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Surface expression using the AIDA autotransporter:  Towards live vaccines and whole-cell biocatalysis
KTH, School of Biotechnology (BIO), Bioprocess Technology.ORCID iD: 0000-0002-3314-6060
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , 49 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2011:25
Keyword [en]
AIDA-autotransporter, Escherichia coli, live vaccines, surface expression
National Category
Bioprocess Technology
URN: urn:nbn:se:kth:diva-48575ISBN: 978-91-7501-182-0OAI: diva2:458075
2011-12-12, FA32, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 14:00 (English)
Vinnova: BIO-AMINESSIDA Vietnam: Production of viral proteins for vaccine development
QC 20111123Available from: 2011-11-23 Created: 2011-11-21 Last updated: 2011-11-23Bibliographically approved
List of papers
1. Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus
Open this publication in new window or tab >>Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus
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2011 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10, 22- p.Article in journal (Refereed) 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.

National Category
Other Industrial Biotechnology
urn:nbn:se:kth:diva-34209 (URN)10.1186/1475-2859-10-22 (DOI)000290528500001 ()2-s2.0-79953801229 (ScopusID)
QC 20110615Available from: 2011-06-15 Created: 2011-05-30 Last updated: 2013-05-16Bibliographically approved
2. Optimisation of surface expression using the AIDA autotransporter
Open this publication in new window or tab >>Optimisation of surface expression using the AIDA autotransporter
2011 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10Article in journal (Refereed) Published
Abstract [en]

Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

National Category
Bioprocess Technology
urn:nbn:se:kth:diva-34435 (URN)10.1186/1475-2859-10-72 (DOI)000295830400001 ()2-s2.0-80052833041 (ScopusID)

QC 20111103

Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2013-05-16Bibliographically approved

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