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Title: Plant-based strategies aimed at expressing a functional human adenosine deaminase at high levels
Author: Singhabahu, S. A. D. Sanjeewa
ISNI:       0000 0004 2733 6305
Awarding Body: University of East London
Current Institution: University of East London
Date of Award: 2012
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An inherited disorder, ADA deficiency, is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants and tobacco BY-2 cell suspensions a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and terminator. In an attempt to maximise the yield various recombined gene constructs containing apoplast targeting sequences were tested along with different translational regulatory sequences such as TMV omega and RUBISCO untranslated regions. Tobacco plants and BY-2 cells transformed with cytosolic constructs showed levels of recombinant ADA of up to 80 ng mg-1 TSP. By comparison, transgenic calli expressing constructs containing apoplast-directing signals showed higher levels of recombinant ADA expression of up to 115 ng mg-1 TSP. The most significant ADA activities were measured in transgenic BY-2 cell suspensions, however. Where, incorporation of a signal for arabinogalactan addition at the C-terminus of the recombinant ADA gene, targeted for secretion, produced a maximum yield of approximately 13 mg L-1. Representing a 336-fold increase over ADA activities recorded in a BY-2 suspension transformed with a cytosolic counterpart.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral