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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 LU ; Moparthi, Vamsi LU ; Miklovyte, Egle; Moparthi, Lavanya; Gorecki, Kamil LU ; Leiding, Thom LU ; Peterson Årsköld, Sindra LU and Hägerhäll, Cecilia LU (2010) In Protein Science 19(8). p.1445-1460
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 to other available fusion-protein tagging... (More)
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 to 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Bacillus subtilis, Escherichia coli, MrpD, MrpA, NuoN, NuoM, NuoL, NuoH, oxidoreductase, NADH:quinone, covalently bound heme, cytochrome c, fusion proteins
in
Protein Science
volume
19
issue
8
pages
1445 - 1460
publisher
The Protein Society
external identifiers
  • wos:000280481300001
  • pmid:20509166
  • scopus:77955105850
ISSN
1469-896X
DOI
10.1002/pro.424
language
English
LU publication?
yes
id
0965dde2-cbb9-4184-86ad-011c093417ff (old id 1609780)
date added to LUP
2010-06-10 16:54:09
date last changed
2018-05-29 12:12:30
@article{0965dde2-cbb9-4184-86ad-011c093417ff,
  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 to 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.},
  author       = {Gustavsson, Tobias and Trane, Maria and Moparthi, Vamsi and Miklovyte, Egle and Moparthi, Lavanya and Gorecki, Kamil and Leiding, Thom and Peterson Årsköld, Sindra and Hägerhäll, Cecilia},
  issn         = {1469-896X},
  keyword      = {Bacillus subtilis,Escherichia coli,MrpD,MrpA,NuoN,NuoM,NuoL,NuoH,oxidoreductase,NADH:quinone,covalently bound heme,cytochrome c,fusion proteins},
  language     = {eng},
  number       = {8},
  pages        = {1445--1460},
  publisher    = {The Protein Society},
  series       = {Protein Science},
  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},
  url          = {http://dx.doi.org/10.1002/pro.424},
  volume       = {19},
  year         = {2010},
}