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| 2. |
- van den Berg, Susanne, et al.
(författare)
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Improved solubility of TEV protease by directed evolution.
- 2006
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Ingår i: Journal of biotechnology. - Amsterdam : Elsevier BV. - 0168-1656 .- 1873-4863. ; 121:3, s. 291-8
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Tidskriftsartikel (refereegranskat)abstract
- The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.
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| 3. |
- Bandmann, Nina, 1971-
(författare)
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Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bacterium Escherichia coli (E. coli) is in many situations an ideal host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve sufficiently high product quantities. However, there are several factors that may limit this host’s ability to produce large amounts of heterologous proteins in a soluble and native form. For many applications a high purity of the recombinant protein is demanded, which implies a purification strategy where the product efficiently can be isolated from the complex milieu of host cell contaminants. In this thesis, different strategies based on both rational and combinatorial genetic engineering principles have been investigated, aiming at improving and facilitating recombinant E. coli protein production and purification. One objective was to improve the PEG/salt aqueous two-phase system (ATPS) purification process of the lipase cutinase, by increasing the selectivity of the protein for the system top-phase. Peptide tags, with varying properties, were designed and genetically fused to the C-terminal end of ZZ-cutinase. Greatly increased partitioning values were observed for purified protein variants fused to tryptophan containing peptide tags, particularly a (WP)4 peptide. The partitioning properties of the ZZ-cutinase-(WP)4 protein were also retained when added to the ATPS directly from an E. coli total cell disintegrate, emphasizing the applicability of this genetic engineering strategy for primary protein purification in ATPSs. Further on, a combinatorial library approach using phage display technology was investigated as a tool for identification of peptide tags capable of improving partitioning properties of ZZ-cutinase in an ATPS. Repeated ATPS-based partitioning-selection cycles of a large phagemid (pVIII) peptide library, resulted in isolation of phage particles preferentially decorated with peptides rich in tyrosine and proline residues. Both a peptide corresponding to a phage library derived peptide sequence as well as peptides designed based on information of amino acid appearance frequencies in later selection rounds, were shown to improve partitioning several-fold when genetically fused to the C-terminal end of ZZ-cutinase. From the two- to four–fold increased production yields observed for these fusion proteins compared to ZZ-cutinase-(WP)4, it was concluded that the selection system used allowed for selection of desired peptide properties related to both partitioning and E. coli protein production parameters. Bacterial protein production is affected by several different mRNA and protein sequence-related features. Attempts to address single parameters in this respect are difficult due to the inter-dependence of many features, for example between codon optimization and mRNA secondary structure effects. Two combinatorial expression vector libraries (ExLib1 and ExLib2) were constructed using a randomization strategy that potentially could lead to variations in many of these sequence-related features and which would allow a pragmatic search of vector variants showing positive net effects on the level of soluble protein production. ExLib1 was constructed to encode all possible synonymous codons of an eight amino acid N-terminal extension of protein Z, fused to the N-terminal of an enhanced green fluorescent reporter protein (EGFP). In ExLib2, the same eight positions were randomized using an (NNG/T) degeneracy code, which could lead to various effects on both the nucleotide and protein level, through the introduction of nucleotide sequences functional as e.g. alternative ribosome binding or translation initiation sites or as translated codons for an Nterminal extension of the target protein by a peptide sequence. Flow cytometric analyses and sorting of library cell cultures resulted in isolation of clones displaying several-fold increases in whole cell fluorescence compared to a reference clone. SDS-PAGE and western blot analyses verified that this was a result of increases (up to 24-fold) in soluble intracellular ZEGFP product protein content. Both position specific codon bias effects and the appearance of new ribosomal binding sites in the library sequences were concluded to have influenced the protein production. To explore the possibility of applying the same combinatorial library strategy for improving soluble intracellular production of heterologous proteins proven difficult to express in E. coli, three proteins with either bacterial (a transcriptional regulator (DntR)) or human (progesterone receptor ligand binding domain (PRLBD) and 11-β Hydroxysteroid dehydrogenase type I (11-β)) origin, were cloned into the ExLib2 library. Flow cytometric sorting of libraries resulted in isolation of DntR library clones showing increased soluble protein production levels and PR-LBD library clones with up to ten-fold increases in whole cell fluorescence, although the product under these conditions co-separated with the insoluble cell material.
