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Plant Molecular Biology

, Volume 81, Issue 3, pp 259–272 | Cite as

Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana

  • Benjamin Zeitler
  • Antonie Bernhard
  • Helge Meyer
  • Michael Sattler
  • Hans-Ulrich Koop
  • Christian LindermayrEmail author
Article

Abstract

Antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. We designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. Especially peptide SP1-1 is highly active with a MIC value of 0.1 μg/ml against Xanthomonas vesicatoria, Pseudomonas corrugata and Pseudomonas syringae pv syringae. However, for commercial applications high amounts of peptide are necessary. The synthetic production of peptides is still quite expensive and, depending on the physico-chemical features, difficult. Therefore we developed a plant/tobacco mosaic virus-based production system following the ‘full virus vector strategy’ with the viral coat protein as fusion partner for the designed antimicrobial peptide. Infection of Nicotiana benthamiana plants with such recombinant virus resulted in production of huge amounts of virus particles presenting the peptides all over their surface. After extraction of recombinant virions, peptides were released from the coat protein by chemical cleavage. A protocol for purification of the antimicrobial peptides using high resolution chromatographic methods has been established. Finally, we yielded up to 0.025 mg of peptide per g of infected leaf biomass. Mass spectrometric and NMR analysis revealed that the in planta produced peptide differs from the synthetic version only in missing of N-terminal amidation. But its antimicrobial activity was in the range of the synthetic one. Taken together, we developed a protocol for plant-based production and purification of biologically active, hydrophobic and positively charged antimicrobial peptide.

Keywords

Antimicrobial peptides Tobacco mosaic virus Designed peptide Fusion protein Plant transformation Molecular farming 

Notes

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2012_9996_MOESM1_ESM.ppt (1.2 mb)
Supplementary material 1 (PPT 1248 kb)
11103_2012_9996_MOESM2_ESM.doc (78 kb)
Supplementary material 2 (DOC 78 kb)

