| 1. |
- Kazakov, Sergey V., et al.
(författare)
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Light Scattering Study of the Antibody-Poly(methacrylic acid) and Antibody-Poly(acrylic acid) Conjugates in Aqueous Solutions
- 2001
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Ingår i: Macromolecular Bioscience. - 1616-5187. ; 1:4, s. 157-163
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Tidskriftsartikel (refereegranskat)abstract
- The effect of the conformational state of the polymer coil on the properties of protein-polymer conjugates has been studied for the conjugates of antibody (monoclonal antibody from 6C5 clone against inactivated rabbit muscle glyceraldehyde-3-phosphate dehydrogenase; Ab) with poly(methacrylic acid) (PMAA) or poly-(acrylic acid) (PAA). The pH-dependencies of molecular properties and structural parameters of aqueous solutions (radius of gyration, intensity of scattered light, hydrodynamic diameter, and polydisperisty index) of Ab, PMAA, and PAA, have been studied using static and dynamic light scattering techniques. While free Ab aggregates in solution and precipitates at its isoelectric point, the covalent attachment of a charged polymer to Ab prevents its association and shifts the precipitation point towards more acidic values (from pH 5.95 for Ab to pH ∼ 4.8 for Ab- PMAA). The predominant role of the conformational status of the polymer in the process of conjugate precipitation has been considered. Contrary to the precipitation of Ab-PMAA, the formation of stable colloidal particles was suggested for Ab-PAA at pH < 4.8. In the conjugates, polymer chains surround the protein globule in an extremely compact manner while Ab significantly affects the polymer conformation. The essentially larger hydrodynamic radii of conjugates, when compared with their radii of gyration, confirm the strong interaction of conjugates with solvent molecules.
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| 2. |
- Ivinova, Olga, et al.
(författare)
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Influence of complexing polyanions on the thermostability of basic protiens.
- 2003
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Ingår i: Macromolecular Bioscience. - : Wiley. - 1616-5195 .- 1616-5187. ; 3:3-4, s. 210-215
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Tidskriftsartikel (refereegranskat)abstract
- Lysozyme (Lyz), chymotrypsinogen (Cht), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as model proteins capable of forming water-soluble polyelectrolyte complexes with linear synthetic polyanions. The complex formation with sodium poly(methacrylate) (PMA), sodium poly(acrylate) (PAA), sodium poly(anetholsulfonate) (PAS), and potassium poly(vinylsulfate) (PVS) markedly reduced the temperature of protein denaturation, Tmax, as determined by differential scanning calorimetry (DSC). The effect of sodium poly(styrenesulfonate) (PSS) on Lyz was so drastic that the protein melting peak was not observed at all during DSC measurements. The temperature shift, most pronounced for Lyz, increased upon substitution of the polyanions according to the following series: PMA < PVS < PAA < PAS < PSS. Decomposition of the complexes by addition of either sodium chloride or poly(N-ethyl-4-vinylpyridinium) cation completely restored the initial Tmax of the protein (except for PSS and PAS). The complex formation slightly affected the enzyme activity up to temperatures close to Tmax of the polyanion-protein complex. On further heating, the activity of the complex decreased steeply, whereas the free enzyme maintained a high activity. The data obtained strongly suggest that the protein-polyelectrolyte interactions in solution, while leaving the thermostability and activity of the proteins practically unaffected over a rather wide temperature range, result in the effective denaturation of proteins once a certain critical temperature is achieved. This finding appears to be crucial for further development of immobilized enzymes in biotechnology and essential for understanding mechanisms and principles of the functioning of proteins immobilized on charged matrices in vivo.
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| 3. |
- Izumrudov, Vladimir, et al.
(författare)
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Factors Controlling Phase Separation in Water-Salt Solutions of DNA and Polycations.
