PMID:20509166

Citation	Gustavsson, T, Trane, M, Moparthi, VK, Miklovyte, E, Moparthi, L, Górecki, K, Leiding, T, Arsköld, SP and 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 Sci. 19:1445-60
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.
Links	PubMed PMC2923498 Online version:10.1002/pro.424
Keywords	Animals; Bacillus subtilis/genetics; Bacillus subtilis/metabolism; Cytochromes c/chemistry; Cytochromes c/genetics; Cytochromes c/metabolism; Electron Transport Complex I/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Models, Molecular; Oxidation-Reduction; Protein Conformation; Protein Subunits/chemistry; Protein Subunits/genetics; Protein Subunits/metabolism; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BACSU:MRPA		GO:0015385: sodium:hydrogen antiporter activity	ECO:0000315:		F		Figure 5. Panel A: MprA null mutants are unable to grow in the sodium rich environments compared to both wild type and cells with MprA placed back into the cells via a plasmid.	complete CACAO 7944
BACSU:MRPA	enables	GO:0015385: sodium:proton antiporter activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
BACSU:MRPD		GO:0015385: sodium:hydrogen antiporter activity	ECO:0000315:		F		Figure 5. Panel B: MprD null mutants are unable to grow in the sodium rich environments compared to both wild type cells and cells with MprD placed back into the cells via a plasmid.	complete CACAO 7945
BACSU:MRPD	enables	GO:0015385: sodium:proton antiporter activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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