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Aug 1, 2008 (Vol. 28, No. 14)

Making Antibiotics Obsolete in Fermentation

Using Bacterial Selection Modules for Protein Production in E.coli

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    Figure 1

    Plasmid instability is a very real concern in recombinant protein or DNA manufacturing. Typically the biomanufacturing process in prokaryotes requires the use of bacterial plasmids as vectors to carry the gene of interest to be overexpressed. The growth of plasmid-bearing cells is significantly reduced relative to plasmid-free hosts, simply because protein production represents a significant burden on cellular metabolism. As both the yield and the production reproducibility of recombinant molecules are lowered by this fact, it is a big concern in the biopharmaceutical industry.

    Antibiotic-resistant genes are the most common selectable markers used in fermentation to prevent plasmid-free cells from overgrowing the culture. Antibiotics, however, are expensive compounds and they can contaminate the biomass or production product. Consequently, when using antibiotics it has to be demonstrated that the final product is antibiotic-free. The assessment of residual antibiotic levels and, if necessary, their removal are costly procedures. As a result, the current trend in the biomanufacturing industry is to forgo antibiotics in the production process altogether.

    Not surprisingly, several other methods to minimize the risk of plasmid-free cells overtaking the culture have been developed. One of the most promising relies on the use of postsegretional killing genes (so called poison/antidote genes or selection modules) that induce host killing upon plasmid loss.

    Delphi Genetics has designed a stabilization system called Staby™ based on the use of selection modules naturally found in plasmids, bacterial chromosomes, and bacteriophages. A selection module is typically organized as an operon and composed of two genes: The ccdB selection gene codes for a small stable protein that is toxic for E. coli, whereas the ccdA antidote gene codes for a small unstable protein that neutralizes the toxic protein both transcriptionaly and via protein-protein interactions.

    The Staby system is based on the ccd module. This module was chosen, because the ccdB toxin is active only in enterobacteriaceae. The antidote gene has been separated from the selection gene, locating the former on the expression plasmid and the latter in the chromosome of the expression strain. Expression of the selection gene is under the control of a promoter that is strongly repressed in the presence of the plasmid. When the plasmid is lost, the antidote protein is degraded, and the production of the poison is induced causing cell death (Figure 1).

    This system allows for stabilization of the plasmid without the use of antibiotics. Furthermore it ensures that during protein expression, every bacterium is carrying the expression plasmid thus enhancing the overall yield of any given protein expression process. If some bacteria lose the vector, they will not obtain a selective advantage but will die instead.

    This stabilization technology solves the problem of plasmid instability and insures that upon induction, 100% of the bacteria will produce the recombinant protein leading to higher yields of the target protein and less background caused by unwanted proteins. Thus, the production of the protein of interest is higher and purer.

  • Maximizing Titer

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    Figure 2

    To maximize protein expression, the Staby system was combined with T7 polymerase technology for the over-expression of recombinant proteins; the gene of interest is under the control of the T7 promoter. This combination is called StabyExpress. The pStaby1 plasmid contains the gene encoding the antidote protein (ccdA) that counteracts the action of the ccdB protein. The SE1 strain, in which the ccdB selection gene is inserted in the chromosome to stabilize the pStaby1 plasmid, has been engineered by Delphi Genetics as a dedicated bacterial strain for protein expression and is derived from BL21(DE3).

    Consequently, IPTG has to be used to induce protein expression. The use of an auto-inducible medium to initiate protein expression when an appropriated cell density has been reached is an alternative strategy.

    StabyExpress has proven useful for the overexpression of recombinant proteins that had been difficult to produce before and for which the attained protein titers had also been poor.

    To establish the validity of this system, the overexpression of a 22.5 kD human protein was attempted (Figure 2). The fermentation was performed in 75-liter fermentors using 50 liters of medium. Using StabyExpress, the plasmid was stabilized before and after the induction period. As a result of the stabilization, 100% of the induced bacteria harbored the expression plasmid.

    The expression difference between a classical system (Lane 4) and StabyExpress (Lanes 2 and 3) was visible on gel. After 90 minutes of induction, significantly more of the desired protein is produced when the expression plasmid is stabilized. After purification, the yield of the protein of interest was estimated to be >600 mg/L using StabyExpress, approximately four times higher than the yield achieved with a conventional system without stabilization.

    Moreover, overproduction of the antidote is not detectable, and only a negligible amount of amino acids is immobilized for the functioning of the stabilization system. Conversely, as the ccdB selection gene is transcriptionaly silenced when the plasmid is present, only an undetectable amount of the toxic protein is produced during the fermentation process.

    Generally speaking, overall yields are consistently higher when using StabyExpress. As an example, we attempted to overexpress the nontoxic enterobacterial protein TraG, which is involved in conjugation and mobilization processes. We deliberately chose this protein to establish the gains achievable with the StabyExpress system purely based on the stabilization issue. We compared the yield in recombinant protein production using the StabyExpress system without the use of antibiotics with an unmodified pET/BL21(DE3) with ampicilin.

    After protein gel electrophoresis, the amounts of TraG protein that were produced in both conditions were quantified and compared. After two hours of induction, significantly more TraG protein (and a lower background) was produced using the stabilized system (Figure 3, lane 4) compared to the antibiotic-dependent classical system (Figure 3, lane 5).

    StabyExpress ensures that only plasmid-bearing clones can grow, whereas every clone losing the plasmid will inevitably die. This feature guarantees the highest possible yields. As the stabilization is purely based on genetic elements, StabyExpress can be used in any culture medium even synthetically defined media, which are devoid of complex compounds like yeast extract or soy peptone.

  • Stability Is Key

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    Figure 3

    To further evaluate the efficiency of the stabilization system, we compared the effect of growth and protein expression on plasmid stability in the absence of antibiotics using a conventional expression system (BL21DE3 with a pET series vector) and StabyExpress (SE1 with pStaby vector).

    After 30 generations, bacterial cultures were plated on nonselective and selective media to quantify, respectively, the total population of bacteria and the subpopulation of plasmid-bearing bacteria before and after an induction period of two hours. The experiment was repeated three times, and the standard deviation was calculated.

    The StabyExpress system is insensitive to instability induced by the overexpression (Figure 4). Two hours after induction of protein production, no cell had escaped the stabilization system, as 100% of the cells exhibited the plasmid construct. In contrast, the conventional system experienced a population shift from an already moderate (31%) to a low (0.14%) proportion of plasmid-bearing bacteria.

    As stabilization relies on the presence of only two genetic elements, one on the plasmid the other in the expression strain, the stabilization technology is readily adaptable into any existing E. coli-based expression platform (Figure 5). In addition, Delphi Genetics can customize any vectors and strains by adding the stabilization cassette into the vector and the selection gene into the chromosome of the chosen expression strain. The technology is also available as kits.

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    Figure 4
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    Figure 5

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