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.
Successful over-expression of a heterologous protein in the conventional prokaryotic host Escherichia coli is a highly unpredictable process and remains to be a major bottleneck for the biotechnological industry (1). Upon completion
of translating the polypeptide from mRNA, many proteins require additional post-translational modifications such as phosphorylation,
glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and proteolysis just to name a few. These modifications are catalyzed by a set of dedicated enzymes
which need to be regulated both in space (different compartments of the cell) and time. Further chaperoning by a dedicated
set of proteins may still also be required for a protein to achieve its correctly folded active state (2).
(IMAGE COURTESY OF AUTHOR)
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.
THE NATURE OF A DISULFIDE BOND
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).