High yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions


Sanjeewa Singhabahu, John George and David Bringloe


Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind a human ADA cDNA has been inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA, led to a recombinant protein yield of approximately 16 mg Lāˆ’1, 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct.