| 11. |
- 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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| 12. |
- Clausen, Per Axel, et al.
(författare)
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Simultaneous extraction of di(2-ethylhexyl) phthalate and nonionic surfactants from house dust. Concentrations in floor dust from 15 Danish schools.
- 2003
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 986:2, s. 179-190
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Tidskriftsartikel (refereegranskat)abstract
- Static extraction, supercritical fluid extraction (SFE), pressurized liquid extraction (PLE) and Soxhlet extraction were compared for simultaneous extraction of di(2-ethylhexyl) phthalate (DEHP) and nonionic surfactants from house dust. Homogenized office floor dust from a vacuum cleaner dust bag ("standard dust") was used for the evaluation. One portion of the extracts was used for analysis of nonionic surfactants with LC–MS and another portion was used for DEHP analysis with GC–MS. The extraction yield of DEHP was comparable for all the methods whereas SFE and PLE were the most efficient extraction techniques for the nonionic surfactants. The PLE extraction was found most suitable as a routine method for simultaneous extraction of both types of compounds and was used in a field study of floor dust from 15 Danish schools. The mean concentration of DEHP in the school dust samples was ~4 times higher than observed in other studies of dust from homes in different countries. The concentrations of nonionic surfactants were one order of magnitude lower than soap and linear alkylbenzene sulfonates measured in other studies of floor dust from offices and other public buildings. However, for the first time nonionic surfactants have been identified in house dust.
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| 13. |
- Collén, Anna, et al.
(författare)
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Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)phosphate aqueous two-phase system
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 943:1, s. 55-62
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Tidskriftsartikel (refereegranskat)abstract
- Endoglucanases (EGI) (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)(2), (WP)(4) or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)(4) extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)(4) fused to the catalytic module and a short sequence of the linker [EGI(core-P5)(WP)(4)] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)(4) tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate.
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| 14. |
- Collén, Anna, et al.
(författare)
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Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems - Effect of fusion localization on endoglucanase I of Trichoderma reesei
- 2001
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 910:2, s. 275-284
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.
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| 15. |
- Dainiak, Maria, et al.
(författare)
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Direct capture of product from fermentation broth using a cell-repelling ion exchanger.
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 942:1-2, s. 123-131
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Tidskriftsartikel (refereegranskat)abstract
- A new technique for treating anion exchangers has been proposed allowing direct capture of the fermentation product, shikimic acid directly from the cell-containing fermentation broth. A layer of hydrophilic polymer, poly(acrylic acid) (PAA) has been physically adsorbed on the anion exchanger followed by a covalent cross-linking of PAA. The PAA layer is penetrable for small molecules despite being negatively charged as PAA is, but the polymer layer repels large negatively charged structures like cell debris and cells preventing them from adsorption to the chromatographic matrix. The binding capacity for pure shikimic was about 81 mg/ml adsorbent for both cross-linked PAA-Amberlite and native Amberlite in the fluidized mode of column operation. Binding capacity dropped to 17 and 15 mg per ml adsorbent, respectively, when using filtrated fermentation broth and to about 10 mg/ml adsorbent for cross-linked PAA-Amberlite when using directly the fermentation broth containing cells. Native Amberlite cannot be used for the direct capture of shikimic acid due to the immediate clogging of the column and the collapse of the expanded bed. The cross-linked PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth.
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| 16. |
- 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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| 17. |
- 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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| 18. |
- Gustavsson, Per-Erik, et al.
(författare)
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Continuous superporous agarose beds in radial flow columns
- 2001
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Ingår i: Journal of chromatography. A. - 0021-9673. ; 925:1-2, s. 69-78
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Tidskriftsartikel (refereegranskat)abstract
- Continuous superporous agarose beds constitute a new support material for chromatography, biocatalysis and electrophoresis. The bed consists of a single piece of agarose gel, homogeneously transected by flow-carrying pores, which easily can be varied in the range of 10–100 μm. In this work, large diameter beds (60 mm) were prepared and used in specially designed radial flow columns. The basic chromatographic properties of the beds were investigated by size-exclusion chromatography experiments. In an affinity chromatography application one bed was derivatized with Cibacron Blue 3GA and used for the purification of lactate dehydrogenase from a crude bovine heart extract. In a biotransformation application one bed was provided with immobilized β-galactosidase and used in the production of lactose-free milk.
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| 19. |
- 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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| 20. |
- 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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