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

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DOIResolve DOI: http://doi.org/10.1016/j.pep.2005.04.010
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TypeArticle
Journal titleProtein Expression and Purification
Volume45
Issue1
Pages8898; # of pages: 11
Subjectpleurocidin; antimicrobial peptide; peptide expression
AbstractThe 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.
Publication date
PublisherElsevier
Copyright noticeCopyright © 2005 Elsevier Inc. All rights reserved.
LanguageEnglish
AffiliationNational Research Council Canada; NRC Institute for Marine Biosciences; Aquatic and Crop Resource Development
Peer reviewedYes
NRC number42496
1485
NPARC number3538195
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Record identifier89d5a900-b201-4d52-a5c6-6f706f07bb8d
Record created2009-03-01
Record modified2016-05-09
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