| 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. |
- Becker, Kristian, et al.
(författare)
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Multipurpose peptide tags for protein isolation.
- 2008
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1202:1, s. 40-46
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Tidskriftsartikel (refereegranskat)abstract
- A multifunctional peptide tag (HYDHYD) consisting of histidine, tyrosine and aspartate residues was fused to the N-terminal ends of green fluorescent protein (GFP), lactate dehydrogenase (LDH) and human hemoglobin (Hb), proteins which were subjected to ion-exchange chromatography (IEC), aqueous two-phase system partition, immobilized metal-ion affinity chromatography (IMAC), and hydrophobic interaction chromatography (HIC). Tagged GFP was retained significantly longer (>1 column volume) in both HIC and IEC. It exhibited 3x greater partition in favor of the hydrophobic phase in a two-phase system and 96% could be bound to an IMAC column which did not bind native GFP.
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| 3. |
- Becker, Kristian, et al.
(författare)
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Probing protein surface accessibility of amino acid substitutions using hydrophobic interaction chromatography.
- 2008
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1215:1-2, s. 152-155
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Tidskriftsartikel (refereegranskat)abstract
- Hydrophobic interaction chromatography (HIC) has been used to determine the influence of amino acid substitutions on protein retention and thereby their accessibility on the protein surface. The retentions of mutants of green fluorescent protein (GFPuv) and human hemoglobin (Hb) were studied on multimodal HIC media and compared to the hydrophobicities from known hydrophobicity scales with respect to the accessible surface area. For GFPuv, the theoretical and experimental results of three hydrophobicity scales correlated well (R(2)>0.85), which clearly indicate that the results can be used for protein retention prediction as well as probing surface properties of protein variants.
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| 4. |
- Bernaudat, Florent, et al.
(författare)
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Rapid evaluation of nickel binding properties of His-tagged lactate dehydrogenases using surface plasmon resonance
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1066:1-2, s. 219-224
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Tidskriftsartikel (refereegranskat)abstract
- The use of surface plasmon resonance (SPR), for the comparison of metal binding properties of polyhistidine tags, was evaluated. Six different tags containing various number of histidines, either none (tags n and t), three (tags H(3)A(3) and H(2)AHA(2)H) or six (tags H-6 and HiS(6)) were genetically fused to the N-terminal of lactate dehydrogenase (LDH). The binding ability of these constructs to nickel ions, immobilised with nitrilotriacetic acid (NTA), was tested both by conventional immobilised metal ion affinity chromatography (IMAC) and SPR. The relative binding strengths of the tags to nickel were identical using both methods (n approximate to t < HA(2)HA(2)H < H(3)A(3) < HiS(6) < H-6) confirming the value of the SPR technique for investigating metal-protein interactions. Protein modelling has also proved to be useful in supporting the experimental results.
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| 5. |
- 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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| 6. |
- Hajizadeh, Solmaz, et al.
(författare)
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Composite imprinted macroporous hydrogels for haemoglobin purification from cell homogenate
- 2018
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1534, s. 22-31
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Tidskriftsartikel (refereegranskat)abstract
- Purification of haemoglobin (Hb) has been studied for many years due to its ability to act as an oxygen carrier and its possible use in urgent clinical treatment. In this study, different types of chromatography columns were developed for Hb purification. Two of them showed satisfactory results as affinity chromatography columns and were thus studied more extensively. The affinity adsorbents were prepared by molecular imprinting techniques. In the first case, Pickering emulsion polymerization was used to prepare affinity adsorbents based on molecular imprinting technology. The imprinted particles were immobilized via covalent bonds on the surface of cryogel, a macroporous hydrogel produced by free radical polymerization under sub-zero temperature. In the second case, the affinity sites for Hb were formed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The dynamic binding capacity of the composite cryogel with the immobilized particles and the directly imprinted acrylamide cryogel was found to be 5.2 mg/g and 3.6 mg/g, respectively. The affinity columns showed high selectivity towards Hb in spite of the presence of serum albumin as well as other interfering substances in non-clarified cell homogenates. The maximum capacity in batch mode, the fluid flow and other physical and chemical properties of these columns were investigated.
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| 7. |
- Hanora, Amro, et al.
(författare)
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Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1087:1-2, s. 38-44
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Tidskriftsartikel (refereegranskat)abstract
- A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries. (c) 2005 Elsevier B.V. All rights reserved.
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| 8. |
- Johansson, Hans-Olof, et al.
(författare)
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Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems.
- 2012
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1233, s. 30-35
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Tidskriftsartikel (refereegranskat)abstract
- Phase diagrams of poly(ethylene glycol)/polyacrylate/Na(2)SO(4) systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coli can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coli homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na(2)SO(4)-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH.
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| 9. |
- Kallberg, Kristian, et al.
(författare)
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Application of a pH responsive multimodal hydrophobic interaction chromatography medium for the analysis of glycosylated proteins.
- 2011
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Ingår i: Journal of chromatography. A. - : Elsevier BV. - 1873-3778 .- 0021-9673. ; 128:5, s. 678-683
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Tidskriftsartikel (refereegranskat)abstract
- Protein glycosylation has significant effects on the structure and function of proteins. The efficient separation and enrichment of glycoproteins from complex biological samples is one key aspect and represents a major bottleneck of glycoproteome research. In this paper, we have explored pH multimodal hydrophobic interaction chromatography to separate glycosylated from non-glycosylated forms of proteins. Three different proteins, ribonuclease, invertase and IgG, have been examined and different glycoforms have been identified. The media itself shows strong responsiveness to small variations in pH, which makes it possible to fine-tune the chromatographic conditions according to the properties of the protein isolated. Optimal glycoprotein separation has been obtained at pH 4. The pH responsive multimodal HIC medium in contrast to conventional HIC media is able to resolve contaminating DNA.
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| 10. |
- Matos, Tiago, et al.
(författare)
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Binding and elution behavior of small deoxyribonucleic acid fragments on a strong anion-exchanger multimodal chromatography resin.
- 2013
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1302, s. 40-44
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Tidskriftsartikel (refereegranskat)abstract
- The separation behavior of small single-stranded from double-stranded DNA molecules has been determined on a multimodal (mixed-mode) chromatography system. The resin used is a strong anion exchanger which also modulates hydrophobic recognition. The intrinsic differences between single- and double-stranded DNAs concerning charge, hydrophobicity and three-dimensional structure render this form of MMC suitable for separation of the different nucleic acid molecules. All DNAs tested bound strongly to the resin and they could be eluted with increasing NaCl concentrations. Each homopolymeric ssDNA sample resulted in a base-specific elution pattern when using a linear NaCl gradient. The elution order was poly(dA)
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