Abstract
Answering questions about proteins’ structures and functions in the new era of systems biology and genomics requires the development of new methods for heterologous production of numerous proteins from newly sequenced genomes. Cytochromes c – electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain – are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. However, significant advances in expression of recombinant cytochromes c have been made during the last decade. It has been shown that a single gene cluster, ccmA–H, is responsible for cytochrome c maturation in Escherichia coli under anaerobic conditions and that constitutive co-expression of this cluster under aerobic conditions is sufficient to provide heme incorporation in many different types of cytochromes c, regardless of their origin, as long as the nascent polypeptide is translocated to the periplasm. Using conditions that result in sub-maximal protein induction can dramatically increase the yield of mature protein. The intrinsic peroxidase activity of hemes can be used as a highly selective and sensitive detection method of mature cytochromes in samples resolved by gel electrophoresis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Notes
- 1.
By definition, cytochromes c are electron transfer proteins in which heme is covalently bound to the polypeptide. This definition excludes proteins that contain covalently bound heme(s) but have functions other than electron transfer, such as nitrite reductase or a recently described family of sensor proteins (5, 6). However, the E. coli expression system described here has been shown to provide covalent heme attachment in many different types of proteins, regardless of their function. For the purpose of brevity, the term “cytochrome c” will be used throughout this chapter but all approaches described here should be applicable to all proteins containing covalently bound hemes.
- 2.
pET-22b(+) has the T7 promoter (see Section 3.1 below).
- 3.
Abbreviations: CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; CMC, critical micelle concentration; IPTG, isopropyl-β-d-thiogalactoside; MBP, maltose-binding protein; TEV, tobacco etch virus; TM, transmembrane.
- 4.
It should be noted that theoretically predicted pI is less accurate for cytochromes than for other proteins as heme group contains iron that can be in oxidation state +2 or +3, depending on the redox potential of the given heme (which is determined by many factors, including the heme environment, its ligands, and the degree of its exposure to solvent) and two carboxylic groups, whose pI can vary significantly, depending on their environment and degree of exposure to solvent.
References
Mathews, F. S. (1985) The structure, function and evolution of cytochromes. Prog Biophys Mol Biol 45, 1–56.
Thöny-Meyer, L. (1997) Biogenesis of respiratory cytochromes in bacteria. Microbiol Mol Biol Rev 61, 337–376.
Pollock, W. B., Chemerika, P. J., Forrest, M. E., Beatty, J. T., Voordouw, G. (1989) Expression of the gene encoding cytochrome c 3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein. J Gen Microbiol 135, 2319–2328.
Grisshammer, R., Oeckl, C., Michel, H. (1991) Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis. Biochim Biophys Acta 1088, 183–190.
Londer, Y. Y., Dementieva, I. S., D’Ausilio, C. A., Pokkuluri, P. R., Schiffer, M. (2006) Characterization of a c-type heme-containing PAS sensor domain from Geobacter sulfurreducens representing a novel family of periplasmic sensors in Geobacteraceae and other bacteria. FEMS Microbiol Lett 258, 173–181.
Pokkuluri, P. R., Pessanha, M., Londer, Y. Y., Wood, S. J., Duke, N. E. C., Wilton, R., Catarino, T., Salgueiro, C. A., Schiffer, M. (2008) Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: implications for signal transduction. J Mol Biol 377, 1498–1517.
Thöny-Meyer, L., Fischer, F., Kunzler, P., Ritz, D., Hennecke, H. (1995) Escherichia coli genes required for cytochrome c maturation, J Bacteriol 177, 4321–4326.
Page, M. D., Sambongi, Y., Ferguson, S. J. (1998) Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria. Trends Biochem Sci 23, 103–108.
Thöny-Meyer, L. (2000) Haem-polypeptide interactions during cytochrome c maturation, Biochim Biophys Acta 1459, 316–324.
Kranz, R., Lill, R., Goldman, B., Bonnard, G., Merchant, S. (1998) Molecular mechanisms of cytochrome c biogenesis: three distinct systems. Mol Microbiol 29, 383–396.
Grove, J., Tanapongpipat, S., Thomas, G., Griffiths, L., Crooke, H., Cole, J. (1996) Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. Mol Microbiol 19, 467–481.
Arslan, E., Schulz, H., Zufferey, R., Kunzler, P., Thöny-Meyer, L. (1998) Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb 3 oxidase in Escherichia coli. Biochem Biophys Res Commun 251, 744–747.
Londer, Y. Y., Pokkuluri, P. R., Erickson, J., Orshonsky, V., Schiffer, M. (2005) Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c. Protein Expr Purif 39, 254–260.
Londer, Y. Y., Pokkuluri, P. R., Orshonsky, V., Orshonsky, L., Schiffer, M. (2006) Heterologous expression of dodecaheme “nanowire” cytochromes c from Geobacter sulfurreducens. Protein Expr Purif 47, 241–248.
Sanders, C., Lill, H. (2000) Expression of prokaryotic and eukaryotic cytochromes c in Escherichia coli. Biochim Biophys Acta 1459, 131–138.
Londer, Y. Y., Giuliani, S. E., Peppler, T., Collart, F. R. (2008) Addressing Shewanella oneidensis “cytochromome”: the first step towards high-throughput expression of cytochromes c. Protein Expr Purif 62, 128–137.
Bothmann, H., Pluckthun, A. (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat Biotechnol 16, 376–380.
Schäfer, U., Beck, K., and Müller, M. (1999) Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins. J Biol Chem 274, 24567–24574.
Mavrangelos, C., Thiel, M., Adamson, P. J., Millard, D. J., Nobbs, S., Zola, H., Nicholson, I. C. (2001) Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin. Protein Expr Purif 23, 289–295.
Bothmann, H., Pluckthun, A. (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines. J Biol Chem 275, 17100–17105.
Arie, J. P., Sassoon, N., Betton, J. M. (2001) Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli. Mol Microbiol 39, 199–210.
Gordon, E. H., Steensma, E., Ferguson, S. J. (2001) The cytochrome c domain of dimeric cytochrome cd 1 of Paracoccus pantotrophus can be produced at high levels as a monomeric holoprotein using an improved c-type cytochrome expression system in Escherichia coli. Biochem Biophys Res Commun 281, 788–794.
Meerman, H. J., Georgiou, G. (1994) Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins. Biotechnology (New York) 12, 1107–1110.
Londer, Y. Y., Pokkuluri, P. R., Tiede, D. M., Schiffer, M. (2002) Production and preliminary characterization of a recombinant triheme cytochrome c 7 from Geobacter sulfurreducens in Escherichia coli. Biochim Biophys Acta 1554, 202–211.
Brigé, A., Cole, J. A., Hagen, W. R., Guisez, Y., Van Beeumen, J. J. (2001) Overproduction, purification and novel redox properties of the dihaem cytochrome c NapB, from Haemophilus influenzae. Biochem J 356, 851–858.
Hoover, D. M., Lubkowski, J. (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30, e43.
Feissner R., Xiang, Y., Kranz, R. G. (2003) Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes. Anal Biochem 315, 90–94.
Fernandes, A. P., Couto, I., Morgado, L., Londer, Y. Y., Salgueiro, C. A. (2008) Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli. Protein Expr Purif 59, 182–188.
Siegele, D. A., Hu, J. C. (1997) Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proc Natl Acad Sci USA 94, 8168–8172.
Acknowledgments
The author would like to thank Dr. L. Thöny-Meyer (ETH, Zürich, Switzerland) for plasmid pEC86 carrying the ccm genes; Drs. P.R. Pokkuluri and M. Schiffer (Argonne National Laboratory) and C.A. Salgueiro (Universidade Nova de Lisboa, Lisbon, Portugal) for helpful discussions and encouragement; Dr. T. Khare (Argonne National Laboratory) for help in mastering heme staining procedures; and Drs. T. Evans and P. Weigele (New England Biolabs) for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Londer, Y.Y. (2011). Expression of Recombinant Cytochromes c in E. coli . In: Evans, Jr., T., Xu, MQ. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 705. Humana Press. https://doi.org/10.1007/978-1-61737-967-3_8
Download citation
DOI: https://doi.org/10.1007/978-1-61737-967-3_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61737-966-6
Online ISBN: 978-1-61737-967-3
eBook Packages: Springer Protocols