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Title: Developing new strategies for the production of foreign proteins in higher plant chloroplasts
Author: Michoux, Franck
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2008
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Abstract:
Transformation of the chloroplast genome is a technique that allows for the production of recombinant proteins in plants. Chloroplast transformation allows the very high expression of a transgene while limiting the gene flow to other species. In addition, multiple genes can be transferred in one transformation event and no gene silencing as been shown so far. Despite these advantages, little work has been directed at assessing the commercial feasibility of using the chloroplast as a means of expressing high-value proteins. In this thesis, a new expression system was developed to allow the very high expression of transgenes in the chloroplast under contained conditions. Tobacco plants were transformed with a tobacco chloroplast vector expressing green fluorescent \ protein. Cell suspensions were induced from these leaves. Using a bioreactor, I was able to demonstrate that coupling temporary immersion with a hormonal shift triggered the rapid production of plant tissue whilst retaining a high level of expression of green fluorescent protein. Another aspect of this thesis was to assess the potential of several fusion tags to improve the solubility and purification of various target proteins in transformed tobacco chloroplasts. The main proteins studied were a mannanase from coffee, which is involved in the detergent, pulp and, more recently, the bioethanol industry, as well as alpha defensin 1 peptide, which has potential therapeutic value in the treatment of several diseases such as HIV and Herpes. N- and C-terminal constructions were created with oleosin, dehydrin, fibrillin, maltose-binding protein and glutathione-S-transferase as tags. Constructs were first evaluated in Escherichia coli before being integrated into the tobacco plastome. Apart from oleo sin, all fusion proteins were successfully expressed in transplastomic tobacco. My work has identified dehydrin, GST and MBP as promising affinity-tags to be used in chloroplast transformation experiments. Finally, I describe the development of experimental tools and procedures for the transformation of the chloroplast genome of coffee, which is one of the world's major cash crops. For this, coffee-specific vectors were created and direct somatic embryogenesis employed to propagate transformed tissue.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.556624  DOI: Not available
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