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| 3. |
- Kepka, C., et al.
(författare)
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Pilot-scale extraction of an intracellular recombinant cutinase from E-coli cell homogenate using a thermoseparating aqueous two-phase system
- 2003
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 103:2, s. 165-181
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Tidskriftsartikel (refereegranskat)abstract
- A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 1 to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO50PO50 (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)4 tag was used for enhanced target protein partitioning to the EO50PO50 phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO50PO50 phase from the first step was supplemented with a non-ionic detergent (C12-18EO5) and heated to the cloud point (CP) temperature (45 degreesC). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.
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| 6. |
- Nordén, Bengt, 1945, et al.
(författare)
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Binding of methyl green to deoxyribonucleic acid analyzed by linear dichroism
- 1977
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Ingår i: Chemical Physics Letters. - : Elsevier BV. - 0009-2614. ; 50:3, s. 508-512
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Tidskriftsartikel (refereegranskat)abstract
- From linear dichroism data a distinct structure of the strongest complex between the methyl green (MG) dication and the B-form DNA is concluded. MG is not intercalated but binds (probably to hydrophobic surfaces in the major groove) with its x and z axes at an angle to the local helix axis of 48° and 90°, respectively. At higher occupation density, exciton coupling between transition dipoles of adjacent molecules is visualized in the linear and circular dichroism spectra. The final spectra may be understood in terms of a right-handed (MG)N helix in the major groove of the double helix.
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| 7. |
- Nordén, Bengt, 1945, et al.
(författare)
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High-sensitivity linear dichroism as a tool for equilibrium analysis in biochemistry- stability constant of DNA-ethidiumbromide complex
- 1976
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Ingår i: Biophysical Chemistry. - : Elsevier BV. - 1873-4200 .- 0301-4622. ; 4:2, s. 191-198
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Tidskriftsartikel (refereegranskat)abstract
- A stoichiometrical application of a sensitive method for linear dichroism (LD) detection is suggested for biochemical purposes. The complex formation between a binding site on a polynucleotide and a ligand may be studied with high precision if the following conditions are fulfilled: (1) The polymer can be given a fixed degree of orientation. (2) The site has a specific orientation with respect to the orientation axis of the polymer (e.g., intercalation). (3) The ligand has an anisotropic optical absorption property.The method was applied to studying the complex between DNA and ethidiumbromide, which was detected by LD with precision of ± 0.5 × 10−7 M in a 4 × 10−4 M DNA solution, i.e., 0.1% occupation of the total site concentration can be detected. The complsxation could be explained by a single type of site (n = 0.14 ± 0.01 sites per nucleotide residue) and a stability constant K1 = (2.5 ± 1) × 105 M−1 at 0.2 M ionic strength.From the specific LD an average angle 60° was concluded between the helix axis and the long axis of the ethidiumbromide molecule.This value formally contradicts the Watson-Crick model or the intercalation model but may be explained by extension and deformation effects on the xhain by the flow.
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| 8. |
- Nordén, Bengt, 1945, et al.
(författare)
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Linear Dichroism as a Tool for Characterizing Strand Separation in DNA
- 1977
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Ingår i: Chemica Scripta. - 0004-2056. ; 12, s. 15-17
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Tidskriftsartikel (refereegranskat)abstract
- Linear dichroism (LD) in combination with studies of UV absorption and circular dichroism is suggested as a method for characterizing denaturation and reassociation phenomena in DNA. Due to its strong dependence on the chain stiffness, LD sensitively reflects partial strand separations. In reassociated DNA, LD provides an index of the amount of ordered structure while hyperchromicity gives a measure of the amount of base-pairs reformed.
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| 9. |
- Nordén, Bengt, 1945, et al.
(författare)
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Linear dichroism studies of binding site structures in solution: Complexes between DNA and basic arylmethane dyes
- 1978
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Ingår i: Biophysical Chemistry. - : Elsevier BV. - 1873-4200 .- 0301-4622. ; 8:1, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect lo optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K1 ca 105 M−1; site density per nucleotide base n1 ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50° to the local DNA helix axis. The complex with fuchsin is weaker (104M−1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C2v symmetry) XXXre angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. Tne optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler's hydrol blue, a perpendicular orientation was observed, possibly due to intercaiation.
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- Nordén, Bengt, 1945, et al.
(författare)
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Optical studies on complexes between DNA and pseudoisocyanine
- 1976
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Ingår i: Biophysical Chemistry. - : Elsevier BV. - 1873-4200 .- 0301-4622. ; 6:1, s. 31-45
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Tidskriftsartikel (refereegranskat)abstract
- Linear dichroism (LD) results when pseudoisocyanine = PIC (1, 1-diethyl-2,2'-cyanine iodide) binds to flow-oriented DNA. LD may be used to follow the complexation both stoichiometrically and structurally, since when specified to unit complex concentration LD provides a measure of the average orientation of the absorbing transition dipole.Two different types of complexes can be distinguished: I. One strong, ionic-strength insensitive complex with monomeric PIC with an orientation indicating intercalation. II. Several weaker complexes of electrostatic nature (only observed at I
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