Oxidative Folding of Proteins in Escherichia Coli - The author highlights novel strains and methods that have recently been shown to express multidisulfide bonded proteins. This article is part of a s

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Oxidative Folding of Proteins in Escherichia Coli
The author highlights novel strains and methods that have recently been shown to express multidisulfide bonded proteins. This article is part of a special section on expression systems.


BioPharm International
Volume 25, Issue 5, pp. 48-54

Cytoplasmic disulfide bond formation


Figure 3
The formation of disulfide bonds is strongly disfavored in the cytoplasm of E. coli (46). This is mainly due to the presence of two reductive pathways, the glutaredoxin and the thioredoxin pathway (see Figure 3) (10). These two pathways maintain a set of reductases in their reduced state, which in turn maintains a set of their substrate proteins cysteines in their reduced state. The reducing potential of the two pathways are received from the cytoplasmic pool of NADPH. NADPH donates its electrons to thioredoxin reductase (trxB) and glutathione reductase (gor), which transfer the electrons ultimately to a set of reductases. It is however possible to form stable disulfide bonds in the cytoplasm. To permit the formation of stable disulfide bonds in the cytoplasm, the reducing power of the glutaredoxin and the thioredoxin pathways need to be diminished. This step can be achieved by knocking out the gor and trxB genes (10, 47). However trxB, gor mutant cells are nonviable as at least one essential protein (ribonucleotide reductase) needs to be maintained in a reduced state (48). Cell viability can be restored by selecting for a suppressor of the lethal phenotype. Such a suppressor was selected and the locus was mapped to a peroxidase named AhpC, which had mutated to lose its function as a peroxidase but had gained the capacity as a disulfide bond reductase (49). It was eventually shown that mutant AhpC* was able to complement the defect in the glutaredoxin pathway by reducing glutathionylated glutaredoxins, which was sufficient reducing power for the cells to gain viability (50).

However, in the absence of thioredoxin reductase, the two thioredoxins in E. coli accumulate in their oxidized forms enabling them to act as disulfide bond formation catalysts, in a reversal of their normal function (51). This final strain FÅ113 had the remarkable capacity to oxidize and form stable disulfide bonds in its cytoplasm (52). FÅ113 was eventually made commercially available under the name Origami (Novagen) and has been used to express numerous disulfide bonded proteins (53–58). In some cases, expression of disulfide bonded proteins in the cytoplasm of Origami was exceptionally successful. For example, collagen prolyl 4-hydrolases yield was ~20 times higher in the cytoplasm of Origami than in the periplasm of the corresponding BL21 wild type strain and 10 times better than when expressed in insect cells (59). However, the efficient formation of correct disulfide bonds is limited in this strain (52).


Figure 4
To enhance disulfide bond isomerization in the cytoplasm of trxB/gor strains, a new protein expression strain was recently engineered and is available under the name SHuffle (New England Biolabs, Ipswitch, MA). This strain encodes a cytoplasmic copy of dsbC, expressed from the strong ribosomal promoter rrnB. Even though DsbC is overexpressed in an oxidative cytoplasm, it is found predominantly in its hemi-reduced active state (data not shown). As this strain has been developed only recently, currently there is only one publication using this strain [60]. For certain disulfide bonded proteins, the disulfide bond isomerase activity of DsbC is essential for their folding. This is the case for the three cysteine containing chitinase from Plasmodium falciparum (see Figure 4). Both E. coli K12 and B versions of this strain were engineered and empirical evidence suggests that the B versions are generally better at production of proteins than the K12 strains.


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