| 1. |
- Andersson, Charlotte, et al.
(författare)
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Enhanced Ribosome and tRNA Contents in Escherichia coli Expressing a Truncated Vitreoscilla Hemoglobin Mutant Analyzed by Flow Field-Flow Fractionation
- 2003
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Ingår i: Biotechnology Letters. - 1573-6776. ; 25:18, s. 1499-1504
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Tidskriftsartikel (refereegranskat)abstract
- The ribosome and tRNA levels of Escherichia coli cells, transformed with a native or mutated Vitreoscilla hemoglobin genes (vhb), were investigated using asymmetrical flow field-flow fractionation (AFFFF). Mutagenesis of vhb by error-prone PCR was carried out to alter the growth behavior of microaerobically cultivated native VHb-expressing E. coli. A VHb mutant, pVMT1, was identified, which was able to reach a remarkably high final A600 of 15, the value of which being 160% higher than that of a VHb control carrying pVHb8 (A600 5.8). AFFFF revealed that cells expressing mutant vhbs showed up to a doubling in the number of active 70S ribosomes cell-1, an almost 3-fold increase in the number of tRNAs cell-1, and up to a 26% increase in the mass fraction of active 70S ribosomes.
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| 2. |
- Andersson, M, et al.
(författare)
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A Novel Selection System for Potato Transformation Using a Mutated AHAS Gene.
- 2003
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Ingår i: Plant Cell Reports. - : Springer Science and Business Media LLC. - 1432-203X .- 0721-7714. ; 22:4, s. 261-267
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Tidskriftsartikel (refereegranskat)abstract
- Acetohydroxyacid synthase (AHAS) is the target enzyme for a number of herbicides. A S653N mutation in the AHAS gene results in an increased tolerance to imidazolinone herbicides. We have investigated the use of the mutated gene as selection gene for potato transformation. This resulted in a transformation system with a very high transformation frequency and low rate of escapes. The mutated AHAS gene was introduced into transformed potato together with a -glucuronidase (GUS) gene. Selection on 0.5 M Imazamox yielded GUS expression in 93–100% of regenerated shoots. Furthermore the mutated AHAS gene was used as selection gene for production of high-amylopectin potato lines. The high transformation frequency was verified and potato lines with the desirable starch quality were obtained.
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| 3. |
- Arvidsson, Pär, et al.
(författare)
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Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 977:1, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylanlino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 mum in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use. (C) 2002 Published by Elsevier Science B.V.
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| 4. |
- Fexby, Sara, et al.
(författare)
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Hydrophobic peptide tags as tools in bioseparation
- 2004
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799. ; 22:10, s. 511-516
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Forskningsöversikt (refereegranskat)abstract
- Hydrophobic interactions are highly selective, and differences in surface hydrophobicities between proteins can be used as an efficient handle to facilitate protein isolation. Aromatic amino acid residues are of particular importance for molecular recognition because they have a key role in several biological functions. The hydrophobicity of a protein can easily be altered with minor genetic modifications, such as site-directed mutagenesis or fusions of hydrophobic peptide tags. An important advantage of hydrophobic peptide tags over traditional affinity tags is the possibility of exploring simple and inexpensive bioseparation materials. Recent results demonstrate the potential of hydrophobic interaction chromatography and aqueous two-phase systems as tools to study relative hydrophobicities of recombinant proteins with only minor alterations. This review focuses on hydrophobic peptide tags as fusion partners, which can be used as important tools in bioseparation.
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| 5. |
- Fexby, Sara, et al.
(författare)
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Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase
- 2002
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Ingår i: Protein Expression and Purification. - 1046-5928. ; 25:2, s. 9-263
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Tidskriftsartikel (refereegranskat)abstract
- The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer (EO30PO70). Five different LDH variants were constructed with N-terminal tags containing tyrosines (Y3 and Y6), tyrosines and prolines (Y3P2 and Y6P2), and only prolines (P2). LDH fused with tags containing tyrosines increased the partitioning coefficient, and the more tyrosines added to the protein, the larger improvement in partitioning. When prolines were added between the tyrosine-rich tag and the protein, a further increased partitioning was obtained. The enhanced partitioning was attributed to the rigid structure of the proline, which in turn led to an increase in the exposure of the tag to the surroundings. The best tyrosine tag, Y6P2, increased the partition coefficient four times and additionally, a higher thermostability was observed.
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| 6. |
- Fexby, Sara, et al.
(författare)
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N-Terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition
- 2004
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 807:1, s. 25-31
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Tidskriftsartikel (refereegranskat)abstract
- The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R-2 = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed. (C) 2004 Elsevier B.V. All rights reserved.
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| 7. |
- Fexby, Sara, et al.
(författare)
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Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems
- 2004
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 20:3, s. 793-798
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Tidskriftsartikel (refereegranskat)abstract
- The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase.
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| 8. |
- Lindberg, Jenny, et al.
(författare)
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Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein
- 2002
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 18:6, s. 1176-1182
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Tidskriftsartikel (refereegranskat)abstract
- In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.
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| 9. |
- Olsson, Peter, et al.
(författare)
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Expression of bovine calmodulin in tobacco plants confers faster germination on saline media
- 2004
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Ingår i: Plant Science. - : Elsevier BV. - 0168-9452. ; 166:6, s. 1595-1604
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin is a Ca2+-dependent regulatory protein in eukaryotic cells involved in a variety of intracellular activities. Many responses to abiotic stresses involve signaling by Ca2+ and Ca2+/calmodulin regulation, including water and salinity stress. To investigate how calmodulin affects germination on media with high concentrations of NaCl, transgenic tobacco plants expressing heterologous calmodulin were generated and characterized. Transgenic tobacco seeds showed significantly shortened germination times on media containing varying salt (120-160 mM) and calcium (3 and 13 mM) concentrations, compared to control seeds. Using media containing 140 mM NaCl and 13 mM CaCl2, the average germination time was shortened by 2-3 days compared to control (P < 0.05). In addition, the germinating transgenic seeds contained higher transient levels of gamma-aminobutyric acid compared to control seeds (P < 0.05). The tobacco Ca2+/calmodulin-regulated glutamate decarboxylase, synthesizing gamma-aminobutyric acid may therefore be stimulated by the heterologous calmodulin. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
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| 10. |
- Pettersson, Henrik, et al.
(författare)
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Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase: Kinetics of a fusion protein of MDH and CS
- 2000
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 267:16, s. 5041-5046
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Tidskriftsartikel (refereegranskat)abstract
- The mechanistic implications of the kinetic behaviour of a fusion protein of mitochondrial malate dehydrogenase and citrate synthase have been reanalysed in view of predictions based on experimentally determined kinetic parameter values for the dehydrogenase and synthase activities of the protein. The results show that the time-course of citrate formation from malate in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is taken to the inhibitory effects of NADH and oxaloacetate on the malate dehydrogenase activity. The effect of aspartate aminotransferase on the coupled reaction is likewise fully consistent with that expected for a free-diffusion mechanism. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses citrate formation from malate by a mechanism involving channelling of the intermediate oxaloacetate.
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