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High yield of Methylophilus methylotrophus cytochrome c'' by coexpression with cytochrome c maturation gene cluster from Escherichia coli

Price, N. J., Brennan, L., Faria, T. Q., Vijgenboom, E., Canters, G. W., Turner, D. L. and Santos, H. (2000) High yield of Methylophilus methylotrophus cytochrome c'' by coexpression with cytochrome c maturation gene cluster from Escherichia coli Protein Expression and Purification, 20, (3), pp. 444-450. (doi:10.1006/prep.2000.1318).

Record type: Article

Abstract

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c'' from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c'' isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and IH-NMR spectroscopy. The success in expressing the mature cytochrome c'' in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship

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More information

Published date: 1 December 2000
Keywords: perpendicular orientation, correct expression, cross-relaxation, cloning, macromolecules, heme, nmr, ferricytochrome-c&#39, &#39, histidine, electron

Identifiers

Local EPrints ID: 19012
URI: http://eprints.soton.ac.uk/id/eprint/19012
ISSN: 1046-5928
PURE UUID: acc41935-fb30-4282-8c18-72bcb3a1f6fc

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Date deposited: 18 Jan 2006
Last modified: 17 Jul 2017 16:34

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Contributors

Author: N. J. Price
Author: L. Brennan
Author: T. Q. Faria
Author: E. Vijgenboom
Author: G. W. Canters
Author: D. L. Turner
Author: H. Santos

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