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| 2. |
- Bonander, Nicklas, 1968, et al.
(författare)
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Optimising yeast as a host for recombinant protein production (review)
- 2012
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029 .- 1064-3745. ; 866, s. 1-9
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Forskningsöversikt (refereegranskat)abstract
- Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.
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| 3. |
- Bonander, Nicklas, 1968, et al.
(författare)
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Optimizing yeast as a host for recombinant protein production
- 2011
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Bok (övrigt vetenskapligt/konstnärligt)abstract
- Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesizing a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, whilst also being quick, easy and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.
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| 4. |
- Bonander, Nicklas, 1968, et al.
(författare)
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Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 8:5, s. Art. no. e64517-
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Tidskriftsartikel (refereegranskat)abstract
- The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.
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| 5. |
- Cartwright, S. P., et al.
(författare)
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Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin
- 2017
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3 Delta, srb5 Delta, gcn5 Delta. and yTHCBMS1 (supplemented with 0.5 mu g/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5' untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10-80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A(2a)R) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described. Results: Polysome profiling experiments were used to analyze the translational state of spt3 Delta, srb5 Delta, gcn5 Delta. and yTHCBMS1 (supplemented with 0.5 mu g/mL doxycycline); all but gcn5 Delta. were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31a Delta., rpl22a Delta., ssf1 Delta. and nop1 Delta.) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3 Delta, srb5 Delta, gcn5 Delta. and yTHCBMS1 (supplemented with 0.5 mu g/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2 alpha. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5' UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3. or wild-type cells, suggesting that high-yielding strains are able to bypass 5' uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories. Conclusions: Regulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.
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| 6. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Evolutionary engineered strains of Saccharomyces cerevisiae for efficient lignocellulosic bioethanol production
- 2014
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Ingår i: 36th Symposium on Biotechnology for Fuels and Chemicals.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic biomass is an abundant raw material that can be utilized to produce ethanol with the help of Saccharomyces cerevisiae; a promising alternative to today’s energy sources. Conversion of lignocellulosic material (cellulose, hemicellulose and lignin) into fermentable sugars including both hexoses and pentoses results in formation of inhibitory compounds such as acetic acid, furan aldehydes and phenolics that are known to inhibit the yeasts’ metabolic processes. The aims of this study were to i) generate S. cerevisiae strains that can readily convert glucose and xylose into ethanol in the presence of inhibitory compounds, and ii) elucidate the underlying genetic changes of importance for the improved properties of the generated strains. For these purposes, a strain of S. cerevisiae containing genes for xylose reductase, xylitol dehydrogenase and xylulokinase was used. The strain was subjected to mutagenesis followed by evolutionary engineering (repetitive batch and chemostat cultivation), which resulted in populations with improved ethanol yield, improved xylose conversion rate and increased inhibitor tolerance. The complex combination of different genetic alterations in the evolved populations will now be revealed using a DNA/RNA sequencing approach. The acquired knowledge of proteins and pathways important for efficient lignocellulosic bioethanol production will then hopefully allow directed engineering for further improvement of yeast performance.
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| 7. |
- Gourdon, Pontus Emanuel, 1978, et al.
(författare)
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Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump
- 2008
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 58:1, s. 103-113
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Tidskriftsartikel (refereegranskat)abstract
- Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0 mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5 mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.
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| 8. |
- Tomas-Pejo, Elia, 1980, et al.
