Team:UNIPV-Pavia

Despite the long and successful history of E. coli as a "protein factory", there are still many limitations, in both quantity and quality, affecting the production process of recombinant proteins. Efficient expression of the recombinant gene can be achieved by improving several steps of the production cycle, in order to obtain a much better yield/cost ratio, especially at industrial scale. We explored different approaches to these manufacturing steps, coming up with several possible improvements:

Self-inducible promoters
Expression of the recombinant gene has to be induced at a desired culture density, in order to ease the burden on the organisms, allowing the cultures to grow undisturbed before initiating production. This is usually achieved by controlling protein expression with inducible promoters: an inducer molecule (usually an expensive chemical compound) is added to the culture at the desired growth phase, thus triggering protein synthesis. A library of self-inducible promoters can be realized and characterized, allowing a degree of control over the time of production without the cost associated to other inducible systems.

Integrative standard vectors for E. coli and yeast
Integration of the recombinant gene or standard part in the genome eliminates the need for antibiotics in cultures for selection, lowering relative costs, and leading to a more stable system; we explored and tested a method that allows us to integrate a part into the genome, with the possibility of building a library of integration sites for both E. coli and yeast (S. cerevisiae).

Self-cleaving affinity tags to easily purify proteins
Purification of the target protein is usually achieved with affinity resins or columns, often amounting to a very large fraction of production costs; while many different approaches to purification have been explored in literature, we wanted to combine two promising techniques: PolyhydroxyAlkanoates production in the cytoplasm and an affinity tag system based on PHA-binding proteins (phasins) and self-cleaving protein segments (inteins). PHA granules covered by tagged proteins can be separated from the lysate by simple mechanical means, once again reducing costs and simplifying the process. Then the target protein can be easily separated by PHA granules through a pH/temperature shock, that triggers the self-cleavage of inteins and the release of purified product.

These solutions are modular, easily combinable and provide useful BioBricks for other applications.