| 1. |
- Aevarsson, Arnthór, et al.
(författare)
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Going to extremes - a metagenomic journey into the dark matter of life
- 2021
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968. ; 368:12
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Forskningsöversikt (refereegranskat)abstract
- The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
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| 2. |
- Ahlqvist, Josefin
(författare)
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A study of DNA interactive proteins and their origin from thermophilic Siphoviridae phages
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis covers the results of studies on the amino acid sequence, three-dimensional structure, and activities of two types of DNA-interactive proteins: Holliday Junction resolving enzyme (represented by Hjc_15-6 isolated from phage Tth15-6) and DNA Polymerase I (represented by PolI_G20c, isolated from phage G20c), including studies on their homologous counterparts. As these proteins originate from two different, but related phages that infect Thermus thermophilus, the studies also include the evolutionary relationship between these and other related phages at genomic level. The novel phage Tth15-6 was isolated, its genome sequenced, and from the sequence data, a large number of genes were selected. The selected genes were cloned and expressed in Escherichia coli and the resulting production levels of soluble target protein were analysed. The DNA polymerase encoded in phage Tth15-6, PolI_15-6, was considered interesting, due to its relatively low sequence conservation compared to previously studied candidates from the same family of polymerases. It was, however, not possible to produce the protein in its soluble form in the selected E. coli production host. Therefore, a gene encoding a homologous protein, PolI_G20c, was identified, cloned and expressed, that resulted in soluble production of the target protein. After purification, PolI_G20c was crystallised, but its structure could not be solved by molecular replacement, using available structures in the Protein Data Bank, PDB. However, a fragment of a homologous protein, ExnV1, was also produced and crystallised. In this case, it was possible to solve the three-dimensional structure, which was subsequently used as a template, allowing the determination of the three-dimensional structure of PolI_G20c. The gene encoding the Holliday junction resolving enzyme, Hjc_15-6, was also cloned, and the enzyme was produced, purified and crystallised. In this case, production of a selenium derivatized variant was successful. Crystallisation of both variants, allowed determination of the three-dimensional structure. Studies were also made on how Hjc_15-6 interacts with branched DNA structures and single peptides in conjunction with polymerases and polymerase reactions. Analysis of the genome sequence of phage Tth15-6, showed that the identified open reading frames in many cases lacked homologues of known function. This sequence divergence makes it difficult to find related candidates of known function using traditional bioinformatics. However, the organisation of genes in the whole genome may be a valuable tool for this purpose. The evolutionary relationship between phage Tth15-6, phage G20c, and other related phages, were also reflected in the deduced amino acid sequences of the enzymes Hjc_15-6 and PolI_G20c. A three-part sequence signature motif of Hjc_15-6 differed from corresponding motif in earlier reported Hj resolving enzymes. It was thus proposed that this novel sequence signature is common among Hj resolving enzymes originating from the related phages. Furthermore, Hjc_15-6 is the first Hj resolving enzyme originating from a phage that could be classified as an archaeal type Hj resolving enzyme, regarding both its amino acid sequence, and its structure. This is the first time an archaeal type Hj resolving enzyme originating from a phage is reported. The structure of PolI_G20c and ExnV1 revealed a new structural motif that has not previously been reported among type 1 polymerases. However, based on the amino acid sequence analysis, this motif may also occur in enzymes from related phages. Finally, the studies show that Hjc_15-6 has unusual features not reported among Hj resolving enzymes elsewhere. Therefore, Hjc_15-6 was studied further, both regarding its capability to cleave DNA oligomers, and its function in conjunction with polymerases and polymerase reactions. It was revealed that Hjc_15-6 is capable of cleaving branched products from isothermal polymerase reactions based on strand replacement. It was also shown that it was possible to induce a polymerase reaction with neither DNA template nor primers present, when Hjc_15-6 was present. Moreover, Hjc_15-6 may form large networks of DNA, something that was studied in real-time under an electron microscope. Hence, Hjc_15-6 may be a valuable tool for DNA origami with a wide variety of applications. This thesis contains four research papers along with introducing chapters for the reader's orientation including brief presentations of the content in the Papers, followed by a chapter of Concluding remarks where the most important findings in the articles are presented, followed by a chapter of Future prospects, Acknowledgements and References.
