Optimising recombinant protein production in E. coli

Osgerby, Alexander (2025). Optimising recombinant protein production in E. coli. University of Birmingham. Ph.D.

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Abstract

Recombinant protein production (RPP), the heterologous overexpression of high-value proteins in more amenable host organisms, is essential for synthesising proteins used in research and with commercial or clinical applications. The use of Escherichia coli, exploited as a host in RPP for over 40 years, is not without caveats; this work has addressed several. Firstly, green fluorescent protein (GFP) was overexpressed from the urea inducible Proteus mirabilis urease promoter Puti101 as a cost-effective alternative to established systems. Optimisation from flask to bioreactor scale indicated that basal GFP expression from Puti101 was low, typically around 2.5-fold higher than that of untransformed cells. Adding urea resulted in an up to 75-fold increase in GFP expression; even concentrations up to 500 mM were shown to have little effect on biomass accumulation. Secondly, many valuable proteins require disulphide bonding for bioactivity, necessitating periplasmic expression in E. coli. Oxidation-resistant GFP derivatives sfGFP and cfSGFP2 were evaluated under RPP-centric conditions to understand their application in improving screening throughput. While the export of GFP to the periplasm was detrimental to host physiology, super-resolution microscopy supported prior indications that periplasmic sfGFP fluoresces. Finally, as periplasmic expression was problematic, our focus shifted to detecting host stress via GFP fusion to the Pspy promoter from the periplasmic chaperone spheroplast protein Y (Spy). Pspy responded to both overexpression of periplasmic recombinant proteins and direct inhibition of translocation machinery. Then, combined with novel repressors, Pspy was exploited as a trigger for negative feedback to greatly improve host physiology during overexpression of poorly tolerated periplasmic recombinant proteins.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):