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A C-terminal glycine suppresses production of pleurocidin as a fusion peptide in Escherichia coli

Par Conseil national de recherches du Canada

DOITrouver le DOI : https://doi.org/10.1016/j.pep.2005.04.010
AuteurRechercher : 1; Rechercher : 1; Rechercher : 2; Rechercher : 1; Rechercher : 1
Affiliation du nom
  1. Conseil national de recherches du Canada. Institut des biosciences marines du CNRC
  2. Conseil national de recherches du Canada. Développement des cultures et des ressources aquatiques
FormatTexte, Article
Sujetpleurocidin; antimicrobial peptide; peptide expression
Résumé
The winter flounder (Pseudopleuronectes americanus) antimicrobial peptide pleurocidin was produced in Escherichia coli using a synthetic gene constructed by PCR. The gene expresses pleurocidin from pET21a fused to the C-terminus of an insoluble carrier peptide. Once expressed, the fusion peptide formed inclusion bodies in the cytoplasm that were collected, solubilized in guanidine–HCl, and chemically cleaved using hydroxylamine at a unique asparaginyl-glycyl dipeptide. This released recombinant pleurocidin (r-pleurocidin), which was purified using ultrafiltration followed by reverse phase chromatography. The r-pleurocidin peptide resolved as a single band (2.7 kDa) when analyzed by Tris–Tricine buffered SDS–PAGE, and its amino acid sequence was confirmed using tandem mass spectrometry. Extending the pleurocidin sequence with a C-terminal glycine (r-pleurocidin-G) suppressed production of the fusion peptide 15-fold. When pleurocidin was extended further to include aspartate (r-pleurocidin-GD), the same effect was observed, and when pleurocidin was extended with aspartate alone, no effect was observed. Expression of fusion peptide containing either r-pleurocidin-G or r-pleurocidin-GD with low concentrations of inductant caused E. coli to enter stationary phase prematurely, but did not affect overall growth rates. A partial production recovery of r-pleurocidin-G was achieved by inducing expression in stationary phase cells. We observed r-pleurocidin-G to have enhanced antimicrobial activity compared with r-pleurocidin, and we propose that this activity interferes with E. coli metabolism during expression. This antimicrobial effect is probably facilitated by residual solubility of the fusion peptide and by a C-terminal cap structure, which stabilizes the r-pleurocidin-G ?-helix that is thought to be important for activity.
Date de publication
Conditions d’utilisation
  • Copyright © 2005 Elsevier Inc. All rights reserved.
Dans
  • Protein Expression and Purification 45, nº 1 (janvier 2006) : 88–98.
Langueanglais
Publications évaluées par des pairsOui
Numéro du CNRCNRCC 42496
1485
Numéro NPARC3538195
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Identificateur de l’enregistrement89d5a900-b201-4d52-a5c6-6f706f07bb8d
Enregistrement créé2009-03-01
Enregistrement modifié2020-04-22
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