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| 4. |
- Ecke, Frauke, et al.
(författare)
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Sublethal Lead Exposure Alters Movement Behavior in Free-Ranging Golden Eagles
- 2017
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Ingår i: Environmental Science and Technology. - : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 51:10, s. 5729-5736
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Tidskriftsartikel (refereegranskat)abstract
- Lead poisoning of animals due to ingestion of fragments from lead-based ammunition in carcasses and offal of shot wildlife is acknowledged globally and raises great concerns about potential behavioral effects leading to increased mortality risks. Lead levels in blood were correlated with progress of the moose hunting season. Based on analyses of tracking data, we found that even sublethal lead concentrations in blood (25 ppb, wet weight), can likely negatively affect movement behavior (flight height and movement rate) of free ranging scavenging Golden Eagles (Aquila chrysaetos). Lead levels in liver of recovered post-mortem analyzed eagles suggested that sublethal exposure increases the risk of mortality in eagles. Such adverse effects on animals are probably common worldwide and across species, where game hunting with lead-based ammunition is widespread. Our study highlights lead exposure as a considerably more serious threat to wildlife conservation than previously realized and suggests implementation of bans of lead ammunition for hunting.
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| 5. |
- Svensson, Maria, 1969, et al.
(författare)
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Teaching and learning system thinking in technology
- 2015
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Ingår i: Plurality and Complementarity of Approaches in Design and Technology Education: PATT29 conference proceedings. Apr 2015, Marseille, France. - Marseille : Presses Universitaires de Provence. - 9782853999946 ; , s. 404-409, s. 404-409
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Konferensbidrag (refereegranskat)abstract
- Complex technological systems have emerged during the last decade as an important strand in technology teaching in several national curricula for compulsory school. However, even though understanding the systemic aspects and connected nature of contemporary society, it remains unclear what such understanding entails in detail, and even more unclear what may constitute good teaching. We present the results from a teaching-learning design project on the topic of large societal and complex technological systems, which are seen as constituted of transformation and transport, acting on matter, energy and information. The main results are a suggested and evaluated plan of teaching developed in collaboration with a team of technology teachers, as well as descriptions of how pupils’ system thinking is constituted in terms of four basic aspects: Resource and intention of the system; System component constitution; Process and transformation in components and system; Network character. In total, a teaching plan spanning four lessons was realised in four different classrooms, with classes’ sizes ranging 15 to 25 pupils in the ages 14 and 15. The teaching design progresses through focusing specific parts of various systems, for example the transformation of polluted water to clean water in a water purification plant as part of the water supply system. There is an emphasis on the function of the part in relation to the system on the one hand, and on how the part is and can be realised technically, taking care to relate the latter to what is taken up in other curricular strands of technology. The last part focuses the examination of technological systems as constituted by interacting and meaningful parts, where their network nature may emerge.
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| 6. |
- Fredriksson, Håkan, et al.
(författare)
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snowBOTS: a mobile robot on snow covered ice
- 2007
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Ingår i: Proceedings of the 13th IASTED International Conference on Robotics and Applications. - Anaheim, Calif. : ACTA Press. - 9780889866850 ; , s. 222-228
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Konferensbidrag (refereegranskat)abstract
- We introduce snowBOTs as a generic name for robots working in snow. This paper is a study on using scan ning range measuring lasers towards an autonomous snow cleaning robot, working in an environment consisting al most entirely of snow and ice. The problem addressed here is using lasers for detecting the edges generated by "the snow meeting the road". First the laser data were filtered using his togram/median to discriminate against falling snowflakes and small objects. Then the road surface was extracted us ing the range weighted Hough/Radon transform. Finally the left and right edges of the road was detected by thresh olding. Tests have been made with a laser on top of a car driven in an automobile test range just south of the Arctic Circle. Moreover, in the campus area, the algorithms were tested in closed loop with the laser on board a robotized wheelchair.
