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.
This is a preview of subscription content, log in via an institution to check access.
References
Angenon G, Van Montagu M, Depicker A (1990) Analysis of the stop codon context in plant nuclear genes. FEBS Lett 271(1–2):144–146
Booth C, Hershfield M, Notarangelo L, Buckley R, Hoenig M, Mahlaoui N, Cavazzana-Calvo M, Aiuti A, Gaspar HB (2006) Management options for adenosine deaminase deficiency; proceedings of the EBMT satellite workshop (Hamburg, March 2006). Clin Immunol 123(2):139–147
Bringloe DH, Dyer TA, Gray JC (1995) Developmental, circadian and light regulation of wheat ferredoxin gene expression. Plant Mol Biol 27:293–306
Chan B, Wara D, Bastian J, Hershfield MS, Bohnsack J, Azen CG, Parkman R, Weinberg K, Kohn DB (2005) Long-term efficacy of enzyme replacement therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). Clin Immunol 117(2):133–143
Dancer JE, Hughes RG, Lindell SD (1997) Adenosine-5′-phosphate deaminase: a novel herbicide target. Plant Physiol 114:119–129
DeBeuckeleer M, Lemmers M, Vos G, Willmitzer L, Montagu M, Schell J (1981) Further insight on the transferred-DNA of octopine crown gall. Mol Gen Genet 183:283–288
Fischer R, Emans N (2000) Molecular farming of pharmaceutical proteins. Transgenic Res 9:279–299
Gallie DR (2002) The 5′-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F’. Nucleic Acids Res 30(15):3401–3411
Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42(6):819–832
Hepburn AG, Clarke LE, Pearson L, White J (1983) The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J Mol Appl Genet 2:315–329
Horsch R, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Kelly MA, Vestling MM, Murphy CM, Hua S, Sumpter T, Fenselau C (1996) Primary structure of bovine adenosine deaminase. J Pharm Biomed Anal 14(11):1513–1519
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol 9:963–967
Medin JA, Hunt L, Gathy K, Evans RK, Coleman MS (1990) Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae. Proc Natl Acad Sci 87:2760–2764
Meins F (2000) RNA degradation and models for post-transcriptional gene silencing. Plant Mol Biol 43:261–273
Meyer P (2000) Transcriptional transgene silencing and chromatin components. Plant Mol Biol 43:221–234
Pelcher L (1995) Selectable/reporter gene for use during genetic engineering of plants and plant cells. United States Patent, 5474929
Polvino WJ, Saravis CA, Sampson CE, Cook RB (1983) Improved protein analysis on nitrocellulose membranes. Electrophoresis 4(5):368–369
Que Q, Wang HY, English JJ, Jorgensen RA (1997) The frequency and degree of co-suppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. Plant Cell 9(8):1357–1368
Schmitz J, Prüfer D, Rohde W, Tacke E (1996) Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence. Nucleic Acids Res 24(2):257–263
Schubert D, Lechtenberg B, Forsbach A, Gils M, Bahadur S, Schmidt R (2004) Silencing in Arabidopsis T-DNA transformants: the predominant role of a gene-specific RNA sensing mechanism versus position effects. Plant Cell 16(10):2561–2572
Shen WJ, Forde BG (1989) Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 17(20):8385
Springer PS, McCombie WR, Sundaresan V, Martienssen RA (1995) Gene trap tagging of PROLIFERA, an essential MCM2-3-5-like gene in Arabidopsis. Science 268:877–880
Stam M, De Bruijn R, Kenter S, Van Der Hoorn RAL, Van Blokland R, Mol JNM, Kooter JM (1997) Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats. Plant J 12:63–82
Vergunst AC, Jansen LET, Hooykaas PJJ (1998) Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucleic Acids Res 26:2729–2734
Wiginton DA, Adrian GS, Hutton JJ (1984) Sequence of human adenosine deaminase cDNA including the coding region and a small intron. Nucleic Acids Res 12(5):2439–2446
Wirth S, Calamante G, Mentaberry A, Bussmann L, Lattanzi M, Barañao L, Bravo-Almonacid F (2004) Expression of active human epidermal growth factor (hEGF) in tobacco plants by integrative and non-integrative systems. Mol Breed 13:23–35
Acknowledgments
S. Singhabahu was funded by a scholarship from the University of East London.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Singhabahu, S., George, J. & Bringloe, D. Expression of a functional human adenosine deaminase in transgenic tobacco plants. Transgenic Res 22, 643–649 (2013). https://doi.org/10.1007/s11248-012-9676-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-012-9676-1