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High yield production process for Shigella outer membrane particles.

Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O, Ferlenghi I, Pesce I, Caboni M, Norais N, Di Cioccio V, Saul A, Gerke C - PLoS ONE (2012)

Bottom Line: Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter.These were highly immunogenic in mice.Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines Institute for Global Health, Siena, Italy.

ABSTRACT
Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.

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Related in: MedlinePlus

Electron microscopy of Shigella sonnei ΔtolR ΔgalU GMMA.GMMA were isolated from the culture supernatant of S. sonnei –pSS ΔtolR ΔmsbB by TFF, prepared for negative staining, and viewed by electron microscopy revealing the presence of well-organized membrane vesicles with a diameter of about 30–60 nm. Bar length  = 100 nm.
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pone-0035616-g003: Electron microscopy of Shigella sonnei ΔtolR ΔgalU GMMA.GMMA were isolated from the culture supernatant of S. sonnei –pSS ΔtolR ΔmsbB by TFF, prepared for negative staining, and viewed by electron microscopy revealing the presence of well-organized membrane vesicles with a diameter of about 30–60 nm. Bar length  = 100 nm.

Mentions: The preparation of GMMA generated from S. sonnei –pSS ΔtolR ΔmsbB obtained after the second TFF step was subjected to electron microscopy analysis, revealing the presence of well-organized membrane vesicles with a diameter of about 30–60 nm (Fig. 3) which is consistent with the reported average size of 40±20 nm of outer membrane particles produced by E. coli tol-pal mutants [11].


High yield production process for Shigella outer membrane particles.

Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O, Ferlenghi I, Pesce I, Caboni M, Norais N, Di Cioccio V, Saul A, Gerke C - PLoS ONE (2012)

Electron microscopy of Shigella sonnei ΔtolR ΔgalU GMMA.GMMA were isolated from the culture supernatant of S. sonnei –pSS ΔtolR ΔmsbB by TFF, prepared for negative staining, and viewed by electron microscopy revealing the presence of well-organized membrane vesicles with a diameter of about 30–60 nm. Bar length  = 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368891&req=5

pone-0035616-g003: Electron microscopy of Shigella sonnei ΔtolR ΔgalU GMMA.GMMA were isolated from the culture supernatant of S. sonnei –pSS ΔtolR ΔmsbB by TFF, prepared for negative staining, and viewed by electron microscopy revealing the presence of well-organized membrane vesicles with a diameter of about 30–60 nm. Bar length  = 100 nm.
Mentions: The preparation of GMMA generated from S. sonnei –pSS ΔtolR ΔmsbB obtained after the second TFF step was subjected to electron microscopy analysis, revealing the presence of well-organized membrane vesicles with a diameter of about 30–60 nm (Fig. 3) which is consistent with the reported average size of 40±20 nm of outer membrane particles produced by E. coli tol-pal mutants [11].

Bottom Line: Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter.These were highly immunogenic in mice.Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines Institute for Global Health, Siena, Italy.

ABSTRACT
Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.

Show MeSH
Related in: MedlinePlus