title: Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana.
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.
authors.affiliation: Institute of Biochemical Plant Pathology, Helmholtz Zentrum Munich, German Research Center for Environmental Health, 85764, Munich, Neuherberg, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Zeitler
authors.forename: Antonie
authors.initials: A
authors.lastname: Bernhard
authors.forename: Helge
authors.initials: H
authors.lastname: Meyer
authors.forename: Michael
authors.initials: M
authors.lastname: Sattler
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Koop
authors.forename: Christian
authors.initials: C
authors.lastname: Lindermayr
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D036022:Capsid Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D007136:Immunoglobulins / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D058634:Molecular Farming / Q000379:methods*; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D016973:Pectobacterium carotovorum / Q000187:drug effects; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000821:virology; D030821:Plants, Genetically Modified; D011549:Pseudomonas / Q000187:drug effects; D011993:Recombinant Fusion Proteins; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014771:Virion / Q000302:isolation & purification; D044184:Xanthomonas vesicatoria / Q000187:drug effects
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D036022:Capsid Proteins; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins; C090698:SP1 antigen