| 1. |
- Blanch, Ewan W, et al.
(författare)
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Raman optical activity characterization of native and molten globule states of equine lysozyme : comparison with hen lysozyme and bovine alpha-lactalbumin
- 2000
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Ingår i: Biopolymers. - 0006-3525 .- 1097-0282. ; 57:4, s. 235-248
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Tidskriftsartikel (refereegranskat)abstract
- Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrated form of alpha helix, is more similar to that in the ROA spectrum of bovine alpha-lactalbumin than hen lysozyme and may be associated with the greater flexibility and calcium-binding ability of equine lysozyme and bovine alpha-lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at approximately 1300 cm(-1) in hen lysozyme that is assigned to an alpha helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at approximately 1305 cm(-1), which may reflect greater heterogeneity in some alpha-helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22 degrees C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium-binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22 degrees C and the apo form at pH 4.5 and 48 degrees C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated alpha helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine alpha-lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at approximately 1551 cm(-1) in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.
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| 2. |
- De Scheerder, Marie-Angélique, et al.
(författare)
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Evaluating predictive markers for viral rebound and safety assessment in blood and lumbar fluid during HIV-1 treatment interruption.
- 2020
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Ingår i: The Journal of antimicrobial chemotherapy. - : Oxford University Press (OUP). - 1460-2091 .- 0305-7453. ; 75:5, s. 1311-20
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Tidskriftsartikel (refereegranskat)abstract
- Validated biomarkers to evaluate HIV-1 cure strategies are currently lacking, therefore requiring analytical treatment interruption (ATI) in study participants. Little is known about the safety of ATI and its long-term impact on patient health.ATI safety was assessed and potential biomarkers predicting viral rebound were evaluated.PBMCs, plasma and CSF were collected from 11 HIV-1-positive individuals at four different timepoints during ATI (NCT02641756). Total and integrated HIV-1 DNA, cell-associated (CA) HIV-1 RNA transcripts and restriction factor (RF) expression were measured by PCR-based assays. Markers of neuroinflammation and neuronal injury [neurofilament light chain (NFL) and YKL-40 protein] were measured in CSF. Additionally, neopterin, tryptophan and kynurenine were measured, both in plasma and CSF, as markers of immune activation.Total HIV-1 DNA, integrated HIV-1 DNA and CA viral RNA transcripts did not differ pre- and post-ATI. Similarly, no significant NFL or YKL-40 increases in CSF were observed between baseline and viral rebound. Furthermore, markers of immune activation did not increase during ATI. Interestingly, the RFs SLFN11 and APOBEC3G increased after ATI before viral rebound. Similarly, Tat-Rev transcripts were increased preceding viral rebound after interruption.ATI did not increase viral reservoir size and it did not reveal signs of increased neuronal injury or inflammation, suggesting that these well-monitored ATIs are safe. Elevation of Tat-Rev transcription and induced expression of the RFs SLFN11 and APOBEC3G after ATI, prior to viral rebound, indicates that these factors could be used as potential biomarkers predicting viral rebound.
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| 3. |
- Fodera, Vito, et al.
(författare)
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Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures
- 2014
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Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 5:18, s. 3254-3258
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Tidskriftsartikel (refereegranskat)abstract
- Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.
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| 4. |
- Malisauskas, Mantas, et al.
(författare)
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Amyloid protofilaments from the calcium-binding protein equine lysozyme : formation of ring and linear structures depends on pH and metal ion concentration
- 2003
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 330:4, s. 879-890
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Tidskriftsartikel (refereegranskat)abstract
- The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 °C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca2+ the protofilaments are present as annular structures with a diameter of 40–50 nm. In the presence of 10 mM CaCl2 the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 °C and 57 °C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70–80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1–80 and 54–125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as -synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.
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| 5. |
- Morozova-Roche, Ludmilla A, et al.