References

  1. Ajikumar PK, Devaky KS (2001) Solid phase synthesis of hydrophobic difficult sequence peptides on BDDMA-PS support. J Pept Sci 7(12):641–649. doi: 10.1002/psc.355 PubMedCrossRefGoogle Scholar
  2. Andreu D, Rivas L (1998) Animal antimicrobial peptides: an overview. Biopolymers 47(6):415–433PubMedCrossRefGoogle Scholar
  3. Asurmendi S, Berg RH, Smith TJ, Bendahmane M, Beachy RN (2007) Aggregation of TMV CP plays a role in CP functions and in coat-protein-mediated resistance. Virology 366(1):98–106. doi: 10.1016/j.virol.2007.03.014 PubMedCrossRefGoogle Scholar
  4. Basaran P, Rodriguez-Cerezo E (2008) Plant molecular farming: opportunities and challenges. Crit Rev Biotechnol 28(3):153–172. doi: 10.1080/07388550802046624 PubMedCrossRefGoogle Scholar
  5. Bendahmane M, Fitchen JH, Zhang G, Beachy RN (1997) Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance. J Virol 71(10):7942–7950PubMedGoogle Scholar
  6. Bendahmane M, Koo M, Karrer E, Beachy RN (1999) Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions. J Mol Biol 290(1):9–20. doi: 10.1006/jmbi.1999.2860 PubMedCrossRefGoogle Scholar
  7. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM (2010) Seed-based expression systems for plant molecular farming. Plant Biotechnol J 8(5):588–606. doi: 10.1111/j.1467-7652.2010.00511.x PubMedCrossRefGoogle Scholar
  8. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3(3):238–250PubMedCrossRefGoogle Scholar
  9. Castro MS, Fontes W (2005) Plant defense and antimicrobial peptides. Protein Pept Lett 12(1):13–18PubMedGoogle Scholar
  10. Chapman SN (1998) Tobamovirus isolation and RNA extraction. Methods Mol Biol 81:123–129. doi: 10.1385/0-89603-385-6:123 PubMedGoogle Scholar
  11. Condron MM, Monien BH, Bitan G (2008) Synthesis and purification of highly hydrophobic peptides derived from the C-terminus of amyloid beta-protein. Open Biotechnol J 2(1):87–93. doi: 10.2174/1874070700802010087 PubMedCrossRefGoogle Scholar
  12. Crimmins DL, Mische SM, Denslow ND (2005) Chemical cleavage of proteins in solution. Curr Protoc Protein Sci Chapter 11:Unit 11 14. doi: 10.1002/0471140864.ps1104s40
  13. Dathe M, Wieprecht T (1999) Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim Biophys Acta 1462(1–2):71–87PubMedGoogle Scholar
  14. Durham AC (1972) Structures and roles of the polymorphic forms of tobacco mosaic virus protein. I. Sedimentation studies. J Mol Biol 67(2):289–305PubMedCrossRefGoogle Scholar
  15. Ehrenfeld N, Gonzalez A, Canon P, Medina C, Perez-Acle T, Arce-Johnson P (2008) Structure-function relationship between the tobamovirus TMV-Cg coat protein and the HR-like response. J Gen Virol 89(Pt 3):809–817. doi: 10.1099/vir.0.83355-0 PubMedCrossRefGoogle Scholar
  16. Eisenberg D (1984) Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem 53:595–623PubMedCrossRefGoogle Scholar
  17. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7(2):152–158. doi: 10.1016/j.pbi.2004.01.007 PubMedCrossRefGoogle Scholar
  18. Fitchen J, Beachy RN, Hein MB (1995) Plant virus expressing hybrid coat protein with added murine epitope elicits autoantibody response. Vaccine 13(12):1051–1057. doi: 10264410X9500075C PubMedCrossRefGoogle Scholar
  19. Fontana A, Gross E (1986) Fragmentation of polypeptides by chemical methods. In: Darbre A (ed) Practical protein chemistry: a handbook. Wiley, Chester, pp 67–120Google Scholar
  20. Fraenkel-Conrat H (1957) Degradation of tobacco mosaic virus with acetic acid. Virology 4(1):1–4. doi: 10042-6822(57)90038-7 PubMedCrossRefGoogle Scholar
  21. Fraenkel-Conrat H, Williams RC (1955) Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components. Proc Natl Acad Sci U S A 41(10):690–698PubMedCrossRefGoogle Scholar
  22. Fraenkel-Conrat H, Singer B, Williams RC (1957) Infectivity of viral nucleic acid. Biochim Biophys Acta 25(1):87–96PubMedCrossRefGoogle Scholar
  23. Fritig B, Heitz T, Legrand M (1998) Antimicrobial proteins in induced plant defense. Curr Opin Immunol 10(1):16–22PubMedCrossRefGoogle Scholar
  24. Garcia-Olmedo F, Molina A, Alamillo JM, Rodriguez-Palenzuela P (1998) Plant defense peptides. Biopolymers 47(6):479–491PubMedCrossRefGoogle Scholar
  25. Gennaro R, Zanetti M (2000) Structural features and biological activities of the cathelicidin-derived antimicrobial peptides. Biopolymers 55(1):31–49. doi: 10.1002/1097-0282(2000)55:1<31:AID-BIP40>3.0.CO;2-9 PubMedCrossRefGoogle Scholar
  26. Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18(11):1151–1155. doi: 10.1038/81132 PubMedCrossRefGoogle Scholar
  27. Gleba Y, Klimyuk V, Marillonnet S (2007) Viral vectors for the expression of proteins in plants. Curr Opin Biotechnol 18(2):134–141. doi: 10.1016/j.copbio.2007.03.002 PubMedCrossRefGoogle Scholar
