| 1. |
- Fexby, Sara, et al.
(författare)
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Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems
- 2004
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 20:3, s. 793-798
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Tidskriftsartikel (refereegranskat)abstract
- The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase.
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| 2. |
- Gonzalez Palmén, Lorena, et al.
(författare)
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A Double Role for a Strictly Conserved Serine : Further Insights into the dUTPase Catalytic Mechanism
- 2008
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Ingår i: Biochemistry. - : American Chemical Society. - 0006-2960 .- 1520-4995. ; 47:30, s. 7863-7874
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Tidskriftsartikel (refereegranskat)abstract
- Ser72 at the active site of the Escherichia coli dUTPase has been mutated to an alanine, and the properties of the mutant have been investigated. The serine is absolutely conserved among the monomeric and trimeric dUTPases (including the bifunctional dCTP deaminase:dUTPases), and it has been proposed to promote catalysis by balancing negative charge at the oxygen that bridges the α- and β-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue α,β-imido-dUTP·Mg, the serine β-OH is indeed hydrogen bonded to the α,β-bridging nitrogen of the analogue. However, in the complex of the Asp90→Asn mutant dUTPase with the true substrate dUTP·Mg, the serine β-OH points in the opposite direction and may form a hydrogen bond to Asn84 at the bottom of the pyrimidine pocket. Here we show that the replacement of the β-OH by hydrogen reduces kcat from 5.8 to 0.008 s-1 but also k-1, the rate of substrate dissociation, from 6.2 to 0.1 s-1 (KM = 6 × 10-9 M). We conclude that the serine β-OH exercises both ground state (GS) destabilization and transition state (TS) stabilization, effects not usually linked to a single residue. With experimental support, we argue that the β-OH destabilizes the GS by imposing conformational constraints on the enzyme and that formation ofthe TS depends on a rotation of the serine side chain that not only relieves the constraints but brings the β-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the β-OH to promote both deamination and hydrolysis in the bifunctional deaminases. We find that the E. coli dUTPase does not catalyze the hydrolysis of the α,β-imido-dUTP·Mg, suggesting that the analogue provides the hydrogen in the bond to the serine β-OH.
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| 3. |
- Carlsson, Magnus L.R., et al.
(författare)
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Expression, Purification and Initial Characterization of Functional α1-Microglobulin (A1M) in Nicotiana benthamiana
- 2020
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.
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| 4. |
- Kanagarajan, Selvaraju, et al.
(författare)
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Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
- 2021
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 184, s. 955-966
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Tidskriftsartikel (refereegranskat)abstract
- Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.
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| 5. |
- Andersson, Anneli, et al.
(författare)
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Continuous and simultaneous determination of venous blood metabolites
- 2017
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Ingår i: Talanta. - : Elsevier BV. - 0039-9140. ; 171, s. 270-274
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Tidskriftsartikel (refereegranskat)abstract
- Metabolic syndrome is associated with cardiovascular disease, type 2 diabetes mellitus (T2DM) and prediabetes. Metabolic syndrome is a cluster of interrelated clinical disorders. Difficulties in regulating glucose levels in blood are implicated in many of these disorders. Lactate, another energy metabolite, is produced under anaerobic conditions and can be used to monitor the balance between aerobic and anaerobic metabolism. Tested together, these metabolite levels can provide pro-diagnostic information that improves patient outcomes. Glucose and lactate were determined continuously and simultaneously in whole blood using a dual-channel thermal biosensor device in which one channel employed glucose oxidase for glucose analysis in comparison with lactate oxidase for lactate analysis in the others. No detectable clogging or interference was observed using venous blood samples. The linear detection range for both the glucose and lactate assays was 0.5–45 mM. The sampling rate of up to 24 samples per hour with assay cycle time of 2.5 min was achieved. Comparative analysis between our device and the HemoCue method showed an excellent correlation. The device was stable for hundreds of injections over a period of 45 days. The broad linear range, fast response and detection sensitivity are satisfactory for the clinical requirements, e.g. for diabetic or cardiovascular patients in intensive care units or surgical operation, where the tight control of blood glucose can decrease morbidity or mortality.
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| 6. |
- Kettisen, Karin, et al.
(författare)
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Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Due to its compatible oxygen-transporting abilities, hemoglobin (Hb) is a protein of interest in the development of artificial oxygen therapeutics. Despite continuous formulation attempts, extracellular Hb solution often exhibits undesirable reactions when applied in vivo. Therefore, protein engineering is frequently used to examine alternative ways of controlling the unwanted reactions linked to cell-free Hb solutions. In this study, three mutants of human fetal hemoglobin (HbF) are evaluated; single mutants αA12D and αA19D, and a double mutant αA12D/A19D. These variants were obtained by site-directed mutagenesis and recombinant production in E. coli, and carry negative charges on the surface of the α-subunit at the designated mutation sites. Through characterization of the mutant proteins, we found that the substitutions affected the protein in several ways. As expected, the isoelectric points (pIs) were lowered, from 7.1 (wild-type) down to 6.6 (double mutant), which influenced the anion exchange chromatographic procedures by shifting conditions toward higher conductivity for protein elution. The biological and physiological properties of HbF could be improved by these small modifications on the protein surface. The DNA cleavage rate associated with native HbF could be reduced by 55%. In addition, the negatively charged HbF mutant had an extended circulation time when examined in a mouse model using top load Hb additions. At the same time, the mutations did not affect the overall structural integrity of the HbF molecule, as determined by small-angle X-ray scattering. In combination with circular dichroism and thermal stability, modest structural shifts imposed by the mutations could possibly be related to changes in secondary structure or reorganization. Such local deformations were too minor to be determined within the resolution of the structural data; and overall, unchanged oxidation and heme loss kinetics support the conclusion that the mutations did not adversely affect the basic structural properties of Hb. We confirm the value of adding negatively charged residues onto the surface of the protein to improve the global functions of recombinant Hb.
