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. / <p>QC 20130516</p>
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-122230 |
| Date | January 2013 |
| Creators | Gustavsson, Martin |
| Publisher | KTH, Bioprocessteknik (stängd 20130101), Stockholm |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | TRITA-BIO-Report, 1654-2312 ; 2013:9 |
Page generated in 0.0022 seconds