title: Expression of a functional human adenosine deaminase in transgenic tobacco plants.
abstract: An inherited disorder, adenosine deaminase 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 a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5' UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves.
authors.affiliation: School of Health, Sport and Bioscience, University of East London, Water Lane, London E15 4LZ, UK.
authors.forename: Sanjeewa
authors.initials: S
authors.lastname: Singhabahu
authors.forename: John
authors.initials: J
authors.lastname: George
authors.forename: David
authors.initials: D
authors.lastname: Bringloe
mesh_terms: D020121:5' Untranslated Regions; D000243:Adenosine Deaminase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015152:Blotting, Northern; D015139:Blotting, Southern; D005822:Genetic Vectors; D006801:Humans; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D011994:Recombinant Proteins; D000243:Adenosine Deaminase