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

High yield of Methylophilus methylotrophus cytochrome c'' by coexpression with cytochrome c maturation gene cluster from Escherichia coli
High yield of Methylophilus methylotrophus cytochrome c'' by coexpression with cytochrome c maturation gene cluster from Escherichia coli
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
perpendicular orientation, correct expression, cross-relaxation, cloning, macromolecules, heme, nmr, ferricytochrome-c&#39, &#39, histidine, electron
1046-5928
444-450
Price, N. J.
9dabf747-c250-4959-9b44-345ab84c999d
Brennan, L.
a178add3-8547-47c8-a03c-17cf2db68a9d
Faria, T. Q.
51051e15-7a28-45e6-97de-050e3132e812
Vijgenboom, E.
d3e2d474-a0ce-4f16-8732-3fb493f49b8a
Canters, G. W.
049469ab-ec5a-4041-9734-4d56e23078bb
Turner, D. L.
158980fd-3487-480b-baba-15102dfb64b4
Santos, H.
d4628582-9c48-4f0a-bdbe-93fc2a2bc0dd
Price, N. J.
9dabf747-c250-4959-9b44-345ab84c999d
Brennan, L.
a178add3-8547-47c8-a03c-17cf2db68a9d
Faria, T. Q.
51051e15-7a28-45e6-97de-050e3132e812
Vijgenboom, E.
d3e2d474-a0ce-4f16-8732-3fb493f49b8a
Canters, G. W.
049469ab-ec5a-4041-9734-4d56e23078bb
Turner, D. L.
158980fd-3487-480b-baba-15102dfb64b4
Santos, H.
d4628582-9c48-4f0a-bdbe-93fc2a2bc0dd

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).

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: 15 Jul 2019 19:28

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