A new generation of E. coli expression hosts and tools for recombinant protein production

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Many biopharmaceuticals are produced in E. coli but current platforms have a number of limitations and cannot produce many potential target products. This project will develop four entirely novel innovations to produce and harvest a wide range of target proteins, delivering new tools and processes that encompass the entire upstream pipeline:

1: Protein synthesis will be driven by a novel set of promoters and inducers that have clear advantages (better inducible control, higher mRNA yield) over currently-used systems. Many of the currently-used inducible promoters for recombinant protein production (RPP) are extremely strong, inherently leaky and present on high-copy number plasmids. RPP often outstrips the ability of the cell to cope, resulting in insoluble aggregates and inclusion body formation.
2: Export to the periplasm will be mediated by an alternative protein export pathway, known as the Tat pathway, that has unique capabilities and clear advantages over the currently-used Sec pathway. A major problem with the Sec pathway is that it transports its substrates in an unfolded state, and cannot handle proteins that fold too quickly or tightly - a significant proportion of potential target molecules. The Tat pathway will instead be exploited to export a wide range of new biotherapeutics in a prefolded form.
3: We will develop and validate a novel method for releasing periplasmic contents which relies on nano-encapsulation of lipids. The method uses a low cost polymer (SMA) which provides a more specific release method than current osmotic shock methods under a wider range of operating conditions.
4: The above innovations will be combined to deliver an integrated platform that is better than the sum of its parts.

The project will be carried out in collaboration with a range of UK companies who will fully validate the new strains and processes.

As described in proposal submitted to TSB