Journal: Scientific Reports
Article Title: A novel secretion and online-cleavage strategy for production of cecropin A in Escherichia coli
doi: 10.1038/s41598-017-07411-5
Figure Lengend Snippet: The technical roadmap for extracellular secretion and online-cleavage of antimicrobial peptides on the surface of Escherichia coli . ( A ) Strategy for generating extracellular fiber-like aggregates based on the E. coli curli system. A curli-deficient mutant of E. coli BL21 (DE3) was constructed by deleting csgBAC and replacing them with a kanamycin resistance gene. pExport 1, encoding a C-M-sup35NM fusion protein, and pETG, directing the overexpression of CsgG, were introduced into strain BL21 (DE3) ∆ csgBAC . The recombinant bacteria were then cultured on YESCA plates supplemented with appropriate antibiotics and inducers at 25 °C for 3 d to produce fiber-like aggregates (blue fibers). The black box shows the composition of the fiber-like aggregates. ( B ) Model of secretion and assembly principle CsgG forms an ungated peptide diffusion channel in the cell outer membrane (OM). C-M-sup35NM fusions firstly pass through the inner membrane (IM) into the periplasm via the Sec-pathway, and are then secreted to the cell surface of BL21 (DE3) ∆ csgBAC in a CsgG- and CsgE-dependent manner. The secreted C-M-sup35NM fusions tend to form fiber-like aggregates because of the amyloid of sup35NM. CsgF associates with the OM and is required for adhesion of the C-M-sup35NM aggregates. ( C ) Schematic diagram of cleavage experiment mediated by Mxe GyrA BL21 (DE3) ∆ csgBAC harboring pExport1 and pETG were cultivated on YESCA plates supplemented with the appropriate antibiotics (100 µg/ml ampicillin; 50 µg/ml kanamycin; 25 µg/ml chloramphenicol) and inducers (0.05% [w/v] L-arabinose, 1 mM IPTG) at 25 °C for 3 d. Subsequently, cells were harvested and resuspended in buffer S1 (20 mM Tris-HCl, 500 mM NaCl, 1 mM disodium edetate (EDTA), pH 8.5) to 10 OD 600 culture/ml, followed by centrifugation at 7500 g for 15 min at 4 °C. The precipitates were washed twice with buffer S1, and resuspended in the same volume of buffer S2 (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA and 40 mM dithiothreitol (DTT), pH 8.5) for cleavage at 4 °C for 12 or 24 h. Cecropin A peptide (soluble) was released and collected by centrifugation at 11000 rpm for 30 min. The M-sup35 fusion (insoluble) formed part of the pellet. The black box shows the composition of the fiber-like aggregates.
Article Snippet: The antimicrobial activity of the bioproduced cecropin A peptide (equivalent to SCe) was determined using MIC tests against E. coli ATCC 25922 with chemically synthesized cecropin A used in controls.
Techniques: Mutagenesis, Construct, Over Expression, Recombinant, Bacteria, Cell Culture, Diffusion-based Assay, Membrane, Centrifugation
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ATCC manufactures this product 
![The technical roadmap for extracellular secretion and online-cleavage of antimicrobial peptides on the surface of <t>Escherichia</t> <t>coli</t> . ( A ) Strategy for generating extracellular fiber-like aggregates based on the E. coli curli system. A curli-deficient mutant of E. coli BL21 (DE3) was constructed by deleting csgBAC and replacing them with a kanamycin resistance gene. pExport 1, encoding a C-M-sup35NM fusion protein, and pETG, directing the overexpression of CsgG, were introduced into strain BL21 (DE3) ∆ csgBAC . The recombinant bacteria were then cultured on YESCA plates supplemented with appropriate antibiotics and inducers at 25 °C for 3 d to produce fiber-like aggregates (blue fibers). The black box shows the composition of the fiber-like aggregates. ( B ) Model of secretion and assembly principle CsgG forms an ungated peptide diffusion channel in the cell outer membrane (OM). C-M-sup35NM fusions firstly pass through the inner membrane (IM) into the periplasm via the Sec-pathway, and are then secreted to the cell surface of BL21 (DE3) ∆ csgBAC in a CsgG- and CsgE-dependent manner. The secreted C-M-sup35NM fusions tend to form fiber-like aggregates because of the amyloid of sup35NM. CsgF associates with the OM and is required for adhesion of the C-M-sup35NM aggregates. ( C ) Schematic diagram of cleavage experiment mediated by Mxe GyrA BL21 (DE3) ∆ csgBAC harboring pExport1 and pETG were cultivated on YESCA plates supplemented with the appropriate antibiotics (100 µg/ml ampicillin; 50 µg/ml kanamycin; 25 µg/ml chloramphenicol) and inducers (0.05% [w/v] L-arabinose, 1 mM IPTG) at 25 °C for 3 d. Subsequently, cells were harvested and resuspended in buffer S1 (20 mM Tris-HCl, 500 mM NaCl, 1 mM disodium edetate (EDTA), pH 8.5) to 10 OD 600 culture/ml, followed by centrifugation at 7500 g for 15 min at 4 °C. The precipitates were washed twice with buffer S1, and resuspended in the same volume of buffer S2 (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA and 40 mM dithiothreitol (DTT), pH 8.5) for cleavage at 4 °C for 12 or 24 h. Cecropin A peptide (soluble) was released and collected by centrifugation at 11000 rpm for 30 min. The M-sup35 fusion (insoluble) formed part of the pellet. The black box shows the composition of the fiber-like aggregates.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4755/pmc05544755/pmc05544755__41598_2017_7411_Fig1_HTML.jpg)
