| 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. |
- 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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| 3. |
- Turesson, Helle, et al.
(författare)
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Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues
- 2010
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Ingår i: American Journal of Botany. - : Wiley. - 0002-9122 .- 1537-2197. ; 97:11, s. 1884-1893
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Tidskriftsartikel (refereegranskat)abstract
- Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.
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| 4. |
- 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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| 5. |
- Christensen, Simon, et al.
(författare)
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Conformational Dynamics of Phytoglobin BvPgb1.2 from Beta vulgaris ssp. vulgaris
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:4
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Tidskriftsartikel (refereegranskat)abstract
- Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (2H, 13C and 15N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1H-15N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins’ roles in planta.
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| 6. |
- Doan, Thuy, et al.
(författare)
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Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli
- 2009
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Ingår i: Journal of Plant Physiology. - : Elsevier BV. - 0176-1617 .- 1618-1328. ; 166:8, s. 787-796
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Tidskriftsartikel (refereegranskat)abstract
- Very long chain primary alcohols are significant components in cuticle waxes of plants. Fatty acyl-CoA reductases (FARs) catalyze the formation of a fatty alcohol from an acyl-CoA. The Arabidopsis (Arabidopsis thaliana) genome contains eight genes homologous to FAR genes from jojoba (Simmondsia chinensis), silk moth, wheat and mouse. Expression of six Arabidopsis FAR homologs in Escherichia coli resulted in production of alcohols from endogenous E. coli fatty acids by five of these genes, confirming that they encode for FAR enzymes. Only a truncated splicing version of the sixth gene was found, and this gene yielded a protein with no FAR activity. The five functional FAR enzymes yielded distinctly different compositions of fatty alcohols when expressed in E. coli, indicating that the different enzymes may be involved in the production of different types of alcohols in plant cells. (C) 2008 Elsevier GmbH. All rights reserved.
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| 7. |
- 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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| 8. |
- Isarankura-Na-Ayudhya, Chartchalerm, et al.
(författare)
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Computational Insights on Sulfonamide Imprinted Polymers
- 2008
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Ingår i: Molecules. - : MDPI AG. - 1420-3049. ; 13:12, s. 3077-3091
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Tidskriftsartikel (refereegranskat)abstract
- Molecular imprinting is one of the most efficient methods for preparing synthetic receptors that possess user defined recognition properties. Despite general success of non-covalent imprinting for a large variety of templates, some groups of compounds remain difficult to tackle due to their structural complexity. In this study we investigate preparation of molecularly imprinted polymers that can bind sulfonamide compounds, which represent important drug candidates. Compared to the biological system that utilizes metal coordinated interaction, the imprinted polymer provided pronounced selectivity when hydrogen bond interaction was employed in an organic solvent. Computer simulation of the interaction between the sulfonamide template and functional monomers pointed out that although methacrylic acid had strong interaction energy with the template, it also possessed high non-specific interaction with the solvent molecules of tetrahydrofuran as well as being prone to self-complexation. On the other hand, 1-vinylimidazole was suitable for imprinting sulfonamides as it did not cross-react with the solvent molecules or engage in self-complexation structures.
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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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