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.
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References
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
Andreu D, Rivas L (1998) Animal antimicrobial peptides: an overview. Biopolymers 47(6):415–433
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
Basaran P, Rodriguez-Cerezo E (2008) Plant molecular farming: opportunities and challenges. Crit Rev Biotechnol 28(3):153–172. doi:10.1080/07388550802046624
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):7942–7950
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
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
Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3(3):238–250
Castro MS, Fontes W (2005) Plant defense and antimicrobial peptides. Protein Pept Lett 12(1):13–18
Chapman SN (1998) Tobamovirus isolation and RNA extraction. Methods Mol Biol 81:123–129. doi:10.1385/0-89603-385-6:123
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
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
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):71–87
Durham AC (1972) Structures and roles of the polymorphic forms of tobacco mosaic virus protein. I. Sedimentation studies. J Mol Biol 67(2):289–305
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
Eisenberg D (1984) Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem 53:595–623
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
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
Fontana A, Gross E (1986) Fragmentation of polypeptides by chemical methods. In: Darbre A (ed) Practical protein chemistry: a handbook. Wiley, Chester, pp 67–120
Fraenkel-Conrat H (1957) Degradation of tobacco mosaic virus with acetic acid. Virology 4(1):1–4. doi:10042-6822(57)90038-7
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):690–698
Fraenkel-Conrat H, Singer B, Williams RC (1957) Infectivity of viral nucleic acid. Biochim Biophys Acta 25(1):87–96
Fritig B, Heitz T, Legrand M (1998) Antimicrobial proteins in induced plant defense. Curr Opin Immunol 10(1):16–22
Garcia-Olmedo F, Molina A, Alamillo JM, Rodriguez-Palenzuela P (1998) Plant defense peptides. Biopolymers 47(6):479–491
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
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
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
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):5818–5822
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
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
Gooding GV Jr, Hebert TT (1967) A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57(11):1285
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):417–423
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
Jervis L, Pierpoint WS (1989) Purification technologies for plant proteins. J Biotechnol 11:161–198
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
Koprowski H, Yusibov V (2001) The green revolution: plants as heterologous expression vectors. Vaccine 19(17–19):2735–2741
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
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
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
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
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
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
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
Marshall SH, Arenas G (2003) Antimicrobial peptides: a natural alternative to chemical antibiotics and a potential for applied biotechnology. Electron J Biotechnol 6(3):271–284
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
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
Nedoluzhko A, Douglas T (2001) Ordered association of tobacco mosaic virus in the presence of divalent metal ions. J Inorg Biochem 84(3–4):233–240
Nelson M, McClelland M (1992) Use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes. Methods Enzymol 216:279–303
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
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
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
Papworth C, Braman J, Wright DA (1996) Site-directed mutagenesis in one day with > 80% efficiency. Strategies 9:3–4
Parish CL, Zaitlin M (1966) Defective tobacco mosaic virus strains: identification of the protein of strain PM1 in leaf homogenates. Virology 30(2):297–302
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
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
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
Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1(1):16–22. doi:10.1038/nprot.2006.4
Scheele RB, Lauffer MA (1967) Acid-base titrations of tobacco mosaic virus and tobacco mosaic virus protein. Biochemistry 6(10):3076–3081
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
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
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
Smith BJ (1994) Chemical cleavage of proteins. Methods Mol Biol 32:297–309. doi:10.1385/0-89603-268-X:297
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
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
Thomma BP, Cammue BP, Thevissen K (2002) Plant defensins. Planta 216(2):193–202
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
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):53–57
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
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
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):4390–4398
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
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):339–345
Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415(6870):389–395
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Zeitler, B., Bernhard, A., Meyer, H. et al. Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana . Plant Mol Biol 81, 259–272 (2013). https://doi.org/10.1007/s11103-012-9996-9
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DOI: https://doi.org/10.1007/s11103-012-9996-9