| 41. |
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| 42. |
- Kamihira, Masamichi, et al.
(författare)
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Integration of extraction with affinity precipitation
- 2000
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Ingår i: Aqueous Two-Phase Systems : Methods and Protocols - Methods and Protocols. - New Jersey : Humana Press. - 9780896035416 - 9781617370670 - 9781592590285 ; , s. 371-379
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Bokkapitel (refereegranskat)abstract
- Conventional aqueous two-phase extraction by spontaneous partitioning has often problems with low specificity. The introduction of an affinity ligand into one of the phases has been attempted to enhance the specificity of protein partitioning (1-7; Chapters 29-31). However, this procedure still has some limitations in the effective removal of impurities as well as recovery and reuse of the ligand. During the past decade, another potentially scalable technique, affinity precipitation, has been studied for the isolation of proteins (8-10). The affinity ligand coupled to a reversibly soluble-insoluble polymer is used to specifically bind the protein in a complex mixture. The precipitation step which can be accomplished by a change in any environmental parameter such as pH, temperature, salinity, and so forth, facilitates the separation of the bound ligand and affinity complex from the unbound components. Although cells and cell debris should be removed before use, recovery and reuse of the ligand are relatively easy.
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| 43. |
- Kazakov, S V, et al.
(författare)
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Characterization of macromolecular solutions by a combined static and dynamic light scattering technique
- 2002
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Ingår i: International Journal of Thermophysics. - 0195-928X. ; 23:1, s. 161-173
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Tidskriftsartikel (refereegranskat)abstract
- The combination of quasi-elastic light scattering (LS) with integrated scattered intensity measurements in the same sample has been applied to study polymer and polymer-protein aqueous solutions. The molecular weight, the radius of gyration, and the second virial coefficient for thermosensitive polymer [poly(N-isopropylacrylamid)] solutions before and after precipitation transition have been obtained using Zimm plot calculations. The precipitation curve (intensity versus temperature dependence) for polymer solutions has been experimentally obtained using the light scattering setup. For the first time the static and dynamic LS properties of aqueous solutions of antibody-poly(methacrylic acid) and antibody-poly(acrylic acid) conjugates and solutions of their components [antibody, poly(methacrylic acid), and poly(acrylic acid)] at different pH values have been measured. In both cases the parallel comparison of the characteristic size variations allowed us to represent novel structural features of scattered particles (macromolecules, associates, aggregates, conjugates, colloidal particles) in studied systems.
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| 44. |
- 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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| 45. |
- Kumar, Ashok, et al.
(författare)
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Affinity fractionation of lymphocytes using a monolithic cryogel.
- 2003
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 283:1-2, s. 185-194
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Tidskriftsartikel (refereegranskat)abstract
- A new type of continuous, supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing specific fractionation and separation of human peripheral blood lymphocytes in a chromatographic format. The affinity adsorbent was used to design a novel cell separation strategy, which was based on the interaction of protein A from Staphylococcus aureus with cells bearing IgG antibodies on the surface. After treating lymphocytes with goat anti-human IgG(H+L), the IgG-positive B-lymphocytes were efficiently separated from T-lymphocytes. Protein A covalently coupled to epoxy activated dimethylacrylamide (DMAA) cryogel matrix specifically bound IgG-bearing B-lymphocytes through the Fc region, while non-bound T-lymphocytes passed through the column. More than 90% of the B-lymphocytes were retained in the column while the cells in the breakthrough fraction were enriched in T-lymphocytes (81%). The viability of the T-lymphocytes isolated was greater than 90%. The bound lymphocytes released by human or dog IgG recovered 60–70% of the B-cells without significantly impairing the cell viability. The technique can be applied in general to cell separation systems where IgG antibodies against specific cell surface markers are available.
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| 46. |
- Kumar, Ashok, et al.
(författare)
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Binding of Cu(II)-poly(N-isopropylacrylamide/vinylimidazole) copolymer to histidine-tagged protein: a surface plasmon resonance study.
