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Title: Directed evolution of bovine enterokinase for high functional expression in E.coli
Author: Lee, W.
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Bovine enterokinase (EK) is a specific protease that has industrial utility for its accurate removal of affinity tags subsequent to affinity-based purification of high-valued biopharmaceuticals. However, recombinant productions in Escherichia coli often required extensive downstream processing steps. Traditional methods rely on the fusion of solubility-enhancing leader peptides to the EK sequence coupled with in vitro autocatalytic cleavage in order to recover the functional enzyme. In contrast, the current work sought to directly express soluble and functional EK by encouraging its targeted secretion into the periplasm. Directed evolution was applied to discover functionally-expressed EK variants. A secondary objective was to use the discovered variants to attempt to understand the soluble expression dynamics of heterologous proteins in E. coli. Rational engineering and directed evolution yielded 288 unique variants after screening ~6500 colonies derived from 3 random mutagenesis and 13 combinatorial libraries. On a "total activity" basis, assay results showed that the top variants discovered were 122-355 fold improved over the pseudo wild-type EK. Most interesting was that characterisation results of 15 selected variants showed that codon-optimisation and expression temperature "effector" perturbations had an effect on a variant's expression, enzyme kinetic, and stability behaviour. For example, a codon-optimised variant WLEK0513 expressed at 37°C exhibited a melting temperature (Tm) of 52.6°C as opposed to 50.8°C for its non-codon-optimised counterpart. Or, the variant's specific expressions at 30C compared to 37C were assessed to be 75 and 363 ug L-1 OD-1 , respectively. Also surprising was the catalytic efficiency (kcat/Km) improvement of variant WLEK0699 from 4394 to 7299 U ng-1 EK mM-1 GD4KNA when expression temperature was raised. These results suggested that the slightly different conformations of the same variant were affected by shifts in soluble expression dynamics. Finally, deductive reasoning hypothesizes that the quality of periplasmic chaperone interactions had a large role in the unanticipated results.
Supervisor: Dalby, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available