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Volume 3
Journal of Pharmacology and Medicinal Chemistry
Nanomedicine 2019
Biotechnology 2019
May 20-21, 2019
May 20-21, 2019 London, UK
4
th
World Biotechnology CONGRESS
Nanomedicine and Nanotechnology
4
th
International Conference on
&
Plant-based strategies aimed at expressing a synthetic human adenosine deaminase at
high levels
David Bringloe
University of East London, UK
A
n 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, synthetic 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
compartmental 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 97 ng mg-1 TSP.
By comparison, transgenic calli expressing constructs containing apoplast-directing signals showed higher levels of recombinant
ADA expression of up to 140 ng mg-1 TSP. The most significant ADA activities, however, were measured in the media of
transgenic BY-2 cell suspensions prepared from transformed calli: where incorporation of a signal for arabinogalactan addition to
ADA, led to a recombinant protein yield of approximately 16 mg L-1. A 336-fold increase over ADA produced by cell suspensions
transformed with a cytosolic construct.
Biography
David Bringloe has completed his PhD and his current research interests involve heterologous gene expression systems and plant
biotechnology, his main focus is to control of foreign gene expression in plants, particularly the production of therapeutic proteins and
now also prions. To date, plant-based strategies have been employed to express various therapeutic enzymes and proteins at high
levels in whole plants and plant cell suspensions.
d.h.bringloe@uel.ac.ukJ Pharmacol Med Cheml, Volume 3