Gibbons, D; (2012) Towards complete release from the E. coli periplasm for improved manufacturing. Doctoral thesis , UCL (University College London).

Abstract

Escherichia coli is used to produce a wide variety of therapeutic proteins, such as Fab fragments. Many such proteins are expressed and retained within the periplasmic region of the cell, and after fermentation the proteins must be recovered for downstream processing using steps such as homogenisation. The aim of this study was to investigate a new way to modify E. coli cells that would increase the levels of release of specific periplasmic proteins of interest into the cell culture broth, in order to allow these proteins to be recovered without the need for lengthy fermentations or the usual downstream processing steps. This thesis describes the construction of plasmid vectors designed to express antisense RNA that would inhibit the translation of cell membrane components including murein lipoprotein (Lpp), the most abundant protein in E. coli and a major component of the outer cell membrane. This was conducted using a strong promoter that could be induced during the cells’ exponential growth, to ensure the expression of the antisense RNA as the cells were growing so that subsequent generations of the cells would have lower levels of the lipoprotein within their cell membrane, and as a result leak a greater proportion of an overexpressed protein, alpha-amylase, into the cell culture broth. The system was also tested with a pair of Fab fragments which were induced with a distinct promoter so that the antisense RNA could be generated as the cells grow, while the Fab production would begin after the exponential growth phase has completed. These strains were tested from shakeflask levels up to 20 L fermentations (with up to 9 L working volumes). The periplasmic proteins examined showed increased levels of release in strains with the antisense plasmids, increasing the levels of release from as low as 10 - 20% in the unmodified strains up to almost 50% in some cases. This was achieved without compromising the growth rates of the cells which make them suitable for industrial fermentations. In summary, this antisense system, can be used to increase release levels of periplasmic proteins although further refinements and research into the use of antisense RNA would be required for the system to achieve full release of periplasmic proteins.

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