| 1. |
- Dainiak, Maria, et al.
(författare)
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Cell chromatography: Separation of different microbial cells using IMAC supermacroporous monolithic columns
- 2005
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:2, s. 644-649
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Tidskriftsartikel (refereegranskat)abstract
- Supermacroporous monolithic columns with CU2+-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10- 100 mu m) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the CU2+-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.
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| 2. |
- Galaev, Igor, et al.
(författare)
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High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1065:2, s. 169-175
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Tidskriftsartikel (refereegranskat)abstract
- Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples. (C) 2005 Elsevier B.V. All rights reserved.
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| 3. |
- Hanora, Amro, et al.
(författare)
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Capture of bacterial endotoxins using a supermacroporous monolithic matrix with immobilized polyethyleneimine, lysozyme or polymyxin B
- 2005
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 118:4, s. 421-433
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial endotoxins (BEs) are integrated part of Escherichia coli, a microorganism widely used for the production of recombinant proteins. BEs should be eliminated in the course of down stream processing of target protein produced by these bacteria. Supermacroporous monolith (continuous bed) columns, so called cryogel columns, with immobilized polyethyleneimine (PEI), polymyxin B (PMB) and lysozyme were employed for BEs capture. Due to the large interconnected pores it was possible to use cryogel columns at flow rates as high as 10 ml/min. The columns packed with Sepharose CL-4B with immobilized PEI, PMB and lysozyme were impossible to use at these high flow rates due to the collapse of the bed. The dynamic capacities of the cryogel columns were nearly independent of the flow rate. In the presence of EDTA, BEs were quantitatively captured from mixtures with a model protein, bovine serum albumin (BSA) at pH 7.2 with practically no protein losses. At pH 3.6 BEs were captured directly from non-clarified E. coli cell lysate resulting in more than 104 times BEs clearance. (C) 2005 Elsevier B.V. All rights reserved.
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| 4. |
- Hanora, Amro, et al.
(författare)
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Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1087:1-2, s. 38-44
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Tidskriftsartikel (refereegranskat)abstract
- A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries. (c) 2005 Elsevier B.V. All rights reserved.
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| 5. |
- Kumar, Ashok, et al.
(författare)
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Affinity binding of cells to cryogel adsorbents with immobilized specific ligands : effect of ligand coupling and matrix architecture
- 2005
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 18:1, s. 84-93
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Tidskriftsartikel (refereegranskat)abstract
- The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.
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| 6. |
- Plieva, Fatima, et al.
(författare)
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Pore structure in supermacroporous polyacrylamide based cryogels
- 2005
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 1:4, s. 303-309
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Tidskriftsartikel (refereegranskat)abstract
- Pore size and thickness of pore walls in macroporous polyacrylamide gels, so-called cryogels (pAAm-cryogels), were controlled by varying the content of monomers in the initial reaction mixture and the cross-linker used. The thickness of pore walls in pAAm-cryogels increased with increasing concentration of monomers in the initial reaction mixture. Pore volume in the supermacroporous pAAm-cryogels was in the range of 70-93% and decreased with increasing concentration of monomers in the reaction feed. The porous structure of the pAAm-cryogels was visualized using environmental scanning electron microscopy (ESEM) that allowed monitoring of the dehydration process in pAAm-cryogels. The accessibility of ligands covalently coupled to the polymer backbone for low molecular weight target, Cu(II) ions, and high molecular weight target, the protein lysozyme, was assessed for pAAm-cryogels produced from feeds with different monomer concentration. The amount of bound Cu(II) ions increased linearly with increasing monomer concentration in the reaction feed, suggesting that all ligands are equally accessible for small targets. On the contrary, lysozyme binding demonstrated a clear maximum at monomer concentration about 18% suggesting that only ligrands present at the surface of pore walls are accessible for high molecular weight targets.
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| 7. |
- Savina, Irina, et al.
(författare)
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Cryostructuration of polymer systems. XXIV. Poly(vinyl alcohol) cryogels filled with particles of a strong anion exchanger: Properties of the composite materials and potential applications
- 2005
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Ingår i: Journal of Applied Polymer Science. - : Wiley. - 1097-4628 .- 0021-8995. ; 95:3, s. 529-538
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Tidskriftsartikel (refereegranskat)abstract
- A composite material produced from a poly(vinyl alcohol) cryogel with entrapped particles of the strong anion-exchange resin Amberlite is presented. The properties of the composite material depended strongly on whether the resin was used in OH- form or Cl- form. The ion-exchange filler in OH- form caused both a significant reinforcement of the composite material and an increase in the gel fusion temperature. These effects were thought to be associated with the additional ionic bonding between the continuous and disperse phases. Beads 200-600 m in size were prepared from the composite material and used in expanded-bed ion-exchange chromatography for the capture of the negatively charged solutes benzoate and lactate from the suspension of negatively charged cells. The plausibility of the approach has been demonstrated on model systems composed of yeast cells and benzoate and with a real fermentation broth produced after lactic acid fermentation.
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