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Title: Protein renaturation and aggregation during dilution refolding
Author: Buswell, A. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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The aim of this thesis was to develop a simple usable kinetic scheme for protein refolding that was not dependent on reactor type, and to estimate the relative effect of mixing on refolding and aggregation. There are few direct comparisons between experiments and simulations for fed-batch or continuous refolding, and only batch-derived kinetic schemes are available for use in these reactors. Their applicability has not been tested in alternative reactor types. The effect of mixing on refolding and aggregation has also largely been ignored. However aggregation is likely to be affected by the efficiency of mixing due to the higher-order dependence of the aggregation reaction and the complex dependence of refolding and aggregation on denaturant concentration. A statistical design refolding experiment was first conducted with recombinant trypsinogen inclusion bodies to assess the effects of various physical refolding parameters. Mixing was shown to be important. The existing batch-derived kinetic scheme for lysozyme refolding was then examined in detail and shown to not predict refolding yield in a fed-batch reactor. An improved kinetic scheme was developed using a novel labelling technique, and stopped-flow classic light scattering to directly measure the aggregation rate constant. The model involves a sequential aggregation mechanism in competition with early commitment to refolding, and gave an improved estimate of lysozyme fed-batch refolding yield. An optimum level of mixing was identified for lysozyme dilution refolding using an oscillating grid reactor. An analysis of a diffusive mixing model combined with the improved kinetic scheme demonstrated the effect of varying mixing length-scales on refolding yield, and partly explained the optimum mixing level identified with the oscillating grid experiments. The key outcomes of this thesis are an improved kinetic scheme for lysozyme refolding, and a better understanding of the effects of mixing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available