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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

2D gel electrophoresis of Shigella sonnei ΔtolR ΔgalU GMMA and immunoblot.A)200 µg of proteins from S. sonnei ΔtolR ΔgalU GMMA were separated in the first dimension on a non linear pH 3–11 gradient, and in the second dimension on a 4–12% polyacrylamide gradient. Visible bands were identified by protein mass fingerprint. OmpA and OmpC were quantified with Image master 2D Platinum 6.0. B) Sera from mice immunized with GMMA from S. sonnei ΔtolR ΔgalU were used to study the subset of proteins present in GMMA that are able to raise antibodies. A 2D gel containing 20 µg of GMMA protein from S. sonnei ΔtolR ΔgalU was blotted and the membrane was incubated with sera from immunized mice with GMMA from S. sonnei ΔtolR ΔgalU in combination with Freund’s adjuvant. Several reactive proteins were identified. The numbers behind the names refer to the position of the proteins in Table 2. C) To verify that the signal observed in the 2D Western blot was due exclusively to antibody raised upon immunization with GMMA, 10 µg of GMMA were separated by 1D SDS-PAGE (12% PA) and stained with Coomassie (1) or transferred to a membrane. Western blots were developed using (2) sera raised against GMMA from S. sonnei ΔtolR ΔgalU as used for the 2D Western blot in B, (3) preimmune serum, (4) sera raised in mice immunized with Freund’s adjuvant or (5) PBS, or (6) secondary antibody only. A signal could only be observed when sera raised against GMMA were used (2).
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pone-0035616-g004: 2D gel electrophoresis of Shigella sonnei ΔtolR ΔgalU GMMA and immunoblot.A)200 µg of proteins from S. sonnei ΔtolR ΔgalU GMMA were separated in the first dimension on a non linear pH 3–11 gradient, and in the second dimension on a 4–12% polyacrylamide gradient. Visible bands were identified by protein mass fingerprint. OmpA and OmpC were quantified with Image master 2D Platinum 6.0. B) Sera from mice immunized with GMMA from S. sonnei ΔtolR ΔgalU were used to study the subset of proteins present in GMMA that are able to raise antibodies. A 2D gel containing 20 µg of GMMA protein from S. sonnei ΔtolR ΔgalU was blotted and the membrane was incubated with sera from immunized mice with GMMA from S. sonnei ΔtolR ΔgalU in combination with Freund’s adjuvant. Several reactive proteins were identified. The numbers behind the names refer to the position of the proteins in Table 2. C) To verify that the signal observed in the 2D Western blot was due exclusively to antibody raised upon immunization with GMMA, 10 µg of GMMA were separated by 1D SDS-PAGE (12% PA) and stained with Coomassie (1) or transferred to a membrane. Western blots were developed using (2) sera raised against GMMA from S. sonnei ΔtolR ΔgalU as used for the 2D Western blot in B, (3) preimmune serum, (4) sera raised in mice immunized with Freund’s adjuvant or (5) PBS, or (6) secondary antibody only. A signal could only be observed when sera raised against GMMA were used (2).

Mentions: GMMA purified by TFF from S. sonnei 53G ΔtolR ΔgalU grown in high density culture were characterized to confirm their integrity and to analyze their protein content. One- and two-dimensional SDS-PAGE of GMMA and densitometry analysis (Fig. 1D and Fig. 4) were used to determine the protein profile and to study relative protein quantities of the most abundant proteins. Most of the Coomassie blue-stained bands and spots were identified using peptide mass fingerprint (Table 2). OmpA and OmpC are known to be among the most abundant proteins present in the outer membrane. In fact, densitometry analysis of GMMA from S. sonnei ΔtolR ΔgalU grown in HTMC and analyzed by 1D SDS-PAGE indicated that OmpA and OmpC together contribute for 45% of the total protein; OmpX, 9%; Slp, 6%; YfiO, 5.6%; TolB, 2.3%; TolC 1.4%; and YaeT, 1.8% (Fig. 1D). With the exception of the predicted periplasmic protein TolB, all of these proteins are predicted to be associated with the outer membrane. YfiO is predicted to be an outer membrane lipoprotein. OmpA, OmpC, OmpX, Slp, TolC, and YaeT are predicted to be outer membrane proteins. Thus, the seven most abundant outer membrane-associated proteins account for approximately 69% of the protein amount in GMMA. Further densitometry analysis after 2D SDS-PAGE determined that there are approximately equal quantities of OmpA and OmpC (OmpA:OmpC is 1∶0.83 by densitometry of a Coomassie blue-stained gel). In order to identify the diverse and less expressed proteins, GMMA were studied by proteolytic digestion and reverse phase liquid chromatography coupled to MS/MS. 61 proteins were identified in total (LC-MS/MS, 1D and 2D SDS-PAGE PMF) (Table 2), with 31 of these proteins predicted to be associated with the outer membrane (Fig. 5). Of these, 14 proteins were predicted to be outer membrane proteins and 17 to be outer membrane lipoproteins. In addition, 16 proteins were predicted to be periplasmic, 8 to be cytoplasmic, 1 to be located in the inner membrane, and for 5 proteins no prediction could be obtained. No inner membrane lipoproteins were predicted. Thus, GMMA generated by the high yield production process are mostly composed of outer membrane-associated and periplasmic proteins as previously seen for outer membrane particles release from cultures at the early logarithmic phase [10], [13].


