| 1. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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Streamlined Preparation of Immobilized Candida antarctica Lipase B
- 2017
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Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 2:12, s. 8674-8677
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Tidskriftsartikel (refereegranskat)abstract
- Candida antarctica lipase B (CalB) was efficiently expressed (6.2 g L-1) in Escherichia coli by utilizing an N-terminal tag cassette and the XylS/Pm expression system in a fed-batch bioreactor; subsequent direct binding to EziG from crude extracts resulted in an immobilized catalyst with superior activity to Novozym 435.
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| 2. |
- Geschwindner, Stefan, et al.
(författare)
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Characterisation of de novo mutations in the C-terminal domain of proprotein convertase subtilisin/kexin type 9.
- 2015
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134.
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Tidskriftsartikel (refereegranskat)abstract
- Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the hepatic low-density lipoprotein receptor (LDL-R) and is therefore a prominent therapeutic target for reducing LDL-cholesterol. The C-terminal domain of PCSK9 is unlikely to be involved in a direct extracellular interaction with the LDL-R. We probed the importance of the C-terminus for the degradation of the LDL-R by designing seven de novo mutants of PCSK9 that fill potential druggable cavities. The mutants were tested for their ability to diminish LDL uptake in human HepG2 cells and for affinity towards a calcium independent mutant of the EGF(A) domain of the human LDL-R. The later was done by a newly developed surface plasmon resonance-based assay format. We identified three mutant proteins (G517R, V610R and V644R) with decreased ability to block LDL uptake into HepG2 cells. These mutations define areas outside the direct interaction area between PCSK9 and the LDL-R that could be targeted to inhibit the PCSK9 triggered degradation of the LDL-R. We also describe the mechanistic rationalisation of the affinity changes seen with the natural occurring human D374Y (gain of function) mutation causing severe hypercholesterolaemia. The action of this mutant is due to a significantly decreased dissociation rate constant, whereas the mutation does not affect the association rate constant.
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| 3. |
- Hermansen, Russell A, et al.
(författare)
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Characterizing selective pressures on the pathway for de novo biosynthesis of pyrimidines in yeast.
- 2015
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Ingår i: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Selection on proteins is typically measured with the assumption that each protein acts independently. However, selection more likely acts at higher levels of biological organization, requiring an integrative view of protein function. Here, we built a kinetic model for de novo pyrimidine biosynthesis in the yeast Saccharomyces cerevisiae to relate pathway function to selective pressures on individual protein-encoding genes.
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| 4. |
- Ishchuk, Olena, 1980, et al.
(författare)
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RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.
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| 5. |
- Koruza, Katarina, et al.
(författare)
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From initial hit to crystal optimization with microseeding of human carbonic anhydrase IX—A case study for neutron protein crystallography
- 2018
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Ingår i: Crystals. - : MDPI AG. - 2073-4352. ; 8:11
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Tidskriftsartikel (refereegranskat)abstract
- Human carbonic anhydrase IX (CA IX) is a multi-domain membrane protein that is therefore difficult to express or crystalize. To prepare crystals that are suitable for neutron studies, we are using only the catalytic domain of CA IX with six surface mutations, named surface variant (SV). The crystallization of CA IX SV, and also partly deuterated CA IX SV, was enabled by the use of microseed matrix screening (MMS). Only three drops with crystals were obtained after initial sparse matrix screening, and these were used as seeds in subsequent crystallization trials. Application of MMS, commercial screens, and refinement resulted in consistent crystallization and diffraction-quality crystals. The crystallization protocols and strategies that resulted in consistent crystallization are presented. These results demonstrate not only the use of MMS in the growth of large single crystals for neutron studies with defined conditions, but also that MMS enabled re-screening to find new conditions and consistent crystallization success.
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| 6. |
- Koruza, Katarina, et al.
(författare)
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Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative
- 2019
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1047-8477. ; 205:2, s. 147-154
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Tidskriftsartikel (refereegranskat)abstract
- Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.
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| 7. |
- Munch-Petersen, Birgitte, et al.
(författare)
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Jure Piskur (1960-2014)
- 2015
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Ingår i: Journal of Genetics and Genomics. - : Elsevier BV. - 1673-8527. ; 42:5, s. 275-277
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 8. |
- Mutahir, Zeeshan, et al.
(författare)
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Gene duplications and losses among vertebrate deoxyribonucleoside kinases of the non-TK1 Family
- 2016
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Ingår i: Nucleosides, Nucleotides and Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 35:10-12, s. 677-690
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Tidskriftsartikel (refereegranskat)abstract
- Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of dCK/deoxyguanosine kinase (dGK)-like enzymes from a frog Xenopus laevis and a bird Gallus gallus. We showed that X. laevis has a duplicated dCK gene and a dGK gene, whereas G. gallus has a duplicated dCK gene but has lost the dGK gene. We cloned, expressed, purified, and subsequently determined the kinetic parameters of the dCK/dGK enzymes encoded by these genes. The two dCK enzymes in G. gallus have broader substrate specificity than their human or X. laevis counterparts. Additionally, the duplicated dCK enzyme in G. gallus might have become mitochondria. Based on our study we postulate that changing and adapting substrate specificities and subcellular localization are likely the drivers behind the evolution of vertebrate dNKs.
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| 9. |
- Orozco Rodriguez, Juan Manuel, et al.
(författare)
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Expression of tomato thymidine kinase 1 by means of the baculovirus expression vector system
- 2016
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Ingår i: Nucleosides, Nucleotides and Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 35:10-12, s. 691-698
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Tidskriftsartikel (refereegranskat)abstract
- Tomato thymidine kinase 1 (ToTK1) is a deoxyribonucleoside kinase (dNK) that has been subject to study because of its potential to phosphorylate the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). The combination of ToTK1 and AZT has been tested in two animal studies for its efficiency and use in suicide gene therapy for malignant glioma. The determination of the 3D structure of ToTK1 might shed light on the structure–function relationships of nucleoside activation by this enzyme and thereby show routes toward further improvement of ToTK1 and other TK1-like dNKs for suicide gene therapy. Here we report the successful expression of both full-length ToTK1 and a C-terminal truncated ToTK1 in Spodoptera frugiperda and Trichoplusia ni insect cells using the baculovirus expression vector system. This constitutes a further step on the road to determine the 3D structure of the first TK1 of plant origin, but also an enzyme with great potential for dNK-mediated suicide gene therapy.
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| 10. |
- Slot Christiansen, Louise, et al.
(författare)
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New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine.
- 2015
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 7:2, s. 966-980
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Tidskriftsartikel (refereegranskat)abstract
- Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT).We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.
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