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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
Author(s) -
Gustavsson Tobias,
Trane Maria,
Moparthi Vamsi K.,
Miklovyte Egle,
Moparthi Lavanya,
Górecki Kamil,
Leiding Thom,
Årsköld Sindra Peterson,
Hägerhäll Cecilia
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.424
Subject(s) - biochemistry , cytochrome , cytochrome c , escherichia coli , antiporters , fusion protein , protein subunit , chemistry , cytochrome b , cytochrome c1 , heme , biology , antiporter , coenzyme q – cytochrome c reductase , mitochondrion , recombinant dna , membrane , enzyme , gene , mitochondrial dna
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.