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Surface Expression of omega-Transaminase in Escherichia coli
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. (Gen Larsson)ORCID-id: 0000-0002-3314-6060
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. (Gen Larsson)
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. (Gen Larsson)ORCID-id: 0000-0002-6979-0069
2014 (engelsk)Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, nr 7, s. 2293-2298Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2014. Vol. 80, nr 7, s. 2293-2298
Emneord [en]
Display, Protein, Autodisplay, Amines
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-122223DOI: 10.1128/AEM.03678-13ISI: 000332840700028Scopus ID: 2-s2.0-84896029013OAI: oai:DiVA.org:kth-122223DiVA, id: diva2:621413
Forskningsfinansiär
Vinnova
Merknad

QC 20140423. Updated from submitted to published.

Tilgjengelig fra: 2013-05-14 Laget: 2013-05-14 Sist oppdatert: 2017-12-06bibliografisk kontrollert
Inngår i avhandling
1. 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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