Novel antibiotic alternatives

a technology of antibiotics and alternatives, applied in the field of new antibiotic alternatives, can solve the problems of not being cost-effective to purify this protein, and death of cellular targeted bacteria

Inactive Publication Date: 2009-09-17
IOWA STATE UNIV RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ion leakage caused by these channels uncouples energy expenditures from growth, causing death in cellular targeted bacteria.
Although colicins have shown potential as alternatives to conventional antibiotics in animal feed, it would not be cost effective to purify this protein from naturally occurring colicin producing E. coli strains, nor to include the levels of these bacteria necessary to obtain an antimicrobial effect in the feed.

Method used

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example 1

Colicin Production, Purification, and Efficacy

Colicin Production and Purification

[0069]Colicin producing E. coli strains, obtained from the National Collection of Type Cultures (Public Health Laboratory Service, London, England), were inoculated into Luria Broth (LB) to a starting OD600˜0.1, and incubated with shaking at 37° C. When the cultures reached an OD600=0.9 colicin production was induced by the addition of 0.2 U Mitomycin C (Sigma) / mL culture. The cell free supernatant was obtained by centrifugation 5.5 h later, and concentrated by ultrafiltration in a stir-cell apparatus (Amicon, Millipore, Bedford, Mass.) across a regenerated cellulose membrane with a 30 kDa cut-off (Millipore). The concentrated sample was then desalted against 10 mM Tris-Cl, pH 8 and purified by ion exchange chromatography. The desalted samples were applied to a column containing Q Sepharose (Amersham Biosciences, Piscataway N.J.), equilibrated with 10 mM Tris-Cl, pH 8.0 (equilibration buffer), and exhau...

example 2

Inhibition of Growth of Escherichia coli 0157:H7 In Vitro

Materials and Methods

Colicin Production and Purification

[0076]Each colicin was produced from a specific colicin-producing E. coli K-12 strain (NC50129-01 containing plasmid pColA-CA31, NC50132-01 containing plasmid pColE1-K53, and NC50145-01 containing plasmid pColN-284) obtained from the National Collection of Type Cultures (Public Health Laboratory Service, London, UK). Cultures were inoculated into Luria-Bertani (LB) broth to an initial optical density of 600 nm (OD600) of approximately 0.1 and incubated in a shaker at 37° C. When the cultures reached OD600=0.9, colicin production was induced by the addition of 0.2 U of mitomycin C per ml of culture (Sigma Chemicals, St. Louis, Mo.). The cell-free supernatant was obtained by centrifugation 5.5 h later and concentrated by ultrafiltration in a stir-cell apparatus (Amicon, Millipore, Bedford, Mass.) across a regenerated cellulose membrane with a 30-kDa cut-off (Millipore). The...

example 3

Recombinant Expression of Colicins

[0088]Yeast expression vectors were constructed and verified by sequencing for Colicin A, B, E1, 1a, and N. Expression and secretion of an active Colicin A by Pichia pastoris were obtained, and confirmed by spot testing of cell-free supernatant of Pichia pastoris expressing Colicin A against E. coli DH5α. In the functional assay (spot testing) 10 μl of cell-free supernatant from transformed P. pastoris showed a clearing of approximately the same size as 1 μg of purified Colicin A from E. coli. Spot 2 showed 1 μl of cell-free supernatant from P. pastoris. Spot 3 showed 5 μl of cell-free supernatant from P. pastoris, and spot 4 showed 10 μl of cell-free supernatant from P. pastoris.

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Abstract

The present invention relates to two recombinant colicin expression systems, one utilizing a yeast expression system that produces a protein that is inexpensive to purify, and the other utilizing a plasmid expression system to be used as a probiotic culture. The recombinant colicins provide effective alternatives to conventional antibiotics and may be used to improve the efficiency of pork production, and the safety of its products.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This is a Divisional Application of U.S. application Ser. No. 11 / 327,713 filed Jan. 6, 2006, which is a Continuation-in-Part Application of PCT / US05 / 08512 filed Mar. 15, 2005 which claims priority to U.S. Provisional Application Ser. No. 60 / 553,209 filed Mar. 15, 2004. This application also relates to U.S. application Ser. No. 12 / 159,235 filed Jun. 26, 2008, which is a National Phase Application of PCT / US07 / 00298 filed Jan. 8, 2007 which claims priority to U.S. application Ser. No. 11 / 327,713 filed Jan. 6, 2006. The disclosures of these applications are specifically incorporated herein by reference in their entirety.BACKGROUND OF THE INVENTION[0002]It is estimated that over 50% of all economic losses in weaned pigs are due to Escherichia coli infections, causing either diarrhea or edema disease. In addition to the E. coli strains responsible for disease in pigs, other E. coli, as well as Salmonella, strains also colonize the intestinal tra...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/16
CPCA23K1/1631A23K1/184C07K14/245C07K14/195A61K38/164A23K20/147A23K50/30A61P31/00A61P31/04
Inventor STAHL, CHAD H.
Owner IOWA STATE UNIV RES FOUND
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