Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies

Abstract

BackgroundThe 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.ResultsTo 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.ConclusionWe 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.

Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.)

“…coli BL21, and the soluble component was obtained. The expression of AMPs as a thioredoxin fusion protein enhances protein quantity and solubility [47]. Numerous studies successfully obtained recombinant AMPs using the Trx-fusion system [46, 47].…”

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

“…Lactic acid bacteria (LAB) comprise a large community of Gram-positive microorganisms, including members of the genera Lactococcus , Lactobacillus , Pediococcus , Streptococcus , Leuconostoc , and Oenococcus , that are used for different applications. Most of these bacteria, which have been used in food production for a long time, are deemed suitable for human consumption, and thus have GRAS status [ 63 , 64 ]. Owing to its simple handling, sequenced genome, and the advancement of genetic techniques in recent years, Lactococcus lactis is the best described species of this group and is characterized as the model organism.…”

Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies

“…Other strategies involving the fusion of StSN1 to the protein Glutathione S-transferase (GST) led to strong expression of the recombinant protein, but also in the form of IB [ 17 , 18 ]. Meiyalaghan et al [ 19 ] expressed StSN1 as a fusion to thioredoxin and obtained adequate quantities of soluble protein. However, the authors did not report the separation from thioredoxin.…”

“…This synthesis involves a laborious work using a combination of solid-phase synthesis, native chemical ligation and a subsequent cysteine/cysteine mediated oxidative folding to form the six internal disulfide bonds [ 5 ]. In addition, Meiyalaghan et al [ 19 ] reported that mature lyophilized commercially synthesized StSN1 could not be solubilized due to the structural nature of the peptides. These observations highlight the difficulty of working with Snakin/GASA peptides.…”

“…Rong et al [ 17 ] produced polyclonal GST-SN1 antibodies in mouse to develop immunoblots for the characterization of transgenic wheat lines. Similarly, Meiyalaghan et al [ 19 ] obtained antibodies against the fusion protein his6-thioredoxin-SN1. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognized the StSN1 peptide that had been expressed in a wheat germ cell-free expression system, but cross reactive bands were also evident [ 19 ].…”