(författare)
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Challenges of strain development and clone selection for bioethanol production from lignocellulose
- 2012
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Ingår i: 2nd Symposium on Biotechnology Applied on Lignocelluloses. Fukuoka, Japan. 14-17 October 2012.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic biomass is one of the most promising raw materials for bioethanol production because it does not compete with food crops and is widely distributed around the world. When using lignocellulosic materials, toxic compounds derived from cellulose, hemicellulose and lignin degradation during pretreatment are also found in the media. It is well known that the most commonly used microorganism in ethanol production is Saccharomyces cerevisisae, however, wild type S. cerevisiae is not able to ferment xylose which could constitute up to 40% of the lignocellulose. Therefore, yeasts strains to be used for second-generation bioethanol production have to cope with challenging conditions that are inherent to the industrial process such as high concentration of inhibitory products, simultaneous use of different carbon sources and growth conditions that are not well controlled. Tolerance to these multiple stresses is likely to be a complex phenotype involving several cellular mechanisms and it could be difficult to perform efficient metabolic engineering. In this context, one of the most promising strategies for developing industrial strains is evolutionary engineering that includes evolution and recombination introducing genetic variability over many generations. Evolved S. cerevisiae strains engineered for xylose fermentation employed in this study have been subjected to targeted engineering for introducing a barcode in order to be able to verify their origin which also provokes random events in the population of cells. Screening after evolution or targeted engineering is challenging because of the high variability introduced during those events. Selection has to be performed carefully in order to select the best clones with best properties for a specific purpose. Furthermore, difficulties in applying novel technics such as next generation sequencing or multiomic analysis in industrial strains result from their genetic complexity such as polyploidy. In this work, mixed populations obtained by evolutionary engineering and different clones obtained after barcoding (Figure 1) are tested and evaluated in ethanol production processes from lignocellulosic hydrolysates. Differences between clones regarding xylose fermentation capability are elucidated.
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| 9. |
- Tomas-Pejo, Elia, 1980, et al.
(författare)
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EVALUATION OF EVOLVED AND BARCODED XYLOSE FERMENTING STRAINS FOR BIOETHANOL PRODUCTION FROM LIGNOCELLULOSE
- 2012
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Ingår i: Science and Technology Day 2012, Chalmers University of Technology, 27th March 2012.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic raw materials for bioethanol production are today the basis for many ethanol production sites around the world. However, the utilization of engineered yeast strains for second generation ethanol production at large-scale can still be improved. Yeasts mainly use the sugars present in the lignocellulosic biomass but, toxic compounds derived from cellulose, hemicellulose and lignin degradation during pretreatment are also found in the media and inhibit yeast growth. Furthermore, wild type Saccharomyces cerevisiae is not able to ferment xylose which could constitute up to 40% of the lignocellulose material. Hence the recombinant yeast strains must be robust and ferment xylose to ethanol with high yields in the presence of inhibitors.In this study, different evolved xylose fermenting Saccharomyces cerevisiae strains have been compared in ethanol production processes from lignocellulosic hydrolysates. The differences between using single cell transformants and mixed populations will be evaluated in terms of ethanol production in large scale bioreactors.Furthermore, we have established a method to barcode the evolved yeast strains in order to be able to verify their origin. It is of outmost importance that after barcoding the original characteristics of a yeast strain are maintained. Those requirements can only be fulfilled by using a dominant selection principle. We have obtained a few hundred transformants that were shown to contain the new unique barcode DNA sequence via DNA isolation and DNA sequencing. The transformed strains must be able to grow on the lignocellulosic material and consume xylose at the same rate as before the transformation which also was tested in this study.
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| 10. |
- Tomas-Pejo, Elia, 1980, et al.
(författare)
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Industrial yeasts strains for biorefinery solutions: Constructing and selecting efficient barcoded xylose fermenting strains for ethanol
- 2014
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Ingår i: Biofuels, Bioproducts and Biorefining. - : Wiley. - 1932-1031 .- 1932-104X. ; 8:5, s. 626-634
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Tidskriftsartikel (refereegranskat)abstract
- There have been intense efforts in the scientific community and industry to engineer S. cerevisiae strains to co-consume xylose and glucose. In this context, this paper describes a process for introducing a barcode into an industrial xylose fermenting yeast strains to ensure quick and reliable identification of patented industrial strains. Furthermore, this work presents a novel procedure for screening and selecting the clones that reflects the characteristics of the industrial process. The key points of efficient selection of high-performing clones are elaborated and discussed. In spite of the unforeseen effects on yeast physiology after the introduction of a new sequence into the genome, we obtained barcoded clones with the same xylose consumption rates and ethanol yields as the cells without the barcode.
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