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| 7. |
- Hammarström, Martin, 1973-
(författare)
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Protein production and purification in structural genomics
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The number of gene products available for structural and functional study is increasing at an unprecedented rate as a result of the successful whole genome sequencing projects. Systematic structure determination of proteins on a genomic scale, called structural genomics, can significantly contribute to the field of protein science and to functional annotation of newly identified genes. This thesis covers different aspects of protein production in Eschericiha coli for structural studies in the context of structural genomics. Protocols have been downscaled and standardized to allow for a rapid assessment of the production characteristics for multiple proteins in parallel under a number of different conditions. Foremost, the ability of different proteins and peptide tags to affect the solubility of the recombinant protein when produced as fusion proteins has been systematically studied. Large differences in the success-rate for production of soluble protein in E. coli were found depending on the fusion partner used, with a more than two-fold increase in the number of proteins produced as soluble when comparing the best and the poorest fusion tags. For different constructs with a histidine tag, commonly used to facilitate protein purification, large differences in yield depending on the design of the expression vector were found. When comparing different fusion proteins produced from identical expression vectors, fusions to the GB1 domain were found to result in the highest yield of purified target protein, on average 25 % higher than any of the other fusions. The suitability for further structural studies was tested at an intermediate scale for proteins that were identified as soluble in the expression screening. For this purpose, protocols for rapid purification and biophysical characterization using nuclear magnetic resonance and circular dichroism spectroscopy were developed and tested on 19 proteins, of which four were structured.
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| 8. |
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| 9. |
- Wahlberg, Elisabet, et al.
(författare)
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An affibody in complex with a target protein: Structure and coupled folding.
- 2003
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 100:6, s. 3185-3190
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Tidskriftsartikel (refereegranskat)abstract
- Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z(SPA-1) affibody has been selected from a phage-displayed library as a binder to protein A. Z(SPA-1) also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z(SPA-1) affibody in its uncomplexed state and determined the solution structure of a Z:Z(SPA-1) protein-protein complex. Uncomplexed Z(SPA-1) behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z(SPA-1). We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z(SPA-1) and Ig Fc, respectively, suggesting that the Z(SPA-1) affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.
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| 10. |
- Östlund, Rickard, et al.
(författare)
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Evaluation of localization and failure of boron alloyed steels with different microstructure compositions
- 2014
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Ingår i: Journal of Materials Processing Technology. - : Elsevier BV. - 0924-0136 .- 1873-4774. ; 214:3, s. 592-598
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Tidskriftsartikel (refereegranskat)abstract
- Within the press hardening technology, where hot sheet blanks are simultaneously formed fixed and quenched, new methods with differential thermal treatment come to light. With controlled tool temperature variation, components with tailored properties can be produced. Automotive components combining high energy absorption and intrusion protection in a crash situation are feasible. In the present work the mechanical properties of three different material qualities, beginning with the same base sheet metal subjected to different thermal histories, are investigated. A strategy for modelling post-necking response and crack initiation using shell elements larger then the typical bandwidth of the localized neck is used. The model relies on a sequence of full field measurements throughout a tensile test; i.e. Digital Speckle Photography(DSP). The full field experimental method allows for evaluation of mechanical and failure properties at different analysis lengths, providing parameters for a model which accounts for shell element size. Additionally the model contains a strain based failure criteria as a function of stress triaxiality. Good correlations between a simulated tensile test and experimental results were found. A detailed metallographic study of the three grades was performed and is presented.
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