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| 3. |
- Ahlqvist, Josefin, et al.
(författare)
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Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies
- 2006
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 122:2, s. 216-225
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Tidskriftsartikel (refereegranskat)abstract
- A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B.. 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.
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| 4. |
- Ahlqvist, Josefin, et al.
(författare)
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Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6
- 2022
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 78:Pt 2, s. 212-227
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Tidskriftsartikel (refereegranskat)abstract
- This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.
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| 5. |
- Ahlqvist, Josefin, et al.
(författare)
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Crystal structure of DNA polymerase I from Thermus phage G20c
- 2022
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 78:Pt 11, s. 1384-1398
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Tidskriftsartikel (refereegranskat)abstract
- This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.
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| 6. |
- Ahlqvist, Josefin, et al.
(författare)
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DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions
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Ingår i: Journal of Biotechnology. - 1873-4863.
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Tidskriftsartikel (refereegranskat)abstract
- The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.
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| 7. |
- Ahlqvist, Josefin, et al.
(författare)
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Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 354:2, s. 229-237
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Tidskriftsartikel (refereegranskat)abstract
- A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved.
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| 8. |
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| 9. |
- Bontidean, Ibolya, et al.
(författare)
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Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection
- 2003
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Ingår i: Biosensors & Bioelectronics. - 1873-4235. ; 18:5-6, s. 547-553
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Tidskriftsartikel (refereegranskat)abstract
- A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described. Synthetic phytochelatin (Glu-Cys)20Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor. The new biosensor was able to detect Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions in concentration range of 100 fM–10 mM, and the order of sensitivity was SZn>SCu>SHg>>SCdSPb. The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.
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| 10. |
- Herraiz-Adillo, Ángel, et al.
(författare)
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Life’s Essential 8 in relation to self-rated health and health-related quality of life in a large population-based sample : the SCAPIS project
- 2024
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Ingår i: Quality of Life Research. - : Springer Nature. - 0962-9343 .- 1573-2649. ; 33, s. 1003-1014
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: To monitor cardiovascular health, in 2022, the American Heart Association (AHA) updated the construct “Life’s Simple 7” (LS7) to “Life’s Essential 8” (LE8). This study aims to analyze the associations and capacity of discrimination of LE8 and LS7 in relation to self-rated health (SRH) and health-related quality of life (HRQoL).Methods: This study from the Swedish CArdioPulmonary bioImage Study (SCAPIS) included 28 731 Swedish participants, aged 50–64 years. Three different scores were derived from the SF-12 questionnaire: 1-item question SRH (“In general, would you say your health is …?”), mental-HRQoL and physical-HRQoL. Logistic regression, restricted cubic splines, and ROC analysis were used to study the associations between the AHA scores in relation to SRH and HRQoL.Results: Compared to those with a LE8 score of 80, participants with a LE8 score of 40 were 14.8 times more likely to report poor SRH (OR: 14.8, 95% CI: 13.0–17.0), after adjustments. Moreover, they were more likely to report a poor mental-HRQoL (OR: 4.9, 95% CI: 4.2–5.6) and a poor physical-HRQoL (OR: 8.0, 95% CI: 7.0–9.3). Area under curves for discriminating poor SRH were 0.696 (95% CI: 0.687–0.704), 0.666 (95% CI: 0.657–0.674), and 0.643 (95% CI: 0.634–0.651) for LE8, LS7 (0–14), and LS7 (0–7), respectively, all p values < 0.001 in the DeLong’s tests.Conclusion: LE8 and LS7 had strong and inverse associations with SRH, mental-HRQoL, and physical-HRQoL, though LE8 had a somewhat higher capacity of discrimination than LS7. The novel LE8, a construct initially conceived to monitor cardiovascular health, also conveys SRH and HRQoL.
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