title: Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.
abstract: BACKGROUND
  The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts.
  
  
  RESULTS
  To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system.
  
  
  CONCLUSIONS
  We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.
authors.forename: Sathiyamoorthy
authors.initials: S
authors.lastname: Meiyalaghan
authors.forename: Julie M
authors.initials: JM
authors.lastname: Latimer
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Kralicek
authors.forename: Martin L
authors.initials: ML
authors.lastname: Shaw
authors.forename: John G
authors.initials: JG
authors.lastname: Lewis
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Conner
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, Private Bag 4704, Christchurch, New Zealand. philippa.barrell@plantandfood.co.nz.
authors.forename: Philippa J
authors.initials: PJ
authors.lastname: Barrell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood*; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000096:biosynthesis / Q000302:isolation & purification; D007275:Injections, Intravenous; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis / Q000302:isolation & purification; D010940:Plant Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013879:Thioredoxins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D000906:Antibodies; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C117071:SN1 protein, Solanum tuberosum; D006639:Histidine; D013879:Thioredoxins