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Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus
KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.ORCID-id: 0000-0002-3314-6060
Vise andre og tillknytning
2011 (engelsk)Inngår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10, s. 22-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

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 beta-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 (His(6)) 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.

sted, utgiver, år, opplag, sider
2011. Vol. 10, s. 22-
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-34209DOI: 10.1186/1475-2859-10-22ISI: 000290528500001Scopus ID: 2-s2.0-79953801229OAI: oai:DiVA.org:kth-34209DiVA, id: diva2:423502
Merknad
QC 20110615Tilgjengelig fra: 2011-06-15 Laget: 2011-05-30 Sist oppdatert: 2017-12-11bibliografisk kontrollert
Inngår i avhandling
1. Surface expression using the AIDA autotransporter:  Towards live vaccines and whole-cell biocatalysis
Åpne denne publikasjonen i ny fane eller vindu >>Surface expression using the AIDA autotransporter:  Towards live vaccines and whole-cell biocatalysis
2011 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation.

  

However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2011. s. 49
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011:25
Emneord
AIDA-autotransporter, Escherichia coli, live vaccines, surface expression
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-48575 (URN)978-91-7501-182-0 (ISBN)
Presentation
2011-12-12, FA32, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 14:00 (engelsk)
Opponent
Veileder
Prosjekter
Vinnova: BIO-AMINESSIDA Vietnam: Production of viral proteins for vaccine development
Merknad
QC 20111123Tilgjengelig fra: 2011-11-23 Laget: 2011-11-21 Sist oppdatert: 2011-11-23bibliografisk kontrollert
2. Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
Åpne denne publikasjonen i ny fane eller vindu >>Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
2013 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements.

 

A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed.

 

Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2013. s. xi, 63
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:9
Emneord
AIDA-I, Autotransport, Biocatalysis, Escherichia coli, Live vaccines, Surface expression
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-122230 (URN)978-91-7501-770-9 (ISBN)
Disputas
2013-06-05, Sal FB42, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20130516

Tilgjengelig fra: 2013-05-16 Laget: 2013-05-14 Sist oppdatert: 2015-06-01bibliografisk kontrollert

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