  28. Goelet P, Lomonossoff GP, Butler PJ, Akam ME, Gait MJ, Karn J (1982) Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A 79(19):5818–5822PubMedCrossRefGoogle Scholar
  29. Goldstein DA, Thomas JA (2004) Biopharmaceuticals derived from genetically modified plants. QJM: Monthly Journal of the Association of Physicians 97(11):705–716. doi: 10.1093/qjmed/hch121 PubMedCrossRefGoogle Scholar
  30. Goodin MM, Zaitlin D, Naidu RA, Lommel SA (2008) Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. Mol Plant Microbe Interact 21(8):1015–1026. doi: 10.1094/MPMI-21-8-1015 PubMedCrossRefGoogle Scholar
  31. Gooding GV Jr, Hebert TT (1967) A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57(11):1285PubMedGoogle Scholar
  32. Holt CA, Hodgson RA, Coker FA, Beachy RN, Nelson RS (1990) Characterization of the masked strain of tobacco mosaic virus: identification of the region responsible for symptom attenuation by analysis of an infectious cDNA clone. Mol Plant Microbe Interact 3(6):417–423PubMedCrossRefGoogle Scholar
  33. Horn ME, Woodard SL, Howard JA (2004) Plant molecular farming: systems and products. Plant Cell Rep 22(10):711–720. doi: 10.1007/s00299-004-0767-1 PubMedCrossRefGoogle Scholar
  34. Jervis L, Pierpoint WS (1989) Purification technologies for plant proteins. J Biotechnol 11:161–198CrossRefGoogle Scholar
  35. Kaiser R, Metzka L (1999) Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds. Anal Biochem 266(1):1–8. doi: 10.1006/abio.1998.2945 PubMedCrossRefGoogle Scholar
  36. Koprowski H, Yusibov V (2001) The green revolution: plants as heterologous expression vectors. Vaccine 19(17–19):2735–2741PubMedCrossRefGoogle Scholar
  37. Kusnadi AR, Nikolov ZL, Howard JA (1997) Production of recombinant proteins in transgenic plants: practical considerations. Biotechnol Bioeng 56(5):473–484. doi: 10.1002/(SICI)1097-0290(19971205)56:5<473:AID-BIT1>3.0.CO;2-F PubMedCrossRefGoogle Scholar
  38. Li Q, Li M, Jiang L, Zhang Q, Song R, Xu Z (2006) TMV recombinants encoding fused foreign transmembrane domains to the CP subunit caused local necrotic response on susceptible tobacco. Virology 348(2):253–259. doi: 10.1016/j.virol.2005.11.013 PubMedCrossRefGoogle Scholar
  39. Lico C, Chen Q, Santi L (2008) Viral vectors for production of recombinant proteins in plants. J Cell Physiol 216(2):366–377. doi: 10.1002/jcp.21423 PubMedCrossRefGoogle Scholar
  40. Lico C, Santi L, Twyman RM, Pezzotti M, Avesani L (2012) The use of plants for the production of therapeutic human peptides. Plant Cell Rep 31(3):439–451. doi: 10.1007/s00299-011-1215-7 PubMedCrossRefGoogle Scholar
  41. Lindbo JA (2007) TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiol 145(4):1232–1240. doi: 10.1104/pp.107.106377 PubMedCrossRefGoogle Scholar
  42. Ma JK, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4(10):794–805. doi: 10.1038/nrg1177 PubMedCrossRefGoogle Scholar
  43. Ma JK, Barros E, Bock R, Christou P, Dale PJ, Dix PJ, Fischer R, Irwin J, Mahoney R, Pezzotti M, Schillberg S, Sparrow P, Stoger E, Twyman RM (2005) Molecular farming for new drugs and vaccines. Current perspectives on the production of pharmaceuticals in transgenic plants. EMBO Rep 6(7):593–599. doi: 10.1038/sj.embor.7400470 PubMedCrossRefGoogle Scholar
  44. Marshall SH, Arenas G (2003) Antimicrobial peptides: a natural alternative to chemical antibiotics and a potential for applied biotechnology. Electron J Biotechnol 6(3):271–284CrossRefGoogle Scholar
  45. McCormick AA, Corbo TA, Wykoff-Clary S, Nguyen LV, Smith ML, Palmer KE, Pogue GP (2006) TMV-peptide fusion vaccines induce cell-mediated immune responses and tumor protection in two murine models. Vaccine 24(40–41):6414–6423. doi: 10.1016/j.vaccine.2006.06.003 PubMedCrossRefGoogle Scholar
  46. Montesinos E (2007) Antimicrobial peptides and plant disease control. FEMS Microbiol Lett 270(1):1–11. doi: 10.1111/j.1574-6968.2007.00683.x PubMedCrossRefGoogle Scholar
  47. Nedoluzhko A, Douglas T (2001) Ordered association of tobacco mosaic virus in the presence of divalent metal ions. J Inorg Biochem 84(3–4):233–240PubMedCrossRefGoogle Scholar
  48. Nelson M, McClelland M (1992) Use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes. Methods Enzymol 216:279–303PubMedCrossRefGoogle Scholar
  49. Nilsson MR, Nguyen LL, Raleigh DP (2001) Synthesis and purification of amyloidogenic peptides. Anal Biochem 288(1):76–82. doi: 10.1006/abio.2000.4887 PubMedCrossRefGoogle Scholar
  50. Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29(2):210–222. doi: 10.1016/j.biotechadv.2010.11.004 PubMedCrossRefGoogle Scholar
  51. Palmer KE, Benko A, Doucette SA, Cameron TI, Foster T, Hanley KM, McCormick AA, McCulloch M, Pogue GP, Smith ML, Christensen ND (2006) Protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing L2 capsid epitopes. Vaccine 24(26):5516–5525. doi: 10.1016/j.vaccine.2006.04.058 PubMedCrossRefGoogle Scholar
