A C-terminal glycine suppresses production of pleurocidin as a fusion peptide in Escherichia coli.
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 alpha-helix that is thought to be important for activity.
Bryksa
Brian C
BC
NRC Institute for Marine Biosciences, National Research Council Canada, Halifax, NS, Canada B3H 3Z1.
MacDonald
Lisa D
LD
Patrzykat
Aleks
A
Douglas
Susan E
SE
Mattatall
Neil R
NR
D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005998:Glycine / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013997:Time Factors
D000890:Anti-Infective Agents; D029941:Fish Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C105851:pleurocidin; D005998:Glycine