Abstract
Overexpression of membrane-bound proteins to obtain milligram quantities for biochemical and biophysical studies is usually a difficult task, especially for those proteins that span the membrane several times (3). An additional difficulty arises when these proteins need to incorporate organic or inorganic cofactors. In order to obtain a functional protein, this requires a host that is able to produce this cofactor or the cofactor has to be added to the growth medium and subsequently taken up by the host cells. Alternatively, the protein can be expressed as inclusion bodies and then refolded in the presence of the cofactor(s), a strategy successfully applied for bacteriorhodopsin and the light-harvesting complex of higher plants (3).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Grisshammer, R., Duckworth, R. and Henderson R. (1993) Expression of a rat neurotensin receptor in Escherichia coli. Biochem. J. 295 (Pt 2), 571–576.
Grisshammer, R., Little, J. and Aharony, D. (1994) Expression of rat NK-2 (neurokinin A) receptor in E. coli. Recept. Chan. 2, 295–302.
Grisshammer, R. and Tate, C. G (1995). Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys. 28, 315–422.
Hauska, G, and Büttner, M. (1997) The cytochrome b 6 f/bc 1-complexes, p. 389–417. In P. Gr ber and M. G (ed.), Bioenergetics. Birkh user Verlag, Basel/Switzerland.
Henninger, T, Anemüller, S. Fitz-Gibbon, S. Miller, J. H. Schäfer, G and Schmidt C. L. (1999) A novel Rieske iron-sulfur protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum: sequencing of the gene, expression in E. coli and characterization of the protein. J. Bioenerg. Biomembr. 31:119–128.
Holton, B., Wu, X. Tsapin, A.I. Kramer, D.M. Malkin, R. and Kallas T (1996) Reconstitution of the 2Fe−2S center and g=1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein. Biochemistry 35:15,485–15,493.
Liebl, U., Sled, V. Brasseur, G. Ohnishi, T. and Daldal F. (1997) Conserved nonliganding residues of the Rhodobacter capsulatus Rieske iron-sulfur protein of the bcl complex are essential for protein structure, properties of the [2Fe-2S] cluster, and communication with the quinone pool. Biochemistry 36:11,675–11,684.
Pils, D., Gregor, W. and Schmetterer G (1997) Evidence for in vivo activity of three distinct respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC6803. FEMS Microbiology Letters 152:83–88.
Rieske, J. S., Hansen, R. E. and Zaugg W. S. (1964) Studies on the electron transfer system. J. Biol. Chem. 239:3017–3030.
Rippka, R., Deruelles, J. Waterbury, J. B. Herdman, M. and Stanier R. Y. 1979. Generic assinments, strainshistories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol 111:1–61.
Sambrook, J., Fritsch, E.F and Maniatis T. 1989. Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Habour Press USA.
Schmidt, C.L., Hatzfeld, O.M. Petersen, A. Link, T. A. and Schäfer G. (1997) Expression of the Sulfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted [2FE−2S] cluster in Escherichia coli. Biochem. Biophys. Res. Commun. 234:283–287.
Schneider, D., Jaschkowitz, K. Seidler, A. and Rögner M. (2000) Overexpression and reconstitution of a Rieske iron-sulfur protein from the cyanobacterium Synechocystis PCC 6803. Indian J. Biochem. Biophys. 37:441–446.
Schneider, D., Skrzypczak, S. Anemueller, S. Schmidt, C. L. Seidler, A. and Rögner M. (2002) Heterogeneous Rieske proteins in the cytochrome b 6 f complex of Synechocystis PCC 6803? J. Biol. Chem. 277:10,949–10,959.
Schöpfer, R. (1993) The pRSET family of T7 promoter expression vectors for Escherichia coli. Gene 124:83–85.
Spehr, V., Schlitt, A. Scheide, D. Guenebaut, V. and Friedrich T. (1999) Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I). Biochemistry 38:16,261–16,267.
Studier, F W. (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219:37–44.
Tucker, J., and Grisshammer R. (1996). Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem. J. 317 (Pt 3):891–899.
Weiner, J. H., Lemire, B.D. Elmes, M.L. Bradley, R. D. and Scraba D.G (1984) Overproduction of fumarate reductase in Escherichia coli induces a novel intracellular lipid-protein organelle. J. Bacteriol. 158:590–596.
Zhang, Z., Huang, L. Shulmeister, V.M. Chi, Y.I. Kim, K.K. Hung, L.W. et al. (1998) Electron transfer by domain movement in cytochrome bc 1. Nature 392:677–684.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Schneider, D., Schmidt, C.L., Seidler, A. (2003). Expression of Membrane-Bound Iron-Sulfur Proteins. In: Selinsky, B.S. (eds) Membrane Protein Protocols. Methods in Molecular Biology, vol 228. Humana Press. https://doi.org/10.1385/1-59259-400-X:37
Download citation
DOI: https://doi.org/10.1385/1-59259-400-X:37
Publisher Name: Humana Press
Print ISBN: 978-1-58829-124-0
Online ISBN: 978-1-59259-400-9
eBook Packages: Springer Protocols