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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

GMMA enrichment and purity after TFF.GMMA were purified from a 5 L fermentation culture of S. sonnei ΔtolR ΔgalU grown in HTMC at 37°C to OD 45 using 2-step TFF. In the first step, the culture supernatant which contains the GMMA was separated from the bacteria using a 0.2 µm filter. The biomass was subjected to 5 diafiltration steps and all filtrates were combined with the initial supernatant to obtain the total permeate. To determine the amount of GMMA in the permeate, GMMA were separated from soluble proteins by ultracentrifugation. After ultracentrifugation, the pellet (GMMA) was resuspended in the initial volume of the centrifuged material to normalize all samples to fermentation volume. Equivalent volumes of the 0.2 µm filtrate before ultracentrifugation (1), the resuspended GMMA pellet (2), and the supernatant of the ultracentrifugation (3) were separated by SDS-PAGE (12% PA) and showed a large amount of soluble proteins (3) in comparison to GMMA-associated proteins (2) to be present in the post 0.2 µm TFF permeate. In the second TFF step, GMMA were separated from soluble proteins using a 0.1 µm filter. The retentate (4) was analyzed by ultracentrifugation as described above and was found to contain almost exclusively GMMA (5) as determined by the strong reduction of soluble proteins (6).
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pone-0035616-g002: GMMA enrichment and purity after TFF.GMMA were purified from a 5 L fermentation culture of S. sonnei ΔtolR ΔgalU grown in HTMC at 37°C to OD 45 using 2-step TFF. In the first step, the culture supernatant which contains the GMMA was separated from the bacteria using a 0.2 µm filter. The biomass was subjected to 5 diafiltration steps and all filtrates were combined with the initial supernatant to obtain the total permeate. To determine the amount of GMMA in the permeate, GMMA were separated from soluble proteins by ultracentrifugation. After ultracentrifugation, the pellet (GMMA) was resuspended in the initial volume of the centrifuged material to normalize all samples to fermentation volume. Equivalent volumes of the 0.2 µm filtrate before ultracentrifugation (1), the resuspended GMMA pellet (2), and the supernatant of the ultracentrifugation (3) were separated by SDS-PAGE (12% PA) and showed a large amount of soluble proteins (3) in comparison to GMMA-associated proteins (2) to be present in the post 0.2 µm TFF permeate. In the second TFF step, GMMA were separated from soluble proteins using a 0.1 µm filter. The retentate (4) was analyzed by ultracentrifugation as described above and was found to contain almost exclusively GMMA (5) as determined by the strong reduction of soluble proteins (6).

Mentions: GMMA were purified from fermentation cultures in a 2-step TFF process. In the first step, the culture supernatant that contains the GMMA was separated from the bacteria using a 0.2 µm filter. In this step, the bacteria remained in the retentate and GMMA transferred into the filtrate. In the second filtration step using a 0.1 µm filter, GMMA were separated from soluble components present in the culture supernatant, including proteins secreted by the bacteria or released by lysis. In this step, GMMA were retained by the filter and collected and concentrated in the retentate whereas soluble proteins passed through the filter. We tested this purification process under two slightly different conditions. Firstly, when the fermentation culture of S. sonnei ΔtolR ΔgalU reached OD 45, the culture was transferred directly from the fermenter to the first TFF and the culture supernatant containing the GMMA was collected. The retained biomass was washed with 5 volumes of PBS buffer (diafiltration) to recover remaining GMMA and the filtrate containing these GMMA was combined with the culture supernatant. In the slightly modified purification process tested, this diafiltration step was omitted. Proteins were quantified by Bradford method. In the purification performed with diafiltration of the biomass the total protein content of the TFF 0.2 µm filtrate was approximately 1.5 g/L of fermentation culture of which 15% was GMMA-associated as determined by separation of the soluble components from the high molecular weight portion (GMMA) via an ultracentrifuge step (Fig. 2 and Table 3). In the second TFF step, GMMA were concentrated in the retentate and washed with five volumes of PBS to remove remaining soluble proteins. As TFF is usually performed under non-sterile conditions, the final retentate was sterilized by filtration through a 0.22 µm filter. An aliquot of the sterilized retentate was subjected to ultracentrifugation to determine the content of GMMA as above. As shown in Fig. 2 most of the proteins in the retentate are GMMA-associated. Protein quantification of the retentate, of the GMMA fraction, and of the supernatant of the ultracentrifugation step (soluble proteins) determined that 90% of all protein present in the retentate was GMMA-associated (Table 3). Thus, soluble proteins were efficiently removed in this step (Fig. 2). GMMA recovery after the 0.1 µm TFF step was 56% of the quantity present after the 0.2 µm cassette. The final yield of GMMA was 120 milligrams of proteins per liter of fermentation (Table 3). In two subsequent tests of the purification method with S. sonnei ΔtolR ΔgalU without diafiltration of the biomass, a lower amount of GMMA was obtained in the 0.2 µm TFF filtrate (Table 3). However, recovery of GMMA in the second TFF step (0.1 µm) was enhanced resulting in an overall similar yield of GMMA with equivalent purity (Table 3 and suppl. figures Fig. S1, Fig. S2). This suggests, firstly, that washing of the biomass increases the recovery GMMA from the fermentation culture, and secondly, that a higher starting concentration might be beneficial for the second TFF step. The 0.1 µm TFF step can likely be optimized to take advantage of the larger amounts of GMMA obtained by diafiltration of the biomass. Fermentations of S. sonnei –pSS ΔtolR ΔmsbB resulted in yields of 140 mg/L from a culture at OD 65 (2.2 mg/L/OD) and 230 mg/L from a culture at OD 80 (2.9 mg/L/OD), demonstrating that the yield of GMMA can be further improved by growing the culture to a higher OD.