(författare)
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Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants
- 2000
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1047-8477 .- 1095-8657. ; 130:2-3, s. 339-351
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Tidskriftsartikel (refereegranskat)abstract
- Wild-type human lysozyme and its two stable amyloidogenic variants have been found to form partially folded states at low pH. These states are characterized by extensive disruption of tertiary interactions and partial loss of secondary structure. Incubation of the proteins at pH 2.0 and 37 degrees C (Ile56Thr and Asp67His variants) or 57 degrees C (wild-type) results in the formation of large numbers of fibrils over several days of incubation. Smaller numbers of fibrils could be observed under other conditions, including neutral pH. These fibrils were analyzed by electron microscopy, Congo red birefringence, thioflavine-T binding, and X-ray fiber diffraction, which unequivocally show their amyloid character. These data demonstrate that amyloidogenicity is an intrinsic property of human lysozyme and does not require the presence of specific mutations in its primary structure. The amyloid fibril formation is greatly facilitated, however, by the introduction of "seeds" of preformed fibrils to the solutions of the variant proteins, suggesting that seeding effects could be important in the development of systemic amyloidosis. Fibril formation by wild-type human lysozyme is greatly accelerated by fibrils of the variant proteins and vice versa, showing that seeding is not specific to a given protein. The fact that wild-type lysozyme has not been found in ex vivo deposits from patients suffering from this disease is likely to be related to the much lower population of incompletely folded states for the wild-type protein compared to its amyloidogenic variants under physiological conditions. These results support the concept that the ability to form amyloid is a generic property of proteins, but one that is mitigated against in a normally functioning organism.
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| 6. |
- Morozova-Roche, Ludmilla A, et al.
(författare)
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Independent nucleation and heterogeneous assembly of structure during folding of equine lysozyme
- 1999
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 289:4, s. 1055-1073
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Tidskriftsartikel (refereegranskat)abstract
- The refolding of equine lysozyme from guanidinium chloride has been studied using hydrogen exchange pulse labelling in conjunction with NMR spectroscopy and stopped flow optical methods. The stopped flow optical experiments indicate that extensive hydrophobic collapse occurs rapidly after the initiation of refolding. Pulse labelling experiments monitoring nearly 50 sites within the protein have enabled the subsequent formation of native-like structure to be followed in considerable detail. They reveal that an intermediate having persistent structure within three of the four helices of the alpha-domain of the protein is formed for the whole population of molecules within 4 ms. Subsequent to this event, however, the hydrogen exchange protection kinetics are complex and highly heterogeneous. Analysis of the results by fitting to stretched exponential functions shows that a series of other intermediates is formed as a consequence of the stepwise assembly of independently nucleated local regions of structure. In some molecules the next step in folding involves the stabilisation of the remaining helix in the alpha-domain, whilst in others persistent structure begins to form in the beta-domain. The formation of native-like structure throughout the beta-domain is itself heterogeneous, involving at least three kinetically distinguishable steps. Residues in loop regions throughout the protein attain persistent structure more slowly than regions of secondary structure. There is in addition evidence for locally misfolded regions of structure that reorganise on much longer timescales. The results reveal that the native state of the protein is generated by the heterogeneous assembly of a series of locally cooperative regions of structure. This observation has many features in common with the findings of recent theoretical simulations of protein folding.
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| 7. |
- Noppe, Wim, et al.
(författare)
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Apparent heterogeneity in the pIII-peptide fusion protein in single-phage clones isolated from peptide libraries
- 2011
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 24:9, s. 721-726
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Tidskriftsartikel (refereegranskat)abstract
- Ligand homogeneity is an important issue in affinity chromatography. Using phages expressing peptides on the pIII protein, a heterogeneity in the binding of monoclonal phages was observed during affinity chromatography on supermacroporous cryogels. Fractions with different apparent binding affinities could be separated by stepwise elution. When these different fractions were re-applied, the respective differences in affinity were retained. However, when phage fractions with different apparent affinities were first amplified, an offspring was generated with again variable affinities. As the sequence of the peptide insert was the same, the heterogeneity must be ascribed to differences in avidity and although no direct evidence could be generated, we hypothesize that this is possibly due to phages displaying different numbers of the same peptide as a consequence of either proteolytic or packaging events during the amplification step in Escherichia coli.
