Optimisation of recombinant protein production in Escherichia coli

Zulkifly, Nurul Asma Hasliza (2024). Optimisation of recombinant protein production in Escherichia coli. University of Birmingham. Ph.D.

Preview

Zulkifly2024PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (9MB) | Preview

Abstract

Currently, recombinant protein production (RPP) has become more crucial in biopharmaceutical industry and E. coli has become the bacteria of choice. This research has been done to find the optimum condition for RPP using E. coli as the host, and looking for an opportunity to be applied industrially. Green fluorescent protein (GFP) was used as a marker to indicate the production of green fluorescence-proteins, which in this study, was applied to TNFα-GFP. TNFα-GFP is recombinant human TNFα fused to GFP. Two promoter systems were used, namely pORT(T7) plasmid which was inserted into bacterial host BL21(T7) and paraBAD plasmid which was recombined into BL21(A) strain. Arabinose was selected to be used as the inducer in this study to enhance protein production. The parameters investigated in this study were the carbon source and its concentration (glycerol and glucose), the growth temperature (30°C and 37°C), and the concentration of inducer (arabinose). Flow cytometry was routinely used to monitor the quantity and folding state of the TNFα-GFP fusion and cell physiology and viability on a single cell level. Further analysis identified three subpopulations with different fluorescence intensity and productivities, existed in the fermentation medium when using pORT(T7) system. In contrast to paraBAD system which had shown the production of only one single population throughout the fermentation. The optimum condition for RPP measured from this study was the usage of glycerol with the concentration of 0.4% (v/v), incubated at 30°C and arabinose concentration induced was 0.015%. The optimum conditions obtained in these experiments were used for the production of GFP and TNFα-GFP in fed-batch fermentation at 5 L scale. It was concluded that with further adaptation, it is possible to use these optimal conditions to be applied for RP production on an industrial scale. The present study also involved the production of soluble and functional Fumarase B (FumB) enzyme, which was achieved via the use of the restriction-free (RF) cloning method to insert the fumB gene into the pET41c plasmid.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):