  52. Papworth C, Braman J, Wright DA (1996) Site-directed mutagenesis in one day with > 80% efficiency. Strategies 9:3–4Google Scholar
  53. Parish CL, Zaitlin M (1966) Defective tobacco mosaic virus strains: identification of the protein of strain PM1 in leaf homogenates. Virology 30(2):297–302PubMedCrossRefGoogle Scholar
  54. Pogue GP, Lindbo JA, Garger SJ, Fitzmaurice WP (2002) Making an ally from an enemy: plant virology and the new agriculture. Annu Rev Phytopathol 40:45–74. doi: 10.1146/annurev.phyto.40.021102.150133 PubMedCrossRefGoogle Scholar
  55. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  56. Sampson WR, Patsiouras H, Ede NJ (1999) The synthesis of ‘difficult’ peptides using 2-hydroxy-4-methoxybenzyl or pseudoproline amino acid building blocks: a comparative study. J Pept Sci 5(9):403–409. doi: 10.1002/(SICI)1099-1387(199909)5:9<403:AID-PSC213>3.0.CO;2-S PubMedCrossRefGoogle Scholar
  57. Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1(1):16–22. doi: 10.1038/nprot.2006.4 PubMedCrossRefGoogle Scholar
  58. Scheele RB, Lauffer MA (1967) Acid-base titrations of tobacco mosaic virus and tobacco mosaic virus protein. Biochemistry 6(10):3076–3081PubMedCrossRefGoogle Scholar
  59. Schillberg S, Fischer R, Emans N (2003) ‘Molecular farming’ of antibodies in plants. Naturwissenschaften 90(4):145–155. doi: 10.1007/s00114-002-0400-5 PubMedGoogle Scholar
  60. Shinmyo A, Kato K (2010) Molecular farming: production of drugs and vaccines in higher plants. J Antibiot (Tokyo) 63(8):431–433. doi: 10.1038/ja.2010.63 CrossRefGoogle Scholar
  61. Simmaco M, Mignogna G, Barra D (1998) Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers 47(6):435–450. doi: 10.1002/(SICI)1097-0282(1998)47:6<435:AID-BIP3>3.0.CO;2-8 PubMedCrossRefGoogle Scholar
  62. Smith BJ (1994) Chemical cleavage of proteins. Methods Mol Biol 32:297–309. doi: 10.1385/0-89603-268-X:297 PubMedGoogle Scholar
  63. Smith ML, Fitzmaurice WP, Turpen TH, Palmer KE (2009) Display of peptides on the surface of tobacco mosaic virus particles. Curr Top Microbiol Immunol 332:13–31. doi: 10.1007/978-3-540-70868-1_2 PubMedCrossRefGoogle Scholar
  64. Takamatsu N, Watanabe Y, Yanagi H, Meshi T, Shiba T, Okada Y (1990) Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector. FEBS Lett 269(1):73–76. doi: 0014-5793(90)81121-4 PubMedCrossRefGoogle Scholar
  65. Thomma BP, Cammue BP, Thevissen K (2002) Plant defensins. Planta 216(2):193–202PubMedCrossRefGoogle Scholar
  66. Turpen TH (1999) Tobacco mosaic virus and the virescence of biotechnology. Philos Trans R Soc Lond B Biol Sci 354(1383):665–673. doi: 10.1098/rstb.1999.0419 PubMedCrossRefGoogle Scholar
  67. Turpen TH, Reinl SJ, Charoenvit Y, Hoffman SL, Fallarme V, Grill LK (1995) Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology (N Y) 13(1):53–57CrossRefGoogle Scholar
  68. van ‘t Hof W, Veerman EC, Helmerhorst EJ, Amerongen AV (2001) Antimicrobial peptides: properties and applicability. Biol Chem 382(4):597–619. doi: 10.1515/BC.2001.072 Google Scholar
  69. Werner S, Marillonnet S, Hause G, Klimyuk V, Gleba Y (2006) Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A. Proc Natl Acad Sci U S A 103(47):17678–17683. doi: 10.1073/pnas.0608869103 PubMedCrossRefGoogle Scholar
  70. Wu L, Jiang L, Zhou Z, Fan J, Zhang Q, Zhu H, Han Q, Xu Z (2003) Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector. Vaccine 21(27–30):4390–4398PubMedCrossRefGoogle Scholar
  71. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55(1):27–55. doi: 10.1124/pr.55.1.2 PubMedCrossRefGoogle Scholar
  72. Yuan JM, Hsiung LM, Gagnon J (1986) CNBr cleavage of the light chain of human complement factor I and alignment of the fragments. Biochem J 233(2):339–345PubMedGoogle Scholar
  73. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415(6870):389–395PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Benjamin Zeitler
    • 1
  • Antonie Bernhard
    • 1
  • Helge Meyer
    • 2
    • 3
  • Michael Sattler
    • 2
    • 3
  • Hans-Ulrich Koop
    • 4
  • Christian Lindermayr
    • 1
    Email author
  1. 1.Institute of Biochemical Plant PathologyHelmholtz Zentrum Munich, German Research Center for Environmental HealthMunich, NeuherbergGermany
  2. 2.Institute of Structural BiologyHelmholtz Zentrum Munich, German Research Center for Environmental HealthMunich, NeuherbergGermany
  3. 3.Munich Center for Integrated Protein Science at Chair of Biomolecular NMR, Department ChemieTechnische Universität MünchenGarchingGermany
  4. 4.Department I, Botany, Faculty of BiologyBiozentrum Ludwig-Maximilians-Universität MünchenPlanegg, MartinsriedGermany

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