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), 444-450. (doi:10.1006/prep.2000.1318).

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Original Publication URL: http://dx.doi.org/10.1006/prep.2000.1318

Description/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

Item Type: Article
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Keywords: perpendicular orientation, correct expression, cross-relaxation, cloning, macromolecules, heme, nmr, ferricytochrome-c&#39, &#39, , histidine, electron
Subjects: Q Science
Q Science > QD Chemistry
Divisions: University Structure - Pre August 2011 > School of Chemistry
ePrint ID: 19012
Date Deposited: 18 Jan 2006
Last Modified: 27 Mar 2014 18:08
URI: http://eprints.soton.ac.uk/id/eprint/19012

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