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1.
  • Balan, S, et al. (författare)
  • Metal chelate affinity precipitation of RNA and purification of plasmid DNA
  • 2003
  • Ingår i: Biotechnology Letters. - Springer. - 0141-5492. ; 25:13, s. 1111-1116
  • Tidskriftsartikel (refereegranskat)abstract
    • The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.
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2.
  • Dainiak, Maria, et al. (författare)
  • Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels
  • 2004
  • Ingår i: Journal of Chromatography A. - Elsevier B.V.. - 0021-9673. ; 1045:1-2, s. 93-98
  • Tidskriftsartikel (refereegranskat)abstract
    • The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 mum) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)(6)-tagged single chain (sc) Fv antibody fragments, (His)(6)-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)(6)-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques. (C) 2004 Published by Elsevier B.V.
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3.
  • Ivanov, Alexander, et al. (författare)
  • Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: a step towards thermosensitive industrial biocatalyst.
  • 2003
  • Ingår i: Biotechnology Progress. - American Chemical Society ; American Institute of Chemical Engineers. - 8756-7938. ; 19:4, s. 1167-1175
  • Tidskriftsartikel (refereegranskat)abstract
    • Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 106 and, presumably, cross-linked protein-polymer aggregates of MW > 107. Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 M) was found to be slightly higher than that of the free enzyme (12 M), and evaluation of Vmax testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from D-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of -lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).
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4.
  • Kumar, Ashok, et al. (författare)
  • Affinity fractionation of lymphocytes using a monolithic cryogel.
  • 2003
  • Ingår i: Journal of Immunological Methods. - Elsevier. - 0022-1759. ; 283:1-2, s. 185-194
  • 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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5.
  • Kumar, Ashok, et al. (författare)
  • Binding of Cu(II)-poly(N-isopropylacrylamide/vinylimidazole) copolymer to histidine-tagged protein: a surface plasmon resonance study.
  • 2003
  • Ingår i: Langmuir. - American Chemical Society. - 0743-7463. ; 19:3, s. 865-871
  • 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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6.
  • Kumar, Ashok, et al. (författare)
  • Metal chelate affinity precipitation: purification of (His)6-tagged lactate dehydrogenase using poly(vinylimidazole-co-N-isopropylacrylamide)copolymers
  • 2003
  • Ingår i: Enzyme and Microbial Technology. - Elsevier. - 0141-0229. ; 33:1, s. 113-117
  • 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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7.
  • Kumar, Ashok, et al. (författare)
  • Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers
  • 2003
  • Ingår i: Biotechnology and Bioengineering. - Wiley Periodicals, Inc.. - 0006-3592. ; 84:4, s. 494-503
  • 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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