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| 2. |
- Berggren, Kristina, et al.
(författare)
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Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers
- 1995
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 718:1, s. 67-79
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Tidskriftsartikel (refereegranskat)abstract
- The partitioning of five well-characterised model proteins, bovine serum albumin (BSA), lysozyme, [beta ]-lactoglobulin A, myoglobin and cytochrome c, in aqueous two-phase systems has been studied. As top phase polymers PEG (polyethylene glycol, 100% EO) and the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymers, Ucon 50-HB-5100 (50% EO, 50% PO) and EO30PO70 (30% EO, 70% PO), respectively, were used. The top phase polymers are increasing in hydrophobicity with increasing content of PO. Reppal PES 200 (hydroxypropyl starch) was used as the bottom phase polymer. Phase diagrams for Reppal PES 200-PEG and Reppal PES 200-EO30PO70 two-phase systems were determined. The partitioning of four salts with different hydrophobicity, and also the effect of the salts on protein partitioning in these systems, was studied. It was found that the partitioning of the salts followed the Hofmeister series. The partitioning of proteins with low surface hydrophobicity, myoglobin and cytochrome c, was little affected by hydrophobic polymers and salts. However, the partitioning of a protein with higher surface hydrophobicity, lysozyme, was strongly affected when polymer hydrophobicity was increased and a hydrophobic counterion was used. A protein with a relatively hydrophobic surface can be partitioned to a phase containing a thermoseparating EO-PO copolymer by using a hydrophobic counterion. The partitioning of lysozyme and cytochrome c in the polymer-water system formed after temperature-induced phase separation was also examined. Both proteins partitioned exclusively to the water phase. A separation of the protein and polymer was obtained by temperature-induced phase separation on the isolated phase containing the EO-PO copolymer. The partitioning data also indicated that the hydroxypropyl starch polymer had a weak negative charge.
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| 3. |
- Carlsson, Mats, et al.
(författare)
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Effects of fused tryptophan rich peptides to a recombinant protein A domain on the partitioning in polyethylene glycol-dextran and Ucon-dextran aqueous two-phase systems
- 1996
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 756:1-2, s. 107-117
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used to construct fusion proteins with tryptophan containing peptides. The peptides and the fusion proteins have been partitioned in aqueous two-phase systems of poly(ethylene glycol) (PEG)-dextran and Ucon-dextran. The studied model protein was ZZT0, where Z is an engineered domain of domain B of staphylococcal protein A. The specially designed hydrophobic peptides, Ala-Trp-Trp-Pro (T1) and (Ala-Trp-Trp-Pro)2 (T2), have been inserted into ZZT0, to give the peptide-protein fusions ZZT1 and ZZT2. In the experimental studies it was found that T1 and T2 preferred the PEG phase and even more the Ucon phase over the dextran phase. For T2 the partitioning was more one sided than for T1. For the fusion proteins, ZZT1 and ZZT2, the partitioning was enhanced into the PEG or Ucon rich phase as compared to ZZT0. The effects were lower than expected from independent contributions to the partition coefficient from the protein and the peptides. A heterogeneous lattice model was used to calculate theoretical peptide and protein partition coefficients. The calculations could reproduce the qualitative features of the experimental data. The model results suggest that a part of these experimentally observed effects is due to a depletion zone, i.e. a zone of reduced polymer concentration around the protein. The experimental results indicate a further reduction of the partition coefficient, beyond that predicted by the lattice calculations. A possible folding of the inserted peptide is discussed as a plausible mechanism for this further reduction in the partition coefficient.
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| 4. |
- Ekblad, Lars, et al.
(författare)
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Aqueous two-phase affinity partitioning of biotinylated liposomes using neutral avidin as affinity ligand
- 1998
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 815:2, s. 189-195
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Tidskriftsartikel (refereegranskat)abstract
- Biotinylated small unilamellar liposomes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using avidin coupled to dextran as affinity ligand. In the absence of affinity ligand more than 90% of the liposomes partitioned in the poly(ethylene glycol)-rich top phase, whereas in its presence more than 95% partitioned in the dextran-rich bottom phase. For this redistribution to occur 10 mM and above of lithium sulphate, or other appropriate salts, had to be added to the two-phase system. Without added salt the liposomes with complexed avidin-dextran instead partitioned in the top phase. An extended mixing time for the system was required for maximum redistribution. Less than two biotin residues per liposome, coupled via a C6 spacer arm, was required to redistribute the liposomes to the bottom phase.
