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

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Nyckelord

Bacillus subtilis

Escherichia coli

MrpD

MrpA

NuoN

NuoM

NuoL

NuoH

oxidoreductase

NADH:quinone

covalently bound heme

cytochrome c

fusion proteins

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art (ämneskategori)

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