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| 8. |
- Noppe, Wim
(författare)
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Bacteriophages and cryogels: A new efficient tool in bioseparation
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Abstract Due to developments in the last decades in the field of protein production, new technologies for bio-separation have evolved. Especially in the field of affinity chromatography, the search for new ligands is of great interest. Besides the antibodies used as affinity ligands, synthetic ligands were introduced. Textile dyes were a first group to be introduced but despite their selectivity and capacity, the use was not very successful due to leakage and toxicity problems. More attention was focused on synthetic peptide ligands which could be synthesized using different techniques. Chemical procedures such as combinatorial chemistry, solid phase chemistry, docking simulation are applied to select peptides with affinity towards a target. By using a biological technique, Phage Display Technology, peptides can also be selected by screening peptide phage libraries towards a target. New protein production technologies have also an influence on down-stream processing techniques. Feeds such as fermentation broth, transgenic milk are particulate feed stocks which implies higher demands from the chromatographic matrices. New matrices and techniques are developed to deal with this challenge. Supermacroporous cryogel monoliths are an excellent tool for this purpose. Due to macropores with the size up to 100 µm, particulate feeds such as fermentation broth, microbial and mammalian cell cultures, milk and even whole blood can flow through the cryogel without any restriction. This thesis describes novel methods of bio-panning and as well as chromatography. Bio-panning in search for new affinity ligands is done by phage display technology. Instead of synthesizing peptides, the bacteriophages expressing a peptide on an envelope protein are used as ligands. This has several advantages compared to the pure peptide.(i) Once the phage is selected, it can be amplified in great quantities at low cost as compared to the synthesis of pure peptide ligands. (ii) By using bacteriophages a very specific ligand is available combining the effects of “affinity” and “avidity” for the optimal capture of target. By combining the cryogel monolith column with bacteriophages as affinity ligands we provided “proof of principle” for this new technique in bio-separation. Two case studies are presented. A selected peptide expressing bacteriophage clone was coupled to a cryogel monolith column. In case study one, human lactoferrin or recombinant human lactoferrin was captured from human milk or transgenic bovine milk respectively. In case study two, von Willebrand factor was captured from plasma and whole blood. In both case the captured protein of interest was recovered with a purity of >95% in a single step chromatographic procedure. Taking advantage of the structural properties of the cryogel monolith a compact bio-panning procedure, Chromato-panning, was developed. Both the screening of phage libraries as the infection of phages in E.coli cells are performed in on-column mode. As a result a significant decrease in time for bio-panning was observed. The procedure is more compact and quicker and only one panning round has to be performed in order to obtain high selective phage clones as compared to conventional bio-panning procedures.
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| 9. |
- Noppe, Wim, et al.
(författare)
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Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries
- 2009
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Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Background: Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library. Results: Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages. Conclusion: Chromato-panning allows combining several steps of the panning procedure resulting in 4-8 fold decrease of total time needed for phage selection.
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| 10. |
- Plieva, Fatima, et al.
(författare)
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Cryogel applications in microbiology.
- 2008
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Ingår i: Trends in Microbiology. - : Elsevier BV. - 1878-4380 .- 0966-842X. ; 16:11, s. 543-551
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Tidskriftsartikel (refereegranskat)abstract
- There is a great demand for improved technologies with regard to rapid processing of nano- and microparticles. The handling of viruses in addition to microbial and mammalian cells requires the availability of appropriate adsorbents. Recent developments in macroporous gels produced at subzero temperatures (known as cryogels) have demonstrated an efficiency for processing cell and virus suspensions, cell separation and cell culture applications. Their unique combination of properties such as macroporosity, tissue-like elasticity and biocompatibility, physical and chemical stability and ease of preparation, renders these materials interesting candidates for a broad range of potential applications within microbiological research. This review describes current applications of macroporous cryogels in microbiology with a brief discussion of future perspectives.
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