Recombinant protein expression often fails from various toxicity challenges associated with cellular production. The most common forms of host incompatibility that lead to toxicity are from overexpression and the formation of inclusion bodies. While there are several tricks used to counter these challenges including lowering copy number, lowering temperature, and using optimized expression strains, navigating these optimizations is usually done through trial and error, wasting time and resources, and in many cases, still not producing the protein of interest.
The Tierra Protein Platform excels at producing toxic proteins. Because our platform is built with cell-free systems (using components, but not a living cell itself), there is no cellular growth to inhibit. Bacteriocins and other inhibitors of cells can be made without inhibiting the host. Overexpression does not result in inclusion bodies since there is no fitness cost and no compartmentalization.
For particularly difficult proteins, the Tierra Protein Platform allows for faster diagnosis steps than in cells. Since cell-free systems are open, additives, such as potentially missing cofactors, can be easily titrated to help increase protein yields. Additionally, the background host can also be optimized. For select cases, we can utilize non E. coli cell-free systems that better match the source of the original protein to improve production.
The result is not only the toxic protein, but also understanding of the root causes of the toxicity. This allows you to move on with your assay having the peace of mind that once the winner is identified, the protein can be scaled for next steps.