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| 5. |
- Gustavsson, Per-Erik, et al.
(författare)
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Continuous superporous agarose beds for chromatography and electrophoresis
- 1999
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Ingår i: Journal of chromatography. A. - 0021-9673. ; 832:1-2, s. 29-39
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Tidskriftsartikel (refereegranskat)abstract
- Continuous agarose beds (monoliths) were prepared by casting agarose emulsions designed to generate superporous agarose. The gel structures obtained were transected by superpores (diameters could be varied in the range 20–200 μm) through which liquids could be pumped. The pore structure and the basic properties of the continuous gel were investigated by microscopy and size exclusion chromatography. The chromatographic behaviour was approximately the same as for beds packed with homogeneous agarose beads with a particle diameter equivalent to the distance between the superpores. In one application, the superporous continuous agarose bed was derivatized with a NAD+ analogue and used in the affinity purification of bovine lactate dehydrogenase from a crude extract. In another application, a new superporous composite gel material was prepared by adding hydroxyapatite particles to the agarose phase. The composite bed was used to separate a protein mixture by hydroxyapatite chromatography. In a third application, the continuous superporous agarose material was used as an electrophoresis gel. Here, a water-immiscible organic liquid was pumped through the superpores to dissipate the joule heat evolved, thus allowing high current densities.
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| 6. |
- Gustavsson, Per-Erik, et al.
(författare)
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Direct measurements of convective fluid velocities in superporous agarose beads
- 1998
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 795:2, s. 199-210
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Tidskriftsartikel (refereegranskat)abstract
- Superporous agarose beads contain two sets of pores, diffusion pores and so-called superpores or flow pores, in which the chromatographic flow can transport substances to the interior of each individual bead [Gustavsson and Larsson, J. Chromatogr. A 734 (1996) 231]. The existence of pore flow may be proven indirectly by the chromatographic performance of beads but it has never been directly demonstrated in a chromatographic bed. In this report, pore flow was directly measured by following the movement of micro-particles (dyed yeast cells) in a packed bed. The passage of the micro-particles through the superpores and through the interstitial pores was followed by a microscope/video camera focused on beads which were situated four layers from the glass wall. The video recordings were subsequently used to determine the convective fluid velocities in both the superpores and the interstitial pores. Experiments were carried out with three different bead size ranges, all of which contained superporous beads having an average superpore diameter of 30 mu m. The superpore fluid velocity as % of interstitial fluid velocity was determined to be 2-5% for columns packed with 300-500-mu m beads (3% average value), 6-12% for columns packed with 180-300 mu m beads (7% average value) and 11-24% for columns packed with 106-180-mu m beads (17% average value). These data were compared to and found to agree with theoretically calculated values based on the Kozeny-Carman equation. In order to observe and accurately measure fluid velocities within a chromatographic bed, special techniques were adopted. Also, precautions were made to ensure that the experimental conditions used were representative of normal chromatography runs. (C) 1998 Elsevier Science B.V.
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| 7. |
- Gustavsson, Per-Erik, et al.
(författare)
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Superporous agarose, a new material for chromatography
- 1996
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Ingår i: Journal of chromatography. A. - : Elsevier BV. - 0021-9673. ; 734:2, s. 231-240
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Tidskriftsartikel (refereegranskat)abstract
- This paper reports on a new type of spherical agarose chromatography particles characterized by two sets of pores, normal diffusion pores, characteristic of all agarose materials and very wide pores, so-called superpores or flow pores. These superpores allow part of the chromatographic flow to pass through each individual particle, which gives improved mass transfer, especially in situations where diffusion is the limiting factor for the overall performance of a chromatographic separation. The particles were prepared by a double emulsification procedure. Observations under a microscope and size-exclusion chromatography were used in order to demonstrate pore flow. The chromatographic behaviour of the new particles was as efficient as that of homogeneous particles which were several times smaller. The agarose particles were derivatized with polyethyleneimine and used for an ion-exchange chromatographic separation of three model proteins. As expected from a perfusion material, the superporous beads resolved the protein mixture more efficiently than homogeneous beads of the same size.
