| 1. |
- Kallberg, Kristian, et al.
(författare)
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Application of a pH responsive multimodal hydrophobic interaction chromatography medium for the analysis of glycosylated proteins.
- 2011
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Ingår i: Journal of chromatography. A. - : Elsevier BV. - 1873-3778 .- 0021-9673. ; 128:5, s. 678-683
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Tidskriftsartikel (refereegranskat)abstract
- Protein glycosylation has significant effects on the structure and function of proteins. The efficient separation and enrichment of glycoproteins from complex biological samples is one key aspect and represents a major bottleneck of glycoproteome research. In this paper, we have explored pH multimodal hydrophobic interaction chromatography to separate glycosylated from non-glycosylated forms of proteins. Three different proteins, ribonuclease, invertase and IgG, have been examined and different glycoforms have been identified. The media itself shows strong responsiveness to small variations in pH, which makes it possible to fine-tune the chromatographic conditions according to the properties of the protein isolated. Optimal glycoprotein separation has been obtained at pH 4. The pH responsive multimodal HIC medium in contrast to conventional HIC media is able to resolve contaminating DNA.
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| 2. |
- Kallberg, Kristian, et al.
(författare)
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Multimodal chromatography: An efficient tool in downstream processing of proteins.
- 2012
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768. ; 7:12, s. 1485-1495
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Tidskriftsartikel (refereegranskat)abstract
- Chromatography has become an indispensable tool for the purification of proteins. Since the regulatory demands on protein purity are expected to become stricter, the need for generating improved resins for chromatographic separations has increased. More advanced scientific investigations of protein structure/function relationships, in particular, have also been a driving force for generating more sophisticated chromatographic materials for protein separations. As a consequence, the development of alternative chromatographic strategies has been very rapid during the past decade and several new ligands have been designed and explored both in the laboratory and in large-scale industrial settings. This review describes some of these efforts using multimodal chromatography, where two or more physicochemical properties are used to enhance the specificity of the interactions between the protein and the ligand on the chromatographic matrix. In addition to experimental studies, computer modeling of ligand-protein binding has improved the design of ligands for protein recognition. The use of descriptors as well as in silico docking methods have been implemented to design multimodal resins in several instances.
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| 3. |
- Kallberg, Kristian
(författare)
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Multimodal Interaction Chromatography for the Investigation and Separation of Proteins
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Minor structural changes on the surface of proteins can have tremendous impact on their biological properties. When producing therapeutic proteins it is therefore important to verify that the generated protein is the desired one. Small variations such as glycosylations or amino acid substitutions can mean the difference between a functional, defective or even toxic product. This thesis describes the characterization and use of a multimodal pH-tunable hydrophobic interaction chromatography media for separating proteins with small structural differences. The media is constructed by attaching copolymer ligands on the surface of spherical agarose support beads. From the multimodal character of the polymeric ligand it is possible to allow several interaction types to be in effect at the same time, a property which makes it sensitive to small protein surface modifications. The chromatography media presented here is mainly hydrophobic but will also participate in electrostatic interactions and form hydrogen bonds. In addition, the media will react to the pH of the surrounding solvent and change its hydrophobicity. At low pH it will be more hydrophobic than at neutral pH. Both pH-dependent behavior and how polymer length effects separation have been explored. The pH-responsiveness of the media provides opportunities to fine-tune the separation process to virtually any protein desirable. This property was used in the work presented here for purifying delicate recombinant hemoglobin. The high selectivity that was obtained from the multimodality was used to separate and investigate the stability of the haptoglobin-hemoglobin complex. Additionally, the media was able to separate glycosylated IgG variants which traditional HIC media could not. This property enabled developing a rapid monitoring method for antibody producing cell systems.
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| 4. |
- Mehmeti-Ajradini, Meliha, et al.
(författare)
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Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer
- 2020
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Ingår i: Life Science Alliance. - 2575-1077. ; 3:11
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Tidskriftsartikel (refereegranskat)abstract
- Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients cotransplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.
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| 5. |
- Mehmeti-Ajradini, Meliha, et al.
(författare)
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Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer
- 2020
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Ingår i: Life Science Alliance. - : LIFE SCIENCE ALLIANCE LLC. - 2575-1077. ; 3:11
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Tidskriftsartikel (refereegranskat)abstract
- Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients co-transplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.
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| 6. |
- Silkstone, Gary G A, et al.
(författare)
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Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin : Implications for blood substitute design
- 2016
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Ingår i: Biochemical Journal. - 0264-6021. ; 473:19, s. 3371-3383
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Tidskriftsartikel (refereegranskat)abstract
- Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic hememediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-Active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the β-subunit (F41). We therefore replaced this residue with a tyrosine (βF41Y, Hb Mequon). The βF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, βF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the β-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in βF41Y. NO bioavailability was enhanced in βF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the β-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-Active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.
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