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Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves

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An Erratum to this article was published on 15 February 2007

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.

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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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Fox JL (2004) Puzzling industry response to ProdiGene fiasco. Nat Biotechnol 21:3–4

    Article  CAS  Google Scholar 

  • Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Janssen BJ, Gardner RC (1990) Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 14:61–72

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Kostal J, Mulchandani A, Gropp KE, Chen W (2003) A temperature responsive biopolymer for mercury remediation. Environ Sci Tech 37:4457–4462

    Article  CAS  Google Scholar 

  • Kusnadi AR, Nikolov ZL, Howard JA (1997) Production of recombinant proteins in transgenic plants: Practical considerations. Biotech Bioeng 56:473–484

    Article  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Meyer DE, Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotech 17:1112–1115

    Article  CAS  Google Scholar 

  • Nature Biotechnology (2004) Drugs in crops—the unpalatable truth. Nat Biotechnol 22:133

    Google Scholar 

  • New Scientist (2005) Too tempting, there is just one problem with edible vaccines. New Sci 2487:3

    Google Scholar 

  • 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

    Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Shimazu M, Mulchandani A, Chen W (2003) Thermally triggered purification and immobilization of elastin-OPH fusions. Biotech Bioeng 81:74–79

    Article  CAS  Google Scholar 

  • Smith TA, Kohorn BD (1991) Direct selection for sequences encoding proteases of known specificity. Proc Natl Acad Sci USA 88:5159–5162

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotech 60:523–533

    CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Jim Brandle.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s11248-007-9071-5

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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

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  • DOI: https://doi.org/10.1007/s11248-006-9026-2

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