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| 8. |
- Gustavsson, Per-Erik, et al.
(författare)
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Superporous agarose as an affinity chromatography support
- 1997
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Ingår i: Journal of chromatography. A. - 0021-9673. ; 776:2, s. 197-203
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Tidskriftsartikel (refereegranskat)abstract
- Superporous agarose beads were used as an affinity support in column chromatography. These beads characteristically possess two sets of pores, normal diffusion pores and flow pores, so-called superpores. The superpores, whose diameter is a substantial fraction of the particle diameter (i.e. 1/3 to 1/10 of the particle diameter), allow part of the chromatographic flow to pass through each individual bead. Consequently, significant improvement in mass transfer is observed in superporous beads as compared with homogeneous beads, especially at high flow-rates [Gustavsson and Larsson, J. Chromatogr. A, 734 (1996) 231–240.]Superporous agarose beads and homogeneous agarose beads were each derivatized with two types of affinity ligands. A NAD+ analogue was used for the purification of bovine lactate dehydrogenase and protein A was used for the adsorption of rabbit IgG. The performances of superporous beads and homogeneous beads were compared. Superporous bead columns derivatized with protein A and NAD+ analogue could be operated 5 times and 3 times, respectively, as fast as corresponding homogeneous bead columns.
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| 9. |
- Gustavsson, Per-Erik, et al.
(författare)
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Superporous agarose beads as a hydrophobic interaction chromatography support
- 1999
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 830:2, s. 275-284
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Tidskriftsartikel (refereegranskat)abstract
- Superporous agarose beads were used as a support for hydrophobic interaction chromatography. These beads have large connecting flow pores in addition to their normal diffusion pores. The flow pores, which are approximately one fifth of the overall diameter of the superporous agarose beads, were earlier shown to give the beads improved mass transfer properties relative to homogeneous agarose beads (Gustavsson and Larsson, J. Chromatogr. A, 734 (1996) 231-240). Superporous agarose beads and homogeneous agarose beads of the same particle size range (106-180 mu m) were derivatized with phenyl groups. The properties of the superporous beads were then compared with the homogeneous beads in the separation of a mixture of three model proteins (ribonuclease A, lysozyme and bovine serum albumin) at various superficial flow velocities from 30 to 600 cm/h. The superporous beads gave satisfactory separation at flow velocities five times higher than was possible for homogeneous beads. The performance of the two types of beads was also compared in the purification of lactate dehydrogenase from a beef heart extract at a superficial flow velocity of 150 cm/h. The superporous beads performed considerably better, leading to twice the purification factor and twice the concentration of the desired product. The results were interpreted using the theoretical treatment given by Carta and Rodrigues (Carta and Rodrigues, Chem. Eng. Sci., 48 (1993) 3927). (C) 1999 Elsevier Science B.V. All rights reserved.
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| 10. |
- Kempe, Henrik, et al.
(författare)
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Simulation of chromatographic processes applied to separation of proteins
- 1999
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 846:1-2, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- The advantages of using a detailed mathematical model for fixed bed chromatography is demonstrated by the personal computer program SIMCHROM. The chromatography model includes axial dispersion in the bulk liquid, external and internal mass transfer resistances and an instationary non-linear adsorption model. Frontal and pulse chromatography can be studied for single and multicomponent systems. The simulation program can easily be used to make parametric evaluations to study the influence of variations in physical, kinetical and operating parameters. The special features of the present intrinsic model is demonstrated by comparing the SIMCHROM results With simulations using simplified lumped models. Experimental data describing affinity chromatography of lysozyme on Cibacron Blue Sepharose CL-6B is used as a model system. The intrinsic model is able to describe variations in the physical, kinetic and operating parameters better than the simplified models. This results in a more reliable prediction of the performance of the chromatography process as well as a better understanding of che underlying mechanisms responsible for the separation. (C) 1999 Elsevier Science B.V. All rights reserved.
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