Successful recombinant production of Allochromatium vinosum cytochrome c′ requires coexpression of cmm genes in heme-rich Escherichia coli JCB712

doi:10.1016/j.bbrc.2004.12.062

Biochemical and Biophysical Research Communications

Volume 327, Issue 3, 18 February 2005, Pages 668-674

https://doi.org/10.1016/j.bbrc.2004.12.062 Get rights and content

Abstract

Cytochrome c′ from the purple photosynthetic bacterium Allochromatium vinosum (CCP) displays a unique, reversible dimer-to-monomer transition upon binding of NO, CO, and CN⁻. This small, four helix bundle protein represents an attractive model for the study of other heme protein biosensors, provided a recombinant expression system is available. Here we report the development of an efficient expression system for CCP that makes use of a maltose binding protein fusion strategy to enhance periplasmic expression and allow easy purification by affinity chromatography. Coexpression of cytochrome c maturase genes and the use of a heme-rich Escherichia coli strain were found to be necessary to obtain reasonable yields of cytochrome c′. Characterization using circular dichroism, UV–vis spectroscopy, and size-exclusion chromatography confirms the native-like properties of the recombinant protein, including its ligand-induced monomerization.

Section snippets

Materials and methods

General methods. DNA sequencing was performed by BaseClear (Leiden, The Netherlands). The concentrations of ampicillin and chloramphenicol used for selection were 100 and 10 μg/mL, respectively. Protein concentrations for MBP-rCCP and rCCP were determined by measuring the absorbance at 399 nm using a molar extinction coefficient of 87,000 M⁻¹ cm⁻¹ per heme [31]. SDS–PAGE was performed on 12% acrylamide/bisacrylamide gels, using Precision Plus Unstained Protein Standard (Bio-Rad, Hercules, CA, USA)

Cloning of expression vector and optimization of protein expression

The low yield that was reported previously for CCP expression in E. coli[24], [25] was the result of low expression levels and a complicated purification procedure that included acid precipitation, two isoelectric focusing steps, and hydrophobic interaction chromatography. In order to minimize the loss during purification we decided to express CCP as a fusion protein with maltose binding protein (MBP), as this allows easy purification on amylose resins. The ccp gene was cloned into the pMAL-p2X

Conclusion

An efficient system was developed for the recombinant expression of cytochrome c′ from A. vinosum. Expression as a fusion protein with maltose binding protein resulted in efficient protein translation, transport to the periplasm, and straightforward purification. Heme incorporation was shown to be the limiting factor for high-level expression of recombinant CCP in E. coli. The highest expression levels were obtained by coexpression of cytochrome c maturation genes in a heme-rich E. coli strain,

Acknowledgments

We thank Prof. Dr. R.J. Kassner (University of Illinois, Chicago, USA) for providing the pBluescript II SK+ plasmid with the gene for CCP, Prof. Dr. L. Thöny-Meyer (ETH, Zürich, Switzerland) for providing plasmid pEC86, Prof. Dr. J. Cole (University of Birmingham, Birmingham, UK) for providing E. coli strain JCB712, J.L.J. van Dongen for support with mass spectometry, and Prof. Dr. E.W. Meijer for general support.

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^☆: Abbreviations: CCP, cytochrome c′ from Allochromatium vinosum; CD, circular dichroism; EDTA, ethylenediaminetetraacetic acid; ESI-MS, electrospray ionization mass spectrometry; IPTG, isopropyl-β-d-thiogalactopyranoside; MBP, maltose binding protein; rCCP, recombinant CCP.

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Successful recombinant production of Allochromatium vinosum cytochrome c′ requires coexpression of cmm genes in heme-rich Escherichia coli JCB712☆

Abstract

Section snippets

Materials and methods

Cloning of expression vector and optimization of protein expression

Conclusion

Acknowledgments

Adv. Protein Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Mol. Biol.

Pharmacol. Ther.

J. Mol. Biol.

Curr. Opin. Struct. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Electrochem. Commun.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

FEMS Microbiol. Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

Kinetics of electron transfer between cytochromes c′ and the semiquinones of free flavin and clostridial flavodoxin

Biochemistry