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)

2D gel electrophoresis of Shigella sonnei ΔtolR ΔgalU GMMA and immunoblot.A)200 µg of proteins from S. sonnei ΔtolR ΔgalU GMMA were separated in the first dimension on a non linear pH 3–11 gradient, and in the second dimension on a 4–12% polyacrylamide gradient. Visible bands were identified by protein mass fingerprint. OmpA and OmpC were quantified with Image master 2D Platinum 6.0. B) Sera from mice immunized with GMMA from S. sonnei ΔtolR ΔgalU were used to study the subset of proteins present in GMMA that are able to raise antibodies. A 2D gel containing 20 µg of GMMA protein from S. sonnei ΔtolR ΔgalU was blotted and the membrane was incubated with sera from immunized mice with GMMA from S. sonnei ΔtolR ΔgalU in combination with Freund’s adjuvant. Several reactive proteins were identified. The numbers behind the names refer to the position of the proteins in Table 2. C) To verify that the signal observed in the 2D Western blot was due exclusively to antibody raised upon immunization with GMMA, 10 µg of GMMA were separated by 1D SDS-PAGE (12% PA) and stained with Coomassie (1) or transferred to a membrane. Western blots were developed using (2) sera raised against GMMA from S. sonnei ΔtolR ΔgalU as used for the 2D Western blot in B, (3) preimmune serum, (4) sera raised in mice immunized with Freund’s adjuvant or (5) PBS, or (6) secondary antibody only. A signal could only be observed when sera raised against GMMA were used (2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368891&req=5

pone-0035616-g004: 2D gel electrophoresis of Shigella sonnei ΔtolR ΔgalU GMMA and immunoblot.A)200 µg of proteins from S. sonnei ΔtolR ΔgalU GMMA were separated in the first dimension on a non linear pH 3–11 gradient, and in the second dimension on a 4–12% polyacrylamide gradient. Visible bands were identified by protein mass fingerprint. OmpA and OmpC were quantified with Image master 2D Platinum 6.0. B) Sera from mice immunized with GMMA from S. sonnei ΔtolR ΔgalU were used to study the subset of proteins present in GMMA that are able to raise antibodies. A 2D gel containing 20 µg of GMMA protein from S. sonnei ΔtolR ΔgalU was blotted and the membrane was incubated with sera from immunized mice with GMMA from S. sonnei ΔtolR ΔgalU in combination with Freund’s adjuvant. Several reactive proteins were identified. The numbers behind the names refer to the position of the proteins in Table 2. C) To verify that the signal observed in the 2D Western blot was due exclusively to antibody raised upon immunization with GMMA, 10 µg of GMMA were separated by 1D SDS-PAGE (12% PA) and stained with Coomassie (1) or transferred to a membrane. Western blots were developed using (2) sera raised against GMMA from S. sonnei ΔtolR ΔgalU as used for the 2D Western blot in B, (3) preimmune serum, (4) sera raised in mice immunized with Freund’s adjuvant or (5) PBS, or (6) secondary antibody only. A signal could only be observed when sera raised against GMMA were used (2).
Mentions: GMMA purified by TFF from S. sonnei 53G ΔtolR ΔgalU grown in high density culture were characterized to confirm their integrity and to analyze their protein content. One- and two-dimensional SDS-PAGE of GMMA and densitometry analysis (Fig. 1D and Fig. 4) were used to determine the protein profile and to study relative protein quantities of the most abundant proteins. Most of the Coomassie blue-stained bands and spots were identified using peptide mass fingerprint (Table 2). OmpA and OmpC are known to be among the most abundant proteins present in the outer membrane. In fact, densitometry analysis of GMMA from S. sonnei ΔtolR ΔgalU grown in HTMC and analyzed by 1D SDS-PAGE indicated that OmpA and OmpC together contribute for 45% of the total protein; OmpX, 9%; Slp, 6%; YfiO, 5.6%; TolB, 2.3%; TolC 1.4%; and YaeT, 1.8% (Fig. 1D). With the exception of the predicted periplasmic protein TolB, all of these proteins are predicted to be associated with the outer membrane. YfiO is predicted to be an outer membrane lipoprotein. OmpA, OmpC, OmpX, Slp, TolC, and YaeT are predicted to be outer membrane proteins. Thus, the seven most abundant outer membrane-associated proteins account for approximately 69% of the protein amount in GMMA. Further densitometry analysis after 2D SDS-PAGE determined that there are approximately equal quantities of OmpA and OmpC (OmpA:OmpC is 1∶0.83 by densitometry of a Coomassie blue-stained gel). In order to identify the diverse and less expressed proteins, GMMA were studied by proteolytic digestion and reverse phase liquid chromatography coupled to MS/MS. 61 proteins were identified in total (LC-MS/MS, 1D and 2D SDS-PAGE PMF) (Table 2), with 31 of these proteins predicted to be associated with the outer membrane (Fig. 5). Of these, 14 proteins were predicted to be outer membrane proteins and 17 to be outer membrane lipoproteins. In addition, 16 proteins were predicted to be periplasmic, 8 to be cytoplasmic, 1 to be located in the inner membrane, and for 5 proteins no prediction could be obtained. No inner membrane lipoproteins were predicted. Thus, GMMA generated by the high yield production process are mostly composed of outer membrane-associated and periplasmic proteins as previously seen for outer membrane particles release from cultures at the early logarithmic phase [10], [13].

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