Expressing an antibacterial protein in bacteria for raising antibodies

Protein Expr Purif. 2004 Jan;33(1):153-9. doi: 10.1016/j.pep.2003.08.026.

Abstract

Magainins are small peptides with broad-spectrum activity against a range of plant and animal microbial pathogens. To detect magainin peptides in applications such as Western blot analysis and enzyme-linked immunosorbent assays, specific antibodies that recognize magainin peptides are required. The production of antibodies against small peptides injected into host animals poses problems with respect to eliciting an adequate immunogenic response due to the small size of the molecules. To increase the immunogenicity of a target peptide, it may be expressed as part of a larger fusion protein. However, expression of an antimicrobial peptide in bacteria may be cytotoxic to the host or subjected to degradation by host-derived peptidases. To overcome these potential problems, we fused the DNA coding sequence of a magainin gene analogue within the sequence of a bacterial thioredoxin gene. The subsequent gene fusion comprising a bacterial thioredoxin gene with a magainin coding sequence ligated at the active site of thioredoxin was successfully translated in a bacterial expression system. The fusion protein was non-toxic to the host bacteria. This represents a novel strategy to express antimicrobial peptides in a bacterial expression system. The fusion protein, purified by molecular size separation, was recovered in a soluble form following electroelution from polyacrylamide gels. Sufficient fusion protein was obtained for injection into rabbits and antibodies were obtained from rabbit sera that selectively recognized magainin peptides in Western blot analysis.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Bacterial / biosynthesis*
  • Antibodies, Bacterial / immunology*
  • Antimicrobial Cationic Peptides / biosynthesis*
  • Antimicrobial Cationic Peptides / genetics
  • Antimicrobial Cationic Peptides / immunology*
  • Antimicrobial Cationic Peptides / isolation & purification
  • Blotting, Western
  • Electrophoresis / methods
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors / genetics
  • Magainins
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / isolation & purification
  • Thioredoxins / biosynthesis
  • Thioredoxins / genetics
  • Xenopus Proteins*

Substances

  • Antibodies, Bacterial
  • Antimicrobial Cationic Peptides
  • Magainins
  • Recombinant Fusion Proteins
  • Xenopus Proteins
  • magainin 2 peptide, Xenopus
  • Thioredoxins