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.