Plant-based strategies aimed at expressing a functional human adenosine deaminase at high levels

PhD Thesis


Singhabahu, S. A. D. Sanjeewa 2012. Plant-based strategies aimed at expressing a functional human adenosine deaminase at high levels. PhD Thesis University of East London School of Health, Sports and Biosciences
AuthorsSinghabahu, S. A. D. Sanjeewa
TypePhD Thesis
Abstract

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.

KeywordsImmunodeficiency; ADA deficiency; Enzyme replacement therapy protocol
Year2012
Digital Object Identifier (DOI)doi:10.15123/PUB.1889
Publication dates
PrintOct 2012
Publication process dates
Deposited15 Apr 2013
Publisher's version
License
CC BY-ND
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https://repository.uel.ac.uk/item/85y85

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