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Oct 15, 2011 (Vol. 31, No. 18)

Protein Bioproduction without Antibiotics

Instrument for Use in E.Coli Designed to Tackle Problems with Antibiotic-Resistant Genes

  • Since the beginning of DNA engineering, plasmid instability has been a significant concern, especially in recombinant protein production. Typically, protein-production processes in prokaryotes require the use of bacterial plasmids as vectors to carry the gene of interest encoding the protein to be overexpressed.

    It is well known that the growth of plasmid-bearing cells is significantly reduced relative to plasmid-free hosts simply because the protein production (corresponding to the gene of interest overexpression) and the vector represent a burden on host metabolism.

    Antibiotic-resistant genes are the selectable markers typically used in fermentation processes to avoid plasmid-free cells overgrowing the culture. Antibiotic and antibiotic markers represent a problem from a regulatory standpoint, and today, only the Kanamycin resistance gene is still tolerated by regulatory authorities. The Kanamycin resistance gene, however, represents a metabolic burden that limits the production yield of the bioproduct of interest.

    The only way to avoid antibiotic-resistance spread in the environment and transfer to pathogenic strains is the complete absence of antibiotic-resistance genes in recombinant constructions. Thus, it is expected that, in the near future, there may be zero tolerance toward antibiotic-based selection and production systems at least in the biopharmaceutical field.

    Several alternatives to antibiotics have been proposed but none of them has been widely adopted. This can be explained by the complexity of the proposed systems, which requires complex or specific media, or nonconventional strains.

    An alternative to antibiotic markers that is less energy consuming for the host metabolism is required for more efficient recombinant bioproduction processes.

    With these challenges in mind, Delphi Genetics developed a stabilization system called StabyExpress™ based on the use of natural bacterial antidote/poison genes and more precisely the ccd operon. This operon was isolated from the F plasmid naturally present in E. coli and encodes two genes (ccdA and ccdB) that are very well characterized.

    The ccdB poison gene codes for a small stable protein, whereas the ccdA antidote gene codes for a small unstable protein that neutralizes the poison protein both transcriptionally and via protein-protein interactions. These genes were chosen because they are not toxic to humans.

    StabyExpress technology takes full advantage of the natural properties of these genetic elements. Indeed, the functions of these genes have been polished by evolution in order to interact very efficiently with E. coli physiology so as to trigger bacterial death in very defined conditions.

    The observed efficiency of the StabyExpress technology comes from this natural evolution. The ccd operon belongs to the poison/antidote bacterial gene family. These loci share common features and are widespread in plasmids and bacterial chromosomes. They represent a natural toolbox for applications in bacterial species presenting an industrial interest.

  • Bacterial Poison/Antidote as an Alternative to Antibiotic Markers

    Click Image To Enlarge +
    Fig.1 The StabyExpress system is based on the use of natural bacterial antidote/poison genes and the ccd operon.

    In the StabyExpress system, the ccdB poison gene has been introduced into the chromosome of E. coli, and the vector contains a copy of the ccdA gene encoding the antidote (Figure 1). When the plasmid is lost, the production of the bacterial toxin is induced, causing bacterial death. Consequently, only bacteria-harboring plasmids are growing and can use nutritional resources. To limit the burden of energy for the host and selection of mutants, the ccdA antidote production was reduced to a maximum (the gene is cloned under a weak constitutive promoter and for all intents and purposes the protein is not detectable).

    Moreover, we used a transcriptional negative feedback loop to avoid ccdB expression when ccdA is present. Since ccdB poison protein is specifically targeting the bacterial DNA gyrase, it is thus not toxic for eukaryotic and human cells.

  • Click Image To Enlarge +
    Figure 2. Researchers can obtain access to StabyExpress technology by purchasing research kits that contain strains and vectors to produce their protein of interest. The purchase of the kits includes a license for research use of the technology.

    Staby technology was combined with a T7 expression system and introduced in research kits. Different laboratories (academic and industrial) have used these kits to produce their protein of interest in E. coli. The system is easy to use and is compatible with any growth medium. It is also possible to integrate StabyExpress technology into any vector: the GetStaby kit contains a ccdA cassette designed for easy combination with any protein-expression system in E. coli (Figure 2).

    In 2009, Delphi Genetics signed a nonexclusive commercial agreement for StabyExpress with Sanofi-Pasteur, which subsequently showed that the technology allows recombinant protein production at industrial scale without the use of antibiotic markers (and antibiotics).

  • Click Image To Enlarge +
    Two conditions have been analyzed: induction at A600=25 (advanced stage of growth), close to standard production, and induction at A600=1 (early stage of growth) to increase the number of cell divisions between induction and end of fermentation and introduce an extended high level of stress over the cells. In normal conditions the plasmid maintenance is increased and productivity doubles. For extended stress condition, the differences between antibiotic-free and standard are dramatic, both in terms of plasmid maintenance and protein productivity.

    Moreover, Sanofi-Pasteur showed that StabyExpress technology improves production yield in fermentors. As shown in the Table, when standard fermentation conditions are applied, the plasmid maintenance is at least equivalent or superior when StabyExpress selection is compared to standard vector (98% versus 90%). More interesting, in these conditions, the amount of expressed antigen increases twofold with StabyExpress (603 mg/L versus 280 mg/L).

    In high-stress conditions, the plasmid loss is extremely rapid with standard vectors as compared to StabyExpress (5% versus 100%), indicating stringent selection of the StabyExpress system.

    In that case, the amount of expressed antigen increases tenfold (36 mg/L versus 350 mg/L). In order to evaluate the potential for scaleup, the fermentation process previously established for standard conditions was transposed at a 30 L scale. Values obtained for both optical densities at different times and plasmid maintenance are almost similar (twofold and tenfold increase).

    Delphi Genetics provides StabyExpress licensees strains that are compliant with cGMP standards in terms of culture quality (animal and GMO free). In addition, strains containing StabyExpress technology have been completely sequenced. This allows industrial researchers to develop an antibiotic-free production process starting from completely genetically characterized material.

  • Conclusion

    Improving yield and quality of recombinant protein production remains a challenge. Necessary improvements include the re-engineering of genetic elements (host and vectors) used in these processes. By using the natural properties of bacterial poison and antidote genes, Delphi Genetics has designed a powerful alternative to antibiotic markers for expression-vector selection in E. coli.

    StabyExpress can be integrated into any E. coli genetic background making it a potential universal alternative to antibiotics/antibiotic markers in this host. In light of new regulatory guidelines limiting the use of antibiotics, this development is significant.

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