High-yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions.
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 with polyethylene glycol (PEG). 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 was 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 L(-1) , a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct.
Human Genetics Unit, Faculty of Medicine, University of Colombo, Kynsey Road, Colombo 8, Sri Lanka.
Sanjeewa
S
Singhabahu
John
J
George
David
D
Bringloe
D000243:Adenosine Deaminase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D030821:Plants, Genetically Modified; D013535:Suspensions; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics*; D014170:Transformation, Genetic
D018076:DNA, Complementary; D013535:Suspensions; D000243:Adenosine Deaminase
adenosine deaminase; apoplast; molecular pharming; plant cell culture; recombinant protein; tobacco