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Heterologous Gene Expression in E.coli pp 123–150Cite as

Expression of Recombinant Cytochromes c in E. coli

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 705))

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.

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Notes

  1. 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. 2.

    pET-22b(+) has the T7 promoter (see Section 3.1 below).

  3. 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. 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.

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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.

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  1. New England Biolabs, Inc., Ipswich, MA, USA

    Yuri Y. Londer

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  1. Yuri Y. Londer
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Correspondence to Yuri Y. Londer .

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  1. , DNA Enzymes Division, New England Biolabs, Inc., County Road 240, Ipswich, 01938, Massachusetts, USA

    Thomas C. Evans, Jr.

  2. , Chemical Biology Division, New England Biolabs, Inc., County Road 240, Ipswich, 01938, Massachusetts, USA

    Ming-Qun Xu

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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

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  • DOI: https://doi.org/10.1007/978-1-61737-967-3_8

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  • Print ISBN: 978-1-61737-966-6

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