| 1. |
- 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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| 2. |
- 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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| 3. |
- Fexby, Sara
(författare)
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HYDROPHOBIC PEPTIDE TAGS - As tools in bioseparation and investigation of recombinant proteins.
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the possibility of using hydrophobic peptide tags as tools for bioseparation and for the investigation of recombinant proteins. The hydrophobicity of a protein can easily be changed by minor modifications, such as fusions of tyrosine-containing peptides. The fusion tags may also increase both stability and expression of the fusion protein. Furthermore, a protein library with randomised pentapeptides was created, and the variants exhibited significant differences in relative hydrophobicity, although the pentapeptides only caused minor alterations in primary structure, size and isoelectric point of the fusion proteins. The changes in relative hydrophobicity were measured with aqueous two-phase systems (ATPSs) and hydrophobic interaction chromatography (HIC), and the results indicated the sensitivity of both methods to slight variations in the primary structure of proteins and their surface hydrophobicity. The results of the techniques correlated well, and the correlation coefficient, R2, was 0.89. Upon comparing the relative hydrophobicity contributions from seventeen LDH variants from thirty-four hydrophobicity scales they were found to correlate fairly well with experimental results from ATPSs and HIC. However, the results from such scales should only be used as a complement to experimental studies, for example, to indicate tag exposure. A novel HIC adsorbent, HBVE/BVE, was developed with polymer grafting techniques. The advantage of the HBVE/BVE medium is the possibility to alter the hydrophobic properties of the medium by only changing the reaction ratio of the two monomers in the grafting procedure. The hydrophobicity can be tuned to improve protein purification.
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| 4. |
- 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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| 5. |
- 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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| 6. |
- Fexby, Sara, et al.
(författare)
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Novel in situ polymerized coatings for hydrophobic interaction chromatography media
- 2007
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1161:1-2, s. 234-241
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Tidskriftsartikel (refereegranskat)abstract
- Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(D polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl vinyl ether. The new HIC media was evaluated using five test proteins (bovine serum albumin, ribonuclease A, (x-chymotrypsinogen A, myoglobin and (x-lactalbumin). The media exhibited classic HIC behavior, predictably controlled hydrophobicity, plus good protein selectivity, capacity (70 mg protein/ml gel) and often total protein recovery. By modifying the degree of matrix hydrophobicity, we could also reduce effects of protein denaturation often seen with conventional HIC and observed as multiple peaks in the chromatograms. Separation of crude protein extracts from Eschericha coli, expressing a green fluorescent protein (GFPuv) and, a more hydrophobic, recombinantly-modified, tyrosine-tagged green fluorescent protein (Y-PYPY-GFPuv), was also performed. These proteins were very stable, exhibited significantly different retention times, and could be used to study the ability of the media to work with complex protein mixtures. Such GFP mutants appear ideal for characterizing the performance of chromatographic media. (c) 2007 Published by Elsevier B.V.
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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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