| 1. |
- Bäcklund, Emma
(författare)
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Impact of glucose uptake rate on recombinant protein production in Escherichia coli
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures. The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.
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| 2. |
- Gustavsson, Martin, 1984-
(författare)
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Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Gustavsson, Martin, 1984-
(författare)
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Surface expression using the AIDA autotransporter : Towards live vaccines and whole-cell biocatalysis
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation. However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.
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| 4. |
- Jarmander, Johan, 1983-
(författare)
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Improved detection and performance of surface expression from the AIDA-I autotransporter
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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