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Process optimization for increased yield of surface-expressed protein in Escherichia coli
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0002-6979-0069
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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.

Place, publisher, year, edition, pages
2014. Vol. 37, no 8, 1685-1693 p.
Keyword [en]
Surface expression, Autotransport, Process optimization, Proteolysis, Live vaccines
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-149969DOI: 10.1007/s00449-014-1141-5ISI: 000339962400023Scopus ID: 2-s2.0-84925884851OAI: oai:DiVA.org:kth-149969DiVA: diva2:743511
Note

QC 20140904

Available from: 2014-09-04 Created: 2014-08-29 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Improved detection and performance of surface expression from the AIDA-I autotransporter
Open this publication in new window or tab >>Improved detection and performance of surface expression from the AIDA-I autotransporter
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Surface expression of recombinant proteins has attracted a lot of attention due to its potential in applications such as enzyme production, vaccine delivery and bioremediation. Autotransporters have been used for surface expression of a variety of proteins, but the expression systems reported in literature have typically been inflexible and incapable of detecting proteolysis, thereby limiting surface expression yield.

In this thesis, a modular surface expression system, utilizing dual tag detection, was therefore created. It was based on the adhesin involved in diffuse adherence (AIDA-I) autotransporter, and was here used to express the model proteins SefA and H:gm on the cell surface of Escherichia coli. Due to the dual tag detection system, proteolysed H:gm could be successfully verified on the cell surface. By optimizing cultivation conditions, surface expression yield of SefA was increased by 300 %, and proteolysis reduced by 33 %. While proteolysis could not be eliminated completely, the work presented in this thesis is a major step towards a general system for surface expression of a wide range of proteins in varied applications.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. 53 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:7
Keyword
surface expression, autotransport, AIDA-I, Escherichia coli, proteolysis, detection tag
National Category
Bioprocess Technology
Identifiers
urn:nbn:se:kth:diva-121561 (URN)978-91-7501-708-2 (ISBN)
Presentation
2013-05-31, AlbaNova, sal FB55, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20130506

Available from: 2013-05-06 Created: 2013-05-02 Last updated: 2014-12-11Bibliographically approved
2. Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
Open this publication in new window or tab >>Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
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
AIDA-I, Autotransport, Biocatalysis, Escherichia coli, Live vaccines, Surface expression
National Category
Bioprocess Technology
Identifiers
urn:nbn:se:kth:diva-122230 (URN)978-91-7501-770-9 (ISBN)
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
3. Strategies for improved Escherichia coli bioprocessing performance
Open this publication in new window or tab >>Strategies for improved Escherichia coli bioprocessing performance
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Escherichia coli has a proven track record for successful production of anything from small molecules like organic acids to large therapeutic proteins, and has thus important applications in both R&D and commercial production. The versatility of this organism in combination with the accumulated knowledge of its genome, metabolism and physiology, has allowed for development of specialty strains capable of performing very specific tasks, opening up opportunities within new areas. The work of this thesis has been devoted to alter membrane transport proteins and the regulation of these, in order for E. coli to find further application within two such important areas.

The first area was vaccine development, where it was investigated if E. coli could be a natural vehicle for live vaccine production. The hypothesis was that the introduction and manipulation of a protein surface translocation system from pathogenic E. coli would result in stable expression levels of Salmonella subunit antigens on the surface of laboratory E. coli. While different antigen combinations were successfully expressed on the surface of E. coli, larger proteins were affected by proteolysis, which manipulation of cultivation conditions could reduce, but not eliminate completely. The surface expressed antigens were further capable of inducing proinflammatory responses in epithelial cells.

The second area was biorefining. By altering the regulation of sugar assimilation, it was hypothesized that simultaneous uptake of the sugars present in lignocellulose hydrolyzates could be achieved, thereby improving the yield and productivity of important bio-based chemicals. The dual-layered catabolite repression was identified and successfully removed in the engineered E. coli, and the compound (R)-3-hydroxybutyric acid was produced from simultaneous assimilation of glucose, xylose and arabinose.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 104 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:9
Keyword
E. coli, Salmonella, surface expression, autotransport, AIDA-I, lignocellulose, glucose, xylose, arabinose, simultaneous uptake, 3HB
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-166387 (URN)978-91-7595-523-0 (ISBN)
Public defence
2015-06-05, FB52, AlbaNova universitetscentrum, Roslagstullsbacken 21, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20150508

Available from: 2015-05-08 Created: 2015-05-08 Last updated: 2015-05-08Bibliographically approved

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