- 2003
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 19:3, s. 865-871
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Tidskriftsartikel (refereegranskat)abstract
- The thermoresponsive copolymer of N-isopropylacrylamide (NIPAM) with 1-vinylimidazole (VI), poly(NIPAM-VI), synthesized by radical polymerization has been used to purify the histidine-tagged green flourescent protein (His-tag GFP) from recombinant E. coli by metal-chelate affinity precipitation. The purified protein was immobilized on the BIAcore sensor chip by carbodiimide coupling. Affinity binding of the Cu(II)-loaded copolymer, poly(NIPAM-VI), to the His-tag GFP-immobilized surface was monitored by surface-plasmon-resonance (SPR) measurements. Complete recovery of the metal copolymer from the surface was achieved with either using the monomer displacer, 200 mM imidazole buffer, or the polymeric displacer, copolymer of poly(NIPAM-VI) (26 mol % VI). The conformation of the copolymer was a critical factor for the metal interactions and hence displacement of the metal copolymer. With the proposed conformation of protein-like copolymers (Wahlund, P.-O.; Galaev, I. Yu.; Kazakov, S. A.; Lozinsky, V. I.; Mattiasson, B. Macromol. Biosci. 2002, 2, 33.), the SPR study confirmed the prediction of exposed imidazole groups in the poly(NIPAM-VI). The complete elution of the affinity-bound metal copolymer was achieved with protein-like copolymer (imidazole groups exposed to the outer solution), and no recovery was obtained with IMAC nonbound copolymer fraction (imidazole groups unexposed). The SPR measurement showed a sharp phase transition of affinity adsorbed thermoresponsive Cu(II)-poly(NIPAM-VI) copolymer at 32 C, thus proposing a sensitive way to determine lower critical solution temperature.
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| 47. |
- Kumar, Ashok, et al.
(författare)
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Metal chelate affinity precipitation: purification of (His)6-tagged lactate dehydrogenase using poly(vinylimidazole-co-N-isopropylacrylamide)copolymers
- 2003
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 33:1, s. 113-117
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Tidskriftsartikel (refereegranskat)abstract
- Affinity precipitation is a highly selective bioseparation method, which has been successfully used in the purification of various enzymes/proteins. The separation of (His)6-tagged lactate dehydrogenase ((His)6-LDH) was carried through affinity precipitation technique using thermoresponsive copolymer poly(vinylimidazole-co-N-isopropyl-acrylamide) (poly(VI-NIPAM)). Nearly quantitative recovery of (His)6-LDH has been achieved using Cu(II)- and Ni(II)-loaded poly(VI-NIPAM) with Cu(II)-poly(VI-NIPAM) being more efficient in enzyme precipitation than Ni(II)-poly(VI-NIPAM). Optimal precipitation of (His)6-LDH was achieved at pH 6 and pH 6.5–7.0 with Cu(II)-poly(VI-NIPAM) and Ni(II)-poly(VI-NIPAM), respectively. Furthermore, the enzyme was purified by affinity precipitation upto 3.5- and 3.1-fold with recoveries of 95 and 82% using Cu(II)- and Ni(II)-loaded copolymers, respectively.
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| 48. |
- Kumar, Ashok, et al.
(författare)
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Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers
- 2003
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:4, s. 494-503
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Tidskriftsartikel (refereegranskat)abstract
- Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His6-scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His6-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 494-503, 2003.
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| 49. |
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| 50. |
- Kumar, R, et al.
(författare)
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Adhesives and plastics based on soy protein products
- 2002
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Ingår i: Industrial Crops and Products. - 0926-6690. ; 16:3, s. 155-172
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Forskningsöversikt (refereegranskat)abstract
- Significance of eco-friendly materials based on easily renewable natural resources, and the finite nature of petrochemical resources, has necessitated the development of polymers from agricultural processing by products such as soy proteins from oil processing. Although, considerable work was done in the early part of last century on polymers based on soy protein, there was almost no activity in this field for the last fifty years. There is a need to critically analyse the available literature on soy protein based polymeric materials. Therefore, an attempt is made to review the state-of-the-art of the polymeric materials with emphasis on adhesives and plastics derived from soy protein, a renewable resource abundantly available in nature.
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