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Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

Nhan NT, Gonzalez de Valdivia E, Gustavsson M, Hai TN, Larsson G - Microb. Cell Fact. (2011)

Bottom Line: FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off.Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins.Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vietnam Institute of Biotechnology (IBT), Vietnamese Academy of Science and Technology (VAST), Hanoi, Vietnam.

ABSTRACT

Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.

Results: Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis.

Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

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Presence of the His6 tag in fusion proteins located in the outer membrane fraction of E. coli. Western blot showing: Lane 1: Positive control; Bacillus Lipase 19.3 kDa [37]; Lane 2: H:gm; Lane 3: SefA; and Lane 4: Negative control: AIDAC. All proteins carry the His6 tag. The molecular sizes of marker proteins are indicated to the left (kDa). Western blot detection with Abcam 6× His tag specific antibody and HisProbe™-HRP.
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Figure 5: Presence of the His6 tag in fusion proteins located in the outer membrane fraction of E. coli. Western blot showing: Lane 1: Positive control; Bacillus Lipase 19.3 kDa [37]; Lane 2: H:gm; Lane 3: SefA; and Lane 4: Negative control: AIDAC. All proteins carry the His6 tag. The molecular sizes of marker proteins are indicated to the left (kDa). Western blot detection with Abcam 6× His tag specific antibody and HisProbe™-HRP.

Mentions: However, since we know that both proteins are present in the OM but that the size of the H:gm fusion protein is lower than expected, we suggest that proteolytic cleavage of the N-terminus, removing the His tag, has taken place. This would mean that conjugation of the antibodies to this tag could not take place and there would consequently be no signal in any of the His-tag-based analytical systems. We therefore performed a new Western blot using antibodies against the His6 tag. As shown in Figure 5, the His-tag is present only in connection with the SefA protein and not in the H:gm protein fraction. The actual orientation of the truncated H:gm protein cannot however be clarified with these data since the truncated forms may also still be facing the environment.


Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

Nhan NT, Gonzalez de Valdivia E, Gustavsson M, Hai TN, Larsson G - Microb. Cell Fact. (2011)

Presence of the His6 tag in fusion proteins located in the outer membrane fraction of E. coli. Western blot showing: Lane 1: Positive control; Bacillus Lipase 19.3 kDa [37]; Lane 2: H:gm; Lane 3: SefA; and Lane 4: Negative control: AIDAC. All proteins carry the His6 tag. The molecular sizes of marker proteins are indicated to the left (kDa). Western blot detection with Abcam 6× His tag specific antibody and HisProbe™-HRP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094208&req=5

Figure 5: Presence of the His6 tag in fusion proteins located in the outer membrane fraction of E. coli. Western blot showing: Lane 1: Positive control; Bacillus Lipase 19.3 kDa [37]; Lane 2: H:gm; Lane 3: SefA; and Lane 4: Negative control: AIDAC. All proteins carry the His6 tag. The molecular sizes of marker proteins are indicated to the left (kDa). Western blot detection with Abcam 6× His tag specific antibody and HisProbe™-HRP.
Mentions: However, since we know that both proteins are present in the OM but that the size of the H:gm fusion protein is lower than expected, we suggest that proteolytic cleavage of the N-terminus, removing the His tag, has taken place. This would mean that conjugation of the antibodies to this tag could not take place and there would consequently be no signal in any of the His-tag-based analytical systems. We therefore performed a new Western blot using antibodies against the His6 tag. As shown in Figure 5, the His-tag is present only in connection with the SefA protein and not in the H:gm protein fraction. The actual orientation of the truncated H:gm protein cannot however be clarified with these data since the truncated forms may also still be facing the environment.

Bottom Line: FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off.Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins.Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vietnam Institute of Biotechnology (IBT), Vietnamese Academy of Science and Technology (VAST), Hanoi, Vietnam.

ABSTRACT

Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.

Results: Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis.

Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

Show MeSH
Related in: MedlinePlus