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 Cterminal 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 1170 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.

Structure and function of the C‐terminal domain of MrpA in the Bacillus subtilis Mrp‐antiporter complex – The evolutionary progenitor of the long horizontal helix in complex I

“…Therefore, a holo‐cytochrome can only be formed when the membrane protein to be tested has the C‐terminus on the periplasmic side of the membrane when the fusion protein is expressed in E. coli , whereas otherwise the apo‐cytochrome can be detected using anti‐cytochrome c 550 antibodies. Therefore, the cytochrome‐tagging strategy is applicable to determine the transmembrane topology of membrane proteins [20] and is also useful to stabilize and enhance the expression of some proteins [18].…”

Revised transmembrane orientation of the NADH:quinone oxidoreductase subunit NuoA

“…We have recently made use of cytochrome c fusion proteins to facilitate the purification and quantification of various complex I subunits from E. coli [15]. A plasmid, pTCH was produced for the convenient tagging of membrane proteins with cytochrome c and a his‐tag for subsequent purification.…”

“…Bacterial strains and plasmids used are listed in Table 1 . The plasmid pTCH was built from pTRC19 that contain the truncated cccA gene, encoding the cytochrome part of cytochrome c 550 from Bacillus subtilis [15]. In pTCH, and a C‐terminally fused his‐tag was added to the cytochrome (Table 1) using the Phusion site‐directed mutagenesis method.…”

“…1), leaving out the last predicted transmembrane helix. The expression of fusion protein was optimized as before (Table 3, [15]). Indeed, this truncated NuoA polypeptide formed a heme containing cytochrome with normal spectroscopic properties ( Fig.…”

“…A comprehensive experimental topology analysis of 714 membrane spanning proteins in the E. coli proteome by fusions to green fluorescent protein (GFP) and alkaline phosphatase (PhoA) fusions successfully determined the location of the C‐termini for 601 proteins, but unfortunately, NuoA was among the few proteins where the orientation was left undecided [13]. In this work, the transmembrane orientation of the NuoA subunit from E. coli complex I was addressed independently, using different fusion protein techniques [14,15]. Cytochrome c fusion proteins comprising truncated and full length NuoA were constructed and analyzed.…”