Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana
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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.
KeywordsAntimicrobial peptides Tobacco mosaic virus Designed peptide Fusion protein Plant transformation Molecular farming
Conflict of interest
The authors declare that they have no conflict of interest.
- 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
- 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
- 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
- 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
- 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
- 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
- Papworth C, Braman J, Wright DA (1996) Site-directed mutagenesis in one day with > 80% efficiency. Strategies 9:3–4Google Scholar
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
- 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
- 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