- 2003
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 19:11, s. 4733-4739
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Tidskriftsartikel (refereegranskat)abstract
- Factors affecting phase separation in water-salt solutions of polyelectrolyte complexes (PECs), formed by DNA and integral or pendant polycations with a quaternary amino group in every monomer unit, have been studied. When no salt was added, quantitative DNA precipitation occurred at a stoichiometric charge ratio, = [+]/[-] 1. In DNA mixtures with poly(N,N'-dimethyldiallylammonium chloride) (PDMDAAC, a pendant polycation), insoluble PECs formed in the range 0.7 < < 2. This suggests the formation of soluble, negatively charged PECs at 0 < < 0.7 and soluble, positively charged PECs at > 2. For different aliphatic ionene bromides (integral polycations), the range of corresponding to insoluble PECs was significantly broader, mainly due to the poor ability of the ionenes to form soluble, positively charged PECs. The range was also relatively broad for poly(N-ethyl-4-vinylpyridinium bromide) (a pendant polycation) and became broader with decreasing degree of polymerization of the polycation. The formation of insoluble PECs was favored by the addition of salt (NaCl), and the effect was more pronounced when decreasing the relative content of the solubilizing component, i.e., the nucleic acid at < 1 and the polycation at > 1. At moderate ionic strength, 0.12 M < [NaCl] < 0.6 M, quantitative precipitation of DNA was attained by addition of PDMAAC in the whole region studied: 1 < < 4.5. The data obtained strongly suggest that phase separation in solutions of DNA-containing PECs follows general rules revealed by studying PECs formed by flexible vinyl polyanions. However, the high rigidity of the DNA double helix appears to be responsible for the key feature revealed in the phase diagrams, i.e., significant broadening of the region for insoluble PECs at the expense of the region in which soluble DNA-containing PECs are formed. This feature may severely limit the application of DNA-containing PECs in medicine and biology but could be beneficial in the development of simple and effective procedures for DNA separation in biotechnology.
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| 4. |
- Wahlund, Per-Olof, et al.
(författare)
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Phase Separations in Water-Salt Solutions of Polyelectrolyte Complexes Formed by RNA and Polycations: Comparison with DNA Complexes.
- 2003
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Ingår i: Macromolecular Bioscience. - : Wiley. - 1616-5195 .- 1616-5187. ; 3:8, s. 404-411
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Tidskriftsartikel (refereegranskat)abstract
- Formation of insoluble polyelectrolyte complexes (PECs) between RNA and polycations was followed by measuring the residual RNA absorbance in the solution after separation of the precipitate. The polycations studied were poly(N,N-dimethyldiallylammonium) chloride (pendant type) and 2,5-ionene bromide (integral type) with quaternary amino groups in every monomer unit. The data obtained were compared with the results of analogous studies of DNA-containing PECs. This study is a part of a project aimed at the specific separation of plasmid DNA from RNA, a major problem in the preparative isolation of plasmid DNA. We thus deliberately chose a heterogenous RNA sample as it represents the RNA present in a real cell extract. In contrast to the exhaustive precipitation of DNA observed at certain values, a significant part of RNA was nonprecipitated at any = [+]/[-], that is, at any ratio of positively charged quaternary amino groups and negatively charged phosphate groups. The addition of sodium chloride increased the nonprecipitated fraction of RNA. DNA, on the other hand, was completely precipitated by both polycations at > 0.7. The less effective precipitation of RNA was probably due to the presence of a considerable fraction of short-chained molecules, incapable of forming a sufficient cooperative system of salt bonds with the polycation. This assumption was supported by a separate experiment, in which the precipitation behavior of RNA fractions of different molecular masses was investigated. The same tendency, while less pronounced, was also ascertained for PECs formed by polycations with DNA fractions of different molecular masses. The possibility of using the revealed differences between DNA and RNA behavior for effective precipitation procedure useful in bioseparation is discussed. The difference in the precipitation behavior of nucleic acids of different molecular masses means there is a possibility for developing an enzymatic assay for DNAase and RNAase activity.
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| 5. |
- Wahlund, Per-Olof, et al.
(författare)
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Polyelectrolyte complexes as a tool for purification of plasmid DNA background and development
- 2004
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 807:1, s. 121-127
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Tidskriftsartikel (refereegranskat)abstract
- The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration. (C) 2004 Elsevier B.V. All rights reserved.
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| 6. |
- Wahlund, Per-Olof, et al.
(författare)
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Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate
- 2004
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 87:5, s. 675-684
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Tidskriftsartikel (refereegranskat)abstract
- The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N,N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production. (C) 2004 Wiley Periodicals, Inc.
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