Abstract
The production of recombinant proteins in plants is an active area of research and many different high-value proteins have now been produced in plants. Tobacco leaves have many advantages for recombinant protein production particularly since they allow field production without seeds, flowers or pollen and therefore provide for contained production. Despite these biosafety advantages recombinant protein accumulation in leaves still needs to be improved. Elastin-like polypeptides are repeats of the amino acids “VPGXG” that undergo a temperature dependant phase transition and have utility in the purification of recombinant proteins but can also enhance the accumulation of recombinant proteins they are fused to. We have used a 11.3 kDa elastin-like polypeptide as a fusion partner for three different target proteins, human interleukin-10, murine interleukin-4 and the native major ampullate spidroin protein 2 gene from the spider Nephila clavipes. In both transient analyses and stable transformants the concentrations of the fusion proteins were at least an order of magnitude higher for all of the fusion proteins when compared to the target protein alone. Therefore, fusions with a small ELP tag can be used to significantly enhance the accumulation of a range of different recombinant proteins in plant leaves.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
De Jaeger G, Scheffer S, Jacobs A, Zambre M, Zobell O, Goossens A, Depicker A, Angenon G (2002) Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat Biotechnol 20:1265–1268
Dohmann K, Wagner B, Horohov DW, Leibold W (2000) Expression and characterization of equine interleukin 2 and interleukin 4. Vet Immunol Immunopathol 77:243–256
Douette P, Navet R, Gerkens P, Levey D, Sluse FE (2005) Escherichia coli fusions carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL. Biochem Biophys Res Commun 333:686–693
Fox JL (2004) Puzzling industry response to ProdiGene fiasco. Nat Biotechnol 21:3–4
Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151
Guda C, Lee S-B, Daniell H (2000) Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts. Plant Cell Rep 19:257–262
Hondred D, Walker JM, Mathews DE, Vierstra RD (1999) Use of ubiquitin fusions to augment protein expression in transgenic plants. Plant Physiol 119:713–723
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
Janssen BJ, Gardner RC (1990) Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 14:61–72
Kapila J, de Rycke R, van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108
Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236:1299–1302
Kostal J, Mulchandani A, Gropp KE, Chen W (2003) A temperature responsive biopolymer for mercury remediation. Environ Sci Tech 37:4457–4462
Kusnadi AR, Nikolov ZL, Howard JA (1997) Production of recombinant proteins in transgenic plants: Practical considerations. Biotech Bioeng 56:473–484
Le HV, Ramanathan L, Labdon J, Mays-Ichinco CA, Syto R, Arai N, Hoy P, Takebe Y, Nagabhushan TL, Trotta P (1988) Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells. J Biol Chem 263:10817–10823
Ma JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van Dolleweerd C, Mostov K, Lehner T (1995) Generation and assembly of secretory antibodies in plants. Science 268:716–719
Ma S, Huang Y, Yin Z, Menassa R, Brandle JE, Jevnikar AM (2004) Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL4. Proc Natl Acad Sci USA 101:5680–5685
Macaulay J (2003) Biopharming: Growing medicine crops. Food Technol 57:20
Magnuson NS, Linzmaier M, Reeves R, An G, HayGlass K, Lee JM (1998) Secretion of biologically active interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture. Prot Express Purif 13:45–52
Mainieri D, Rossi M, Archinti M, Belluci M, De Marchis F, Vavassori S, Pompa A, Arcioni S, Vitale A (2004) Zeolin, a new recombinant storage protein constructed using maize γ-zein and bean phaseolin. Plant Physiol 136:3447–3456
Matsuoka M, Yamamoto N, Kano-Murakami Y, Tanaka Y, Ozeki Y, Hirano H, Kagawa H, Oshima M, Ohashi Y (1987) Classification and structural comparison of full-length cDNAs for pathogenesis-related proteins. Plant Physiol 85:942–946
