Biophysical characterisation of recombinant protein-packaged vesicles from Escherichia coli for research, medical and biotechnology applications

The Vesicle Nucleating peptide (VNp) technology offers a simple peptide tag-based method for high-yield production of functional recombinant proteins, including toxic otherwise insoluble or disulfide-bond containing proteins, in Escherichia coli. These proteins are packaged into membrane-bound vesicles which provide a stable micro-environment for long-term protein storage or simplified downstream processing. The work in this thesis extends this technology's capabilities by successfully optimising the production of recombinant vesicles containing a lumenal VNp cargo and the outer membrane protein OmpX in their membrane, through co-expression. These innovative surface-functionalised vesicles have the potential for targeted drug delivery, using OmpX for ligand display. Furthermore, the co-expression is shown to enhance extracellular yield of some VNp fusions. For the first time, an in-depth analysis of vesicle biophysical characteristics is undertaken comparing VNp and VNp-OmpX vesicles with natural outer membrane vesicles determining their size, membrane structure, lipid composition and the intravesicular concentration of different recombinant fusion proteins. Crucially, all three vesicle types have similar phospholipid compositions and contain lipid A but VNp/VNp-OmpX vesicles have an increased vesicular concentration of the protein of interest. These data advanced the mechanistic understanding of the VNp technology and further proved its advantages over standard recombinant protein production methods.