Despite the long and successful history of E. coli as a "protein
factory", there are still many limitations in 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.
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 to trigger the protein 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.