Hibbert, E.G.; (2004) Engineering & molecular biology approaches to improving trypsin-based bioprocesses. Doctoral thesis , University of London.

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Abstract

This project was designed, in partnership with Eli Lilly, Fegersheim, with the aim of developing a series of methodologies for the mutation and characterisation of trypsin variants with extended substrate specificities for certain amino acid combinations. This library of modified enzymes with enhanced specificity towards 4-aa 1 sequence motifs could then be used as efficient and 'clean' biocatalytic agents. The goal was thus to explore the tailoring of industrial bioprocess enzymes to better suit process criteria, thus lowering the overall cost. It was a principle aim of the project to design a practical method of directed evolution for the breeding of variants with novel or enhanced substrate specificities. The organism on which this work was to be based was the Eli Lilly commercial recombinant bovine trypsinogen production strain, termed ELTRP-1. In order to devise a high-throughput assay suitable for screening a mutant library of 103+ variants, it was essential to fully characterise a method of microwell fermentation, and also to engineer the recombinant protein for solubility, i.e render it available for assaying. A high-throughput screen such as this would have to involve the minimum number of process steps and be entirely microplate-based. A microplate-scale solubilisation and refolding protocol for the r-trypsinogen inclusion bodies was developed that was successful for commercial enzyme but was impractical for use on recombinant inclusion bodies as the nature of the screen disallowed extensive purification, and thus the highly heterologous nature of the solutions appeared to inhibit refolding. Two variants of the enzyme, one with the prosequence removed, were cloned into a pET26b vector, behind a pelB leader, and all available fermentation parameters were experimented with. The construct plasmid was then subjected to a range of mutation rates using the XL1-Red mutator strain and variants were screened for increases in solubility. A series of mutants were obtained which demonstrated over 100 g/ml soluble mature enzyme after microplate fermentation, and enzyme translocation was monitored over 5ml and 100ml scale-up. Observed plasmid instability was due, in part, to the autotoxicity of the expressed enzyme and compounded by the fact that the T7 induction control mechanism of the strain appeared to have been disabled. These problems only manifested when the fermentation was scaled-up above microplate volumes and thus it appeared that the screening of the library for soluble mutant enzymes had been entirely successful by its own specific criteria, i.e. the isolated variants performed as required only when in well-culture.

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