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)

GMMA enrichment and purity after TFF.GMMA were purified from a 5 L fermentation culture of S. sonnei ΔtolR ΔgalU grown in HTMC at 37°C to OD 45 using 2-step TFF. In the first step, the culture supernatant which contains the GMMA was separated from the bacteria using a 0.2 µm filter. The biomass was subjected to 5 diafiltration steps and all filtrates were combined with the initial supernatant to obtain the total permeate. To determine the amount of GMMA in the permeate, GMMA were separated from soluble proteins by ultracentrifugation. After ultracentrifugation, the pellet (GMMA) was resuspended in the initial volume of the centrifuged material to normalize all samples to fermentation volume. Equivalent volumes of the 0.2 µm filtrate before ultracentrifugation (1), the resuspended GMMA pellet (2), and the supernatant of the ultracentrifugation (3) were separated by SDS-PAGE (12% PA) and showed a large amount of soluble proteins (3) in comparison to GMMA-associated proteins (2) to be present in the post 0.2 µm TFF permeate. In the second TFF step, GMMA were separated from soluble proteins using a 0.1 µm filter. The retentate (4) was analyzed by ultracentrifugation as described above and was found to contain almost exclusively GMMA (5) as determined by the strong reduction of soluble proteins (6).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368891&req=5

pone-0035616-g002: GMMA enrichment and purity after TFF.GMMA were purified from a 5 L fermentation culture of S. sonnei ΔtolR ΔgalU grown in HTMC at 37°C to OD 45 using 2-step TFF. In the first step, the culture supernatant which contains the GMMA was separated from the bacteria using a 0.2 µm filter. The biomass was subjected to 5 diafiltration steps and all filtrates were combined with the initial supernatant to obtain the total permeate. To determine the amount of GMMA in the permeate, GMMA were separated from soluble proteins by ultracentrifugation. After ultracentrifugation, the pellet (GMMA) was resuspended in the initial volume of the centrifuged material to normalize all samples to fermentation volume. Equivalent volumes of the 0.2 µm filtrate before ultracentrifugation (1), the resuspended GMMA pellet (2), and the supernatant of the ultracentrifugation (3) were separated by SDS-PAGE (12% PA) and showed a large amount of soluble proteins (3) in comparison to GMMA-associated proteins (2) to be present in the post 0.2 µm TFF permeate. In the second TFF step, GMMA were separated from soluble proteins using a 0.1 µm filter. The retentate (4) was analyzed by ultracentrifugation as described above and was found to contain almost exclusively GMMA (5) as determined by the strong reduction of soluble proteins (6).
Mentions: GMMA were purified from fermentation cultures in a 2-step TFF process. In the first step, the culture supernatant that contains the GMMA was separated from the bacteria using a 0.2 µm filter. In this step, the bacteria remained in the retentate and GMMA transferred into the filtrate. In the second filtration step using a 0.1 µm filter, GMMA were separated from soluble components present in the culture supernatant, including proteins secreted by the bacteria or released by lysis. In this step, GMMA were retained by the filter and collected and concentrated in the retentate whereas soluble proteins passed through the filter. We tested this purification process under two slightly different conditions. Firstly, when the fermentation culture of S. sonnei ΔtolR ΔgalU reached OD 45, the culture was transferred directly from the fermenter to the first TFF and the culture supernatant containing the GMMA was collected. The retained biomass was washed with 5 volumes of PBS buffer (diafiltration) to recover remaining GMMA and the filtrate containing these GMMA was combined with the culture supernatant. In the slightly modified purification process tested, this diafiltration step was omitted. Proteins were quantified by Bradford method. In the purification performed with diafiltration of the biomass the total protein content of the TFF 0.2 µm filtrate was approximately 1.5 g/L of fermentation culture of which 15% was GMMA-associated as determined by separation of the soluble components from the high molecular weight portion (GMMA) via an ultracentrifuge step (Fig. 2 and Table 3). In the second TFF step, GMMA were concentrated in the retentate and washed with five volumes of PBS to remove remaining soluble proteins. As TFF is usually performed under non-sterile conditions, the final retentate was sterilized by filtration through a 0.22 µm filter. An aliquot of the sterilized retentate was subjected to ultracentrifugation to determine the content of GMMA as above. As shown in Fig. 2 most of the proteins in the retentate are GMMA-associated. Protein quantification of the retentate, of the GMMA fraction, and of the supernatant of the ultracentrifugation step (soluble proteins) determined that 90% of all protein present in the retentate was GMMA-associated (Table 3). Thus, soluble proteins were efficiently removed in this step (Fig. 2). GMMA recovery after the 0.1 µm TFF step was 56% of the quantity present after the 0.2 µm cassette. The final yield of GMMA was 120 milligrams of proteins per liter of fermentation (Table 3). In two subsequent tests of the purification method with S. sonnei ΔtolR ΔgalU without diafiltration of the biomass, a lower amount of GMMA was obtained in the 0.2 µm TFF filtrate (Table 3). However, recovery of GMMA in the second TFF step (0.1 µm) was enhanced resulting in an overall similar yield of GMMA with equivalent purity (Table 3 and suppl. figures Fig. S1, Fig. S2). This suggests, firstly, that washing of the biomass increases the recovery GMMA from the fermentation culture, and secondly, that a higher starting concentration might be beneficial for the second TFF step. The 0.1 µm TFF step can likely be optimized to take advantage of the larger amounts of GMMA obtained by diafiltration of the biomass. Fermentations of S. sonnei –pSS ΔtolR ΔmsbB resulted in yields of 140 mg/L from a culture at OD 65 (2.2 mg/L/OD) and 230 mg/L from a culture at OD 80 (2.9 mg/L/OD), demonstrating that the yield of GMMA can be further improved by growing the culture to a higher OD.

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