols