Expression of active recombinant proteins in E. coli which require post-translational modifications, such as disulfide bonds, is difficult, mainly due to the fact that disulfide bond formation in E. coli is compartmentalized to the periplasm and does not have the capacity to express complex multidisulfide bonded eukaryotic proteins. Novel expression strains and procedures are in demand to handle the growing pharmaceutical and biotechnological field. The author highlights novel strains and methods that have recently been shown to express multidisulfide bonded proteins.

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One major form of post-translational modification is the formation of covalent disulfide bonds. Disulfide bonds are more common than appreciated. After the peptide bond, disulfide bonds are the second most common covalent bonds found within proteins (3). It is estimated that one-third of the human proteins reside in the endoplasmic reticulum, of those at least half of them are predicted to have disulfide bonds (3). When expressing an open reading frame (ORF) from an uncharacterized organism or unknown sources (as in the case of environmental DNA libraries), a researcher is challenged to find the correct expression host and condition to express soluble active protein to a satisfactory yield. When attempting to express a protein which requires disulfide bonds for its folding, a sufficient understanding on the mechanism of disulfide bond formation and its subsequent biological roles is essential. This review will attempt to summarize the necessary knowledge to assist the researcher in finding the correct conditions to express a disulfide bonded protein, within the model prokaryotic host E. coli.

Disulfide bonds are formed by the oxidation of thiol groups (SH) found within the side-chains of cysteines. Disulfide bonds play multiple critical roles in proteins stability, function and can be summarized into three major biological groups; structural, signaling, and catalytic.

Redox state of cysteines

Cysteines involved in the formation of structural disulfide bonds decrease the entropy of a protein by restricting conformational possibilities, increasing the proteins thermostability (4). It is therefore possible to create more stable versions of a protein by engineering disulfide bonds into the proteins sequence (5). Not surprisingly, the stabilizing property of disulfide bonds is most likely the reason why secreted proteins, which are outside the chaperone rich environment of the cytoplasm, are rich in disulfide bonds. However, cysteines are uniquely sensitive to their environment and can be readily reduced or oxidized depending on the redox state of their surroundings (6). This feature has been used by many proteins to sense, signal, and regulate the redox state of their environment.

For example, the transcriptional factor OxyR has two redox sensitive cysteines which upon oxidation promote a conformational change, resulting in the activation of OxyR as a transcriptional factor (7). Other signaling disulfide bonds can be found in the two-component signal transduction system of ArcAB and oxidative stress response of RsrA (8, 9). Catalytic cysteines are crucial to the activity of oxidoreductases and are found within the CxxC active site motif, where x is any amino acid. The active site cysteines of reductases such as the cytoplasmic thioredoxin are maintained reduced, whereas those of oxidases such as the periplasmic DsbA are maintained oxidized (10, 11).