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Jan 1, 2014 (Vol. 34, No. 1)

Advances in Protein Expression

  • Click Image To Enlarge +
    A schematic representation provided by Research Center Borstel of the hexa-acylated lipopolysaccharide from E. coli K-12 and the tetra-acylated, endotoxically inactive lipid IVA from ClearColi™. Because ClearColi lacks outer membrane agonists for activating a human Toll-like receptor, it reduces the need for downstream removal of endotoxin.

    Approximately half of approved recombinant biotherapeutics are expressed in Escherichia coli. During downstream processing, it is necessary to remove the organism’s outer membrane, which consists of lipopolysaccharide (LPS), also known as endotoxin.

    LPS is involved in a diverse spectrum of immune-response pathologies.

    Standard methods for removing this toxic component include activated carbon, modified sepharose resins, anion exchange chromatography, surfactants, and ultrafiltration. These methods, which are employed by processors during the final phases of downstream processing, all have their drawbacks. At best they add a process step at significant cost; at worst they affect recovery of active protein.

    An alternative approach, the production of protein in endotoxin-free E. coli cells, was described at a recent Informa Life Sciences BioProduction conference in Dublin. In addition to revealing innovations concerning E. coli, the conference highlighted advances with respect to CHO strains.

    The E. coli work that involved removing endotoxin was carried out by a team of scientists representing the University of Michigan, Research Corporation Technologies (RCT), Research Center Borstel, and Lucigen. This group essentially eliminated the need to remove endotoxin during downstream processing.

    The scientists developed a technology called ClearColi™, cell strains derived from E. coli. These E. coli mutants lack outer membrane agonists for activating the human Toll-like receptor hTLR4/MD-2.

    Of the many ligand-binding receptors on the surfaces of human immune system cells, hTLR4/MD-2 specifically recognizes endotoxin. After binding, the receptor dimerizes, which induces a signal cascade that activates a transcription factor, NF-κB, which controls transcription of several proinflammatory cytokines that are harmful to humans.

    The scientists achieved the construction of ClearColi cells by blocking the synthesis of endotoxically active LPS from the precursor lipid IVA (four-A) through multiple gene deletions. An additional mutation enables viability in the presence of lipid IVA.

    Lipid IVA consists of four fatty acid chains, whereas the mature LPS, which induces the hTLR4/MD-2 pathway, has six acyl chains. By contrast, precursor lipid IVA does not induce the hTLR4/MD-2 pathway, so it is endotoxically inactive.

    A key feature of ClearColi cells, according to Uwe Mamat, Ph.D., a senior research scientist at the Research Center Borstel, is the presence of a so-called ABC transporter with relaxed substrate specificity. “Usually, the LPS transporter is highly specific for the hexa-acylated LPS, but barely moves lipid IVA out of the cell,” explained Dr. Mamat. “The transporter’s ability to transfer lipid IVA out to the cytoplasm is critical for maintaining cell viability.

    “The ClearColi system is a versatile technology,” Mamat continued. “We have tested ClearColi cells with a number of model proteins, all of which were endotoxically inactive.”

  • (Fab’)ulous Yields

    Fab’ fragment expression within the periplasm of E. coli is desirable on several levels. Primarily, the periplasm’s oxidizing environment benefits formation of disulfide bonds, which the reducing nature of the cytoplasm does not. Additionally, the periplasm constitutes as much as 40% of the cell’s volume, proteolytic activity is lower than within the cytoplasm, and proteins that are toxic within the cytoplasm can accumulate with no deleterious effects.

    Mark Ellis, principal scientist at UCB Celltech, has described techniques for generating periplasmic Fab’ yields of 5 g/L, without compromising product quality or cell viability.

    Interestingly, high concentrations of Fab’ do not by themselves compromise viability. “That arises from a lack of resources, when cells deplete sugars and amino acids,” said Ellis, who also spoke at the Dublin conference.

    Particularly when under pressure to overexpress a foreign protein, cells may not have access to molecules that enable them to divide. “The cells are consuming resources faster than normal.”

    Although Ellis uses fed-batch cultures, the only feed ingredient is glycerol. One could also feed amino acids, but that would result in rapid and premature accumulation of biomass. Instead, Ellis knocked out protease genes specific to the periplasm, an approach that leads to higher protein accumulation.

    Similar techniques, he noted, are available for proteins that accumulate within the cytoplasm. In addition, Ellis co-expresses a gene for the isomerase DsbC, which is required for forming disulfide bonds in Fab’. “There is only a finite amount of DsbC in the cell,” he explained. “Once cells start using it to make Fab’, levels are insufficient for the cell’s own disulfide-containing proteins.”

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