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Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101). (Gen Larsson)ORCID iD: 0000-0002-3314-6060
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. , xi, 63 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:9
Keyword [en]
AIDA-I, Autotransport, Biocatalysis, Escherichia coli, Live vaccines, Surface expression
National Category
Bioprocess Technology
Identifiers
URN: urn:nbn:se:kth:diva-122230ISBN: 978-91-7501-770-9 (print)OAI: oai:DiVA.org:kth-122230DiVA: diva2:621423
Public defence
2013-06-05, Sal FB42, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20130516

Available from: 2013-05-16 Created: 2013-05-14 Last updated: 2015-06-01Bibliographically 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, E-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
Identifiers
urn:nbn:se:kth:diva-34209 (URN)10.1186/1475-2859-10-22 (DOI)000290528500001 ()2-s2.0-79953801229 (Scopus ID)
Note
QC 20110615Available from: 2011-06-15 Created: 2011-05-30 Last updated: 2017-12-11Bibliographically 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, E-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.

Keyword
ESCHERICHIA-COLI, BIOTECHNOLOGICAL APPLICATIONS, DISPLAY, BATCH, PROTEINS, PROTEASE
National Category
Bioprocess Technology
Identifiers
urn:nbn:se:kth:diva-34435 (URN)10.1186/1475-2859-10-72 (DOI)000295830400001 ()2-s2.0-80052833041 (Scopus ID)
Note

QC 20111103

Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2017-12-11Bibliographically approved
3. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli
Open this publication in new window or tab >>A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli
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2012 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 11, 118Article in journal (Refereed) Published
Abstract [en]

Background: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H: gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His(6)-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H: gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His(6)-tag facing the medium. Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His(6)-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

Keyword
AIDA, Surface expression, Autotransport, Escherichia coli, Proteolysis, Detection tag
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-107478 (URN)10.1186/1475-2859-11-118 (DOI)000311849500001 ()22943700 (PubMedID)2-s2.0-84865600202 (Scopus ID)
Funder
Sida - Swedish International Development Cooperation Agency
Note

QC 20130108

Available from: 2012-12-12 Created: 2012-12-12 Last updated: 2017-12-07Bibliographically approved
4. Surface Expression of omega-Transaminase in Escherichia coli
Open this publication in new window or tab >>Surface Expression of omega-Transaminase in Escherichia coli
2014 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 7, 2293-2298 p.Article in journal (Refereed) Published
Abstract [en]

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus omega-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

Keyword
Display, Protein, Autodisplay, Amines
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:kth:diva-122223 (URN)10.1128/AEM.03678-13 (DOI)000332840700028 ()2-s2.0-84896029013 (Scopus ID)
Funder
Vinnova
Note

QC 20140423. Updated from submitted to published.

Available from: 2013-05-14 Created: 2013-05-14 Last updated: 2017-12-06Bibliographically approved
5. Process optimization for increased yield of surface-expressed protein in Escherichia coli
Open this publication in new window or tab >>Process optimization for increased yield of surface-expressed protein in Escherichia coli
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2014 (English)In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 37, no 8, 1685-1693 p.Article in journal (Refereed) Published
Abstract [en]

The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.

Keyword
Surface expression, Autotransport, Process optimization, Proteolysis, Live vaccines
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-149969 (URN)10.1007/s00449-014-1141-5 (DOI)000339962400023 ()2-s2.0-84925884851 (Scopus ID)
Note

QC 20140904

Available from: 2014-09-04 Created: 2014-08-29 Last updated: 2017-12-05Bibliographically approved
6. Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli
Open this publication in new window or tab >>Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli
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2015 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, 47Article in journal (Refereed) Published
Abstract [en]

Background: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the β-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. Results: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. Conclusions: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.

Keyword
AIDA-I, Autotransport, Escherichia coli, Live vaccines, Salmonella enterica, Surface expression, Bacteria (microorganisms), Salmonella, Salmonella enteritidis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-167747 (URN)10.1186/s12934-015-0227-3 (DOI)000353617100001 ()2-s2.0-84928551436 (Scopus ID)
Funder
Sida - Swedish International Development Cooperation Agency
Note

QC 20150601

Available from: 2015-06-01 Created: 2015-05-22 Last updated: 2017-12-04Bibliographically approved

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