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| 7. |
- Sakhnini, Laila Ismail, et al.
(författare)
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Designing monoclonal antibody fragment-based affinity resins with high binding capacity by thiol-directed immobilisation and optimisation of pore/ligand size ratio
- 2016
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1468, s. 143-153
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibody (mAb) based affinity resins usually suffer from low binding capacity, most probably as a result of steric hindrance by the large 150 kDa size of the mAb and a random immobilisation approach. The present work investigates the influence of a variety of factors on dynamic binding capacity (DBC) such as pore/ligand size ratio, accessibility of ligand and ligand density. The effect of pore/ligand size ratio was investigated using Fab and scFv fragments on various resins with different pore sizes. The accessibility of the ligand was investigated by a site-directed immobilisation approach, where three C-terminal tags, PPKPPK, FLAG™ and Cys, were introduced into the Fab fragments for immobilisation on resins via amino-, carboxyl- and thiol-groups, respectively. The scFv fragments were tagged at the C-terminal only with FLAG™ to enable a straight forward purification procedure, and were immobilised to resins via amino- and carboxyl-groups. The target protein had a molecular weight (MW) of 50 kDa. A 3-fold higher dynamic binding capacity at 100% breakthrough (DBC100%) was observed for Fab wild-type (wt) on CNBr-activated Sepharose 4 FF relative to mAb on same resin at the same ligand density. However, no major difference in DBC100% was observed between Fab wt and scFv immobilised on CNBr-activated Sepharose 4 FF at the same ligand density. Thus, further increase of pore/ligand size ratio from Fab to scFv on a resin with average pore size of 300 Å, did not seem to be beneficial. Among the tested tags, only the C-terminal Cys tag proved to site-direct the ligands during immobilisation as it allowed the DBC100% to increase 1.6-fold as compared to Fab wt immobilised via amino-groups on CNBr-activated Sepharose 4 FF and Actigel ALD Superflow at the same ligand density. The influence of ligand density was investigated by selecting immobilised Fab Cys on Sulfhydryl-reactive resin. Increasing ligand density from 0.103 to 0.202 μmol/mL resulted in the same utilisation yield (82–85%), whereas a further increase in ligand density from 0.202 to 0.328 μmol/mL resulted in a 20%-unit decrease in utilisation yield and less steep breakthrough curve, suggesting steric hindrance in the pores of the resin. In addition, site-directed affinity ligands resulted in a more pronounced, sigmoid-shaped breakthrough curve, leading to more efficient use of capacity. The highest DBC100% was obtained for Fab Cys on Sulfhydryl-reactive resin and scFv on Actigel ALD Superflow; 11 mg/mL and 10 mg/mL, respectively, as compared to the DBC100% of 0.8 mg/mL for mAb on CNBr-activated Sepharose 4 FF. Pore/ligand size ratio of 3, which was achieved for Fab ligands on the studied resins, was shown to be feasible for capturing a protein in MW of 50 kDa. Totally, a 13.8-fold improvement in DBC100% was achieved with the Fab-based affinity resin coupled via the C-terminal Cys as compared to the mAb-based affinity resin.
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| 8. |
- Silkstone, R S, et al.
(författare)
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The βLys66Tyr Variant of Human Hemoglobin as a Component of a Blood Substitute.
- 2016
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Ingår i: Advances in Experimental Medicine and Biology. - New York, NY : Springer New York. - 0065-2598. ; 876, s. 455-460
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Tidskriftsartikel (refereegranskat)abstract
- It has been proposed that introducing tyrosine residues into human hemoglobin (e.g. βPhe41Tyr) may be able to reduce the toxicity of the ferryl heme species in extracellular hemoglobin-based oxygen carriers (HBOC) by facilitating long-range electron transfer from endogenous and exogenous antioxidants. Surface-exposed residues lying close to the solvent exposed heme edge may be good candidates for mutations. We therefore studied the properties of the βLys66Tyr mutation. Hydrogen peroxide (H2O2) was added to generate the ferryl protein. The ferryl state in βLys66Tyr was more rapidly reduced to ferric (met) by ascorbate than recombinant wild type ((r)wt) or βPhe41Tyr. However, βLys66Tyr suffered more heme and globin damage following H2O2 addition as measured by UV/visible spectroscopy and HPLC analysis. βLys66Tyr differed notably from the (r)wt protein in other ways. In the ferrous state the βLys66Tyr forms oxy, CO, and NO bound heme complexes similar to (r)wt. However, the kinetics of CO binding to the mutant was faster than (r)wt, suggesting a more open heme crevice. In the ferric (met) form the typical met Hb acid-alkaline transition (H2O to (-)OH) appeared absent in the mutant protein. A biphasicity of cyanide binding was also evident. Expression in E. coli of the βLys66Tyr mutant was lower than the (r)wt protein, and purification included significant protein heterogeneity. Whilst, βLys66Tyr and (r)wt autoxidised (oxy to met) at similar rates, the oxygen p50 for βLys66Tyr was very low. Therefore, despite the apparent introduction of a new electron transfer pathway in the βLys66Tyr mutant, the heterogeneity, and susceptibility to oxidative damage argue against this mutant as a suitable starting material for a HBOC.
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| 9. |
- Johansson, Hans-Olof, et al.
(författare)
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Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems.
- 2012
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1233, s. 30-35
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Tidskriftsartikel (refereegranskat)abstract
- Phase diagrams of poly(ethylene glycol)/polyacrylate/Na(2)SO(4) systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coli can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coli homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na(2)SO(4)-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH.
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| 10. |
- 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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