Maximova SN, Dandekar AM, Guiltinan MJ (1998) Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate limiting. Plant Mol Biol 27:549–559
Menassa R, Nguyen V, Jevnikar A, Brandle J (2001) A self-contained system for the field production of plant recombinant interleukin-10. Mol Breed 8:177–185
Menassa R, Zhu H, Karatzas CN, Lazaris A, Richman A, Brandle J (2004) Spider dragline silk in transgenic tobacco leaves: accumulation and field production. Plant Biotech J 2:431–438
Merle C, Perret S, Lacour T, Jonval V, Hudaverdian S, Garrone R, Ruggiero F, Theisen M (2002) Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant. FEBS Lett 515:114–118
Meyer DE, Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotech 17:1112–1115
Nature Biotechnology (2004) Drugs in crops—the unpalatable truth. Nat Biotechnol 22:133
New Scientist (2005) Too tempting, there is just one problem with edible vaccines. New Sci 2487:3
Obregon P, Chargelegue D, Drake PMW, Prada A, Nuttall J, Frigerio L, Ma J K-C (2005). HIV-1 p24-immunoglobin fusion molecule: a new strategy for plant based protein production. Plant Biotechnol J 3:195–207
Raju K, Anwar RA (1987) Primary structures of bovine of bovine elastin a, b, and c deduced from the sequences of cDNA clones. J Biol Chem 262:5755–5762
Richter LJ, Thanavala Y, Arntzen CJ, Mason HS (2000) Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol 18:1167–1171
Rymerson RT, Menassa R, Brandle JE (2002) Tobacco, a platform for the production of recombinant proteins. In: Erickson L, Brandle J, Rymerson RT (eds), Molecular Farming of Plants and Animals for Human and Veterinary Medicine. Kluwer, Amsterdam
Scheller J, Gührs K-H, Grosse F, Conrad U (2001) Production of spider silk proteins in tobacco and potato. Nat Biotechnol 19:573–577
Scheller J, Henggeler D, Viviani A, Conrad U (2004) Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation. Trans Res 13:51–57
Scheller J, Leps M, Conrad U (2006) Forcing single chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides. Plant Biotechnol J 4:243–249
Shimazu M, Mulchandani A, Chen W (2003) Thermally triggered purification and immobilization of elastin-OPH fusions. Biotech Bioeng 81:74–79
Smith TA, Kohorn BD (1991) Direct selection for sequences encoding proteases of known specificity. Proc Natl Acad Sci USA 88:5159–5162
Sojikul P, Buehner N, Mason H (2003) A plant signal peptide-hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells. Proc Natl Acad Sci USA 100:2209–2214
Spiegel H, Schillberg S, Sack M, Holzem A, Nahring J, Monecke M, Liao YC, Fischer R (1999) Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells. Plant Sci 149:63–71
Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333-338
Stiborva H, Kostal J, Mulchandani A, Chen W (2003) One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation. Biotech Bioeng 82:605–611
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotech 60:523–533
Trabbic-Carlson K, Liu L, Kim B, Chilkoti A (2004) Expression and purification of recombinant proteins from Escherichia coli: comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Prot Sci 13:3274–3284
Twyman RM (2004) Host plant, systems and expression strategies for molecular farming. In: Fischer R, Schillberg S (eds) Molecular farming: plant made pharmaceuticals and technical proteins. Wiley-VCH, Wienheim, pp 191–216
Wu K, Malic K, Tian L, Hu M, Martin T, Foster E, Brown D, Miki B (2001) Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP. Mol Genet Genom 265:763–770
Yang YN, Li RG, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551
Author information
Authors and Affiliations
Corresponding author
Additional information
An erratum to this article can be found at http://dx.doi.org/10.1007/s11248-007-9071-5
Rights and permissions
About this article
Cite this article
Patel, J., Zhu, H., Menassa, R. et al. Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves. Transgenic Res 16, 239–249 (2007). https://doi.org/10.1007/s11248-006-9026-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-006-9026-2