Physiological aspects underpinning recombinant protein production in Escherichia coli

Alfasi, Sara Nuri (2011). Physiological aspects underpinning recombinant protein production in Escherichia coli. University of Birmingham. Ph.D.


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Many biopharmaceutical projects require the production of recombinant protein in a bacterial host. Conventional procedures used for recombinant protein production (RPP) involve the rapid synthesis of the target protein. This results in the accumulation of unfolded protein, the induction of the heat shock stress response and bacterial growth arrest. More importantly, the target protein accumulates in inclusion bodies and hence is useless to determine its structure. The immediate impact of this is that both the yield and quality of the target protein are compromised. This thesis reports two generically successful approaches that were developed to overcome this series of stress-induced events in Escherichia coli. Both strategies were developed during the production of a cytoplasmic protein and outer membrane lipoproteins using the pET expression system in the bacterial host, E. coli strain BL21(DE3)*. First, the induction protocol was modified to minimise the stress on the host bacterium. This method relies on the induction of very low levels of the T7 RNA polymerase in BL21* and thus the correspondingly slow synthesis of the target protein. Using this approach, growth and productivity of different types of correctly folded target proteins were sustained for at least 70 h. Secondly, mutant hosts that significantly improve recombinant protein production during conventional protocols were isolated. These improved hosts are resistant to IPTG-induced stress and continue to accumulate high levels of the correctly folded target protein. Key to the stress resistance is the presence of mutations that downregulate the synthesis of T7 RNA polymerase. However, different improved hosts were able to enhance the production of different types of target protein, such as those requiring extensive post-translational modification. The potential for isolating a plethora of improved bacterial hosts that are tailored for the production of different types of recombinant protein is discussed in light of the challenges faced by bioindustry. Procedures enabling the isolation of mutant hosts during the production of GFP-tagged and untagged proteins are reported.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
College/Faculty: Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department: School of Biosciences
Funders: Biotechnology and Biological Sciences Research Council
Subjects: T Technology > TP Chemical technology
Q Science > QH Natural history > QH301 Biology


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