A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits

Tobias Gustavsson; Maria Trane; Vamsi K Moparthi; Egle Miklovyte; Lavanya Moparthi; Kamil Górecki; Thom Leiding; Sindra Peterson Arsköld; Cecilia Hägerhäll

doi:10.1002/pro.424

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits

Tobias Gustavsson, Maria Trane, Vamsi K Moparthi, Egle Miklovyte, Lavanya Moparthi, Kamil Górecki, Thom Leiding, Sindra Peterson Arsköld, Cecilia Hägerhäll

VAR2CSA team

12 Citationer (Scopus)

Abstract

Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c ₅₅₀. Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c ₅₅₀ domain in all the fusion proteins exhibited normal spectra and redox properties, with an E _m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins. Published by Wiley-Blackwell.

Originalsprog	Engelsk
Tidsskrift	Protein Science
Vol/bind	19
Udgave nummer	8
Sider (fra-til)	1445-60
Antal sider	16
ISSN	0961-8368
DOI	https://doi.org/10.1002/pro.424
Status	Udgivet - aug. 2010

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Citationsformater

Gustavsson, T., Trane, M., Moparthi, V. K., Miklovyte, E., Moparthi, L., Górecki, K., Leiding, T., Arsköld, S. P., & Hägerhäll, C. (2010). A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits. Protein Science, 19(8), 1445-60. https://doi.org/10.1002/pro.424

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits. / Gustavsson, Tobias; Trane, Maria; Moparthi, Vamsi K et al.

I: Protein Science, Bind 19, Nr. 8, 08.2010, s. 1445-60.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

Gustavsson, T, Trane, M, Moparthi, VK, Miklovyte, E, Moparthi, L, Górecki, K, Leiding, T, Arsköld, SP & Hägerhäll, C 2010, 'A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits', Protein Science, bind 19, nr. 8, s. 1445-60. https://doi.org/10.1002/pro.424

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title = "A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits",

abstract = "Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c 550. Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c 550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an E m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins. Published by Wiley-Blackwell.",

keywords = "Animals, Bacillus subtilis/genetics, Cytochromes c/chemistry, Electron Transport Complex I/metabolism, Escherichia coli/genetics, Escherichia coli Proteins/chemistry, Membrane Proteins/chemistry, Models, Molecular, Oxidation-Reduction, Protein Conformation, Protein Subunits/chemistry, Recombinant Fusion Proteins/chemistry",

author = "Tobias Gustavsson and Maria Trane and Moparthi, {Vamsi K} and Egle Miklovyte and Lavanya Moparthi and Kamil G{\'o}recki and Thom Leiding and Arsk{\"o}ld, {Sindra Peterson} and Cecilia H{\"a}gerh{\"a}ll",

year = "2010",

month = aug,

doi = "10.1002/pro.424",

language = "English",

volume = "19",

pages = "1445--60",

journal = "Protein Science",

issn = "0961-8368",

publisher = "Wiley-Blackwell",

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T1 - A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits

AU - Gustavsson, Tobias

AU - Trane, Maria

AU - Moparthi, Vamsi K

AU - Miklovyte, Egle

AU - Moparthi, Lavanya

AU - Górecki, Kamil

AU - Leiding, Thom

AU - Arsköld, Sindra Peterson

AU - Hägerhäll, Cecilia

PY - 2010/8

Y1 - 2010/8

N2 - Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c 550. Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c 550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an E m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins. Published by Wiley-Blackwell.

AB - Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c 550. Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c 550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an E m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins. Published by Wiley-Blackwell.

KW - Animals

KW - Bacillus subtilis/genetics

KW - Cytochromes c/chemistry

KW - Electron Transport Complex I/metabolism

KW - Escherichia coli/genetics

KW - Escherichia coli Proteins/chemistry

KW - Membrane Proteins/chemistry

KW - Models, Molecular

KW - Oxidation-Reduction

KW - Protein Conformation

KW - Protein Subunits/chemistry

KW - Recombinant Fusion Proteins/chemistry

U2 - 10.1002/pro.424

DO - 10.1002/pro.424

M3 - Journal article

C2 - 20509166

SN - 0961-8368

VL - 19

SP - 1445

EP - 1460

JO - Protein Science

JF - Protein Science

IS - 8

ER -