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Improved detection and performance of surface expression from the AIDA-I autotransporter
KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
2013 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2013. , s. 53
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:7
Nyckelord [en]
surface expression, autotransport, AIDA-I, Escherichia coli, proteolysis, detection tag
Nationell ämneskategori
Bioprocessteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-121561ISBN: 978-91-7501-708-2 (tryckt)OAI: oai:DiVA.org:kth-121561DiVA, id: diva2:619110
Presentation
2013-05-31, AlbaNova, sal FB55, Roslagstullsbacken 21, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20130506

Tillgänglig från: 2013-05-06 Skapad: 2013-05-02 Senast uppdaterad: 2014-12-11Bibliografiskt granskad
Delarbeten
1. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli
Öppna denna publikation i ny flik eller fönster >>A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli
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2012 (Engelska)Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 11, artikel-id 118Artikel i tidskrift (Refereegranskat) 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.

Nyckelord
AIDA, Surface expression, Autotransport, Escherichia coli, Proteolysis, Detection tag
Nationell ämneskategori
Annan industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-107478 (URN)10.1186/1475-2859-11-118 (DOI)000311849500001 ()22943700 (PubMedID)2-s2.0-84865600202 (Scopus ID)
Forskningsfinansiär
Sida - Styrelsen för internationellt utvecklingssamarbete
Anmärkning

QC 20130108

Tillgänglig från: 2012-12-12 Skapad: 2012-12-12 Senast uppdaterad: 2017-12-07Bibliografiskt granskad
2. Process optimization for increased yield of surface-expressed protein in Escherichia coli
Öppna denna publikation i ny flik eller fönster >>Process optimization for increased yield of surface-expressed protein in Escherichia coli
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2014 (Engelska)Ingår i: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 37, nr 8, s. 1685-1693Artikel i tidskrift (Refereegranskat) 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.

Nyckelord
Surface expression, Autotransport, Process optimization, Proteolysis, Live vaccines
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
urn:nbn:se:kth:diva-149969 (URN)10.1007/s00449-014-1141-5 (DOI)000339962400023 ()2-s2.0-84925884851 (Scopus ID)
Anmärkning

QC 20140904

Tillgänglig från: 2014-09-04 Skapad: 2014-08-29 Senast uppdaterad: 2017-12-05Bibliografiskt granskad

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