The bacterial host Escherichia coli provides an attractive industrial protein production approach in terms of time and cost efficiency. Microbial production technology is straight forward, safe, and well understood. Knowledge of the genetic properties of E. coli is more comprehensive than that of any other host organism. Both the development of E. coli cell lines and the production of protein by the bacterium proceed quickly, often allowing high quantities of protein, and are therefore less cost intensive than protein production by mammalian cells. Furthermore, in contrast to mammalian cell culture, the risk of transmitting viral infections or transmissible spongiform encephalopathies is precluded.
Despite all these advantages, microbial expression systems have their limitations. Post-translational modifications such as phosphorylation and glycosylation are difficult to achieve in a bacterial host but are required for a number of proteins, e.g., full-length antibodies. Also, proteins with large and complex structures (e.g., heterodimers and proteins with disulfide bonds) are often challenging.
As a result, the production of complex, recombinant proteins in E. coli may produce inactive, aggregated proteins (inclusion bodies) that accumulate in cells. Although expression yields and the separation of inclusion bodies from cell debris are efficient, converting inclusion bodies into native, biologically functional proteins requires their solubilization with chaotropic agents and a subsequent refolding step.
Refolding, however, is frequently the rate- and yield-limiting step for protein production and purification. In addition to inclusion bodies, more and more therapeutic proteins are solubly produced in the periplasm of E. coli. In these cases, the native, soluble proteins fold correctly in the oxidizing environment of the periplasm and thus, do not require refolding steps. Protein yields, however, are compromised by the limited space of the periplasm. Moreover, protein purification is strenuous because the whole cell lysate containing the recombinant protein also contains many host cell contaminants.
Wacker has developed an E. coli expression system—ESETEC®—that overcomes the limitations of periplasmic and inclusion-body production processes by efficiently secreting recombinant therapeutic proteins into the culture broth. By secreting the recombinant therapeutic protein extracellularly, the protein is produced in its native form. Furthermore, production yields are not limited by periplasmic volume. Refolding is not required either because the protein is expressed solubly and, since the protein has been secreted, cell disruption is unnecessary. Therefore, secretion reduces the number of necessary downstream processing steps, increases the overall process yield, and thus, lowers process costs (Figure 1).
ESETEC consists of bacterial strains that are derived from the well-characterized E. coli K12 strain of expression plasmids, and of a set of additional elements that help to optimize expression, solubility, and secretion of the target protein.
ESETEC strains are suitable for cGMP production because of their documented history. They facilitate the secretion of recombinant proteins into the culture broth thanks to their modified outer membrane. Wacker’s expression plasmids usually encode the ptac promotor and proprietary signal sequences for the translocation of recombinant proteins to the periplasm.
For translocation, ESETEC uses the Sec pathway—a major pathway of active protein transport from the cytosol across the inner cytoplasmic membrane in bacteria (Figure 2). Sec translocase recognizes the short signal sequences at the N-terminus of the protein and transports the protein across the inner membrane.
Upon transfer, a signal peptidase removes this signal sequence, and the premature recombinant protein is released with its native N-terminus. Then the premature protein is folded, and disulfide bridges are formed. Formation of disulfide bridges is favored in the oxidative environment of the periplasm, in contrast to the reducing conditions of the cytosol.
Moreover, recombinant proteins are less amenable to proteolytic degradation in the periplasm than they are in the cytosol. ESETEC thus achieves high-yield production of correctly folded proteins because these proteins are secreted into the culture broth. This system has been shown to be suitable for different prokaryotic and eukaryotic proteins, for proteins with a wide range of molecular weights and isoelectric points, for fusion as well as native proteins, for proteins with an N-terminal amino acid other than methionine, and for proteins with disulfide bridges.
High yields of up to 11 g/L have been obtained for various proteins. A range of peptides, enzymes, and proteins, such as antibody fragments (Fabs, scFvs) and engineered protein scaffolds (Anticalin®), have been successfully produced. When combining ESETEC with the high-cell-density fermentation technology DENSETEC® (another Wacker technology), optimal space-time yields and high reproducibility are achieved.