title: Production of bioactive wheat puroindoline proteins in Nicotiana benthamiana using a virus-based transient expression system.
abstract: The emergence of antibiotic resistant pathogenic strains of bacteria has necessitated the development of novel antimicrobial agents. The puroindoline A and B (PINA and PINB) proteins of wheat, well-known for their roles in determining the important phenotype of grain texture, are also antimicrobial, making them attractive as natural bio-control agents. However, the biochemical basis of PIN functionality remains unclear due to limitations in expressing them at the required yield and purity and lack of accurate tertiary structure. This study focussed on rapid transient expression of PINs targeted to different subcellular compartments (chloroplast, apoplast, endoplasmic reticulum and cytosol) of Nicotiana benthamiana leaf cells using the deconstructed tobacco mosaic virus-based 'magnICON®' system. The expressed recombinant PINs were characterised by Western blot using the Durotest anti-friabilin antibody, enzyme-linked immunosorbent assays (ELISA) and antimicrobial activity tests. Maximum yield of the His-tagged PINs occurred when targeted to the chloroplast. Both PINs exhibited oligomeric and monomeric forms on gels, but Western blots with the widely used Durotest anti-friabilin antibody identified only oligomeric forms. Only the PINs purified by a hydrophobic interaction method exhibited monomeric forms with the anti-His tag antibody, indicating correct folding. Interestingly, the Durotest antibody did not bind to monomers, suggesting their epitope may be obscured. PINs purified by His-tag affinity purification under native conditions or by the hydrophobic method exhibited antimicrobial activities. The successful in planta expression and optimisation of purification will enable future studies to examine the detailed structure of the PINs and explore novel bio-control applications in health, food and/or agriculture.
authors.affiliation: Faculty of Science, Engineering and Technology, Swinburne University of Technology, Melbourne, Victoria, Australia.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Niknejad
authors.affiliation: School of Biological Sciences, Monash University, Melbourne, Victoria, Australia.
authors.forename: Diane
authors.initials: D
authors.lastname: Webster
authors.affiliation: Faculty of Science, Engineering and Technology, Swinburne University of Technology, Melbourne, Victoria, Australia. Electronic address: mbhave@swin.edu.au.
authors.forename: Mrinal
authors.initials: M
authors.lastname: Bhave
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D002736:Chloroplasts / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli; D005658:Fungi / Q000187:drug effects; D018506:Gene Expression Regulation, Plant; D057927:Hydrophobic and Hydrophilic Interactions; D010641:Phenotype; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014026:Nicotiana / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000235:genetics*; D014908:Triticum / Q000235:genetics / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; C082942:puroindoline protein, Triticum aestivum
keywords: Antimicrobial proteins; Grain texture; Puroindolines; magnICON® system