| 1. |
- Ahlqvist, Josefin, et al.
(författare)
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Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 354:2, s. 229-237
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Tidskriftsartikel (refereegranskat)abstract
- A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved.
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| 2. |
- Andac, M, et al.
(författare)
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Poly(hydroxyethyl methacrylate)-based macroporous hydrogels with disulfide cross-linker
- 2008
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Ingår i: Macromolecular Chemistry and Physics. - : Wiley. - 1521-3935 .- 1022-1352. ; 209:6, s. 577-584
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Tidskriftsartikel (refereegranskat)abstract
- Biodegradable supermacroporous PHEMA cryogels were produced by combining two cross-linkers, poly(ethylene glycol) diacrylate and a newly developed disulfide water soluble crosslinker, N,N'-bis(methacryloyl)-L-cystine. The biodegradable PHEMA cryogels were prepared with gel fraction yields up to 70% and were characterized by highly interconnected pores of micrometer size and good mechanical stability. When subjected to reductive agents like DTT, the biodegradable PHEMA cryogels disintegrated into small pieces. The rate of disintegration was controlled by the crosslinking density in the cryogels and the DTT concentration.
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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. |
- Arvidsson, Pär, et al.
(författare)
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Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent
- 2003
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 986:2, s. 275-290
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Tidskriftsartikel (refereegranskat)abstract
- A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates. The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N,N′-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix having interconnected pores of 10–100 m size. Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His)6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli. The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column. No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column. A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties. Mechanically the cryogel adsorbent is very stable. The continuous matrix could easily be removed from the column, dried at 70 °C and kept in a dry state. After rehydration and reinsertion of the matrix into an empty column, (His)6-LDH was purified as efficiently as on the newly prepared column. The procedure of manufacturing the supermacroporous continuous cryogel is technically simple. Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions. Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts
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| 5. |
- Babac, Ceyhun, et al.
(författare)
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Binding of antibodies to concanavalin A-modified monolithic cryogel
- 2006
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Ingår i: Reactive & Functional Polymers. - : Elsevier BV. - 1873-166X .- 1381-5148. ; 66:11, s. 1263-1271
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Tidskriftsartikel (refereegranskat)abstract
- Binding of human immunoglobulin-G (IgG) from aqueous solutions and human plasma to concanavalin A (Con A) immobilized poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] monolithic cryogel has been studied. Poly(AAm-AGE) cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the monolithic cryogel contains a continuous polymeric matrix having interconnected pores of 10-100 pm size. Con A was immobilized by covalent binding onto poly(AAm-AGE) cryogel via epoxy groups. The maximum IgG adsorption on the Con A-poly(AAm-AGE) cryogels was observed at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the plain poly(AAm-AGE) adsorbents was about 0.25 mg/g. Up to 6.7 mg/g IgG were bound to Con A-poly(AAm-AGE) cryogels from aqueous solutions. The large pore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 25.6 mg/g). Bound IgG was eluted using 2.0 M NaCl with a purity of 85%. Adsorption capacities of other blood proteins were obtained as 1.0 mg/g for fibrinogen and 1.7 mg/g for albumin. The total protein adsorption was determined as 28.6 mg/g. Con A-poly(AAm-AGE) cryogels was used for repetitive adsorption/desorption of IgG molecules without noticeable loss in IgG adsorption capacity after 10 cycles. (c) 2006 Elsevier B.V. All rights reserved.
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| 6. |
- Balan, S, et al.
(författare)
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Metal chelate affinity precipitation of RNA and purification of plasmid DNA
- 2003
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Ingår i: Biotechnology Letters. - 1573-6776. ; 25:13, s. 1111-1116
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Tidskriftsartikel (refereegranskat)abstract
- The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.
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| 7. |
- Baydemir, Goezde, et al.
(författare)
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Bilirubin recognition via molecularly imprinted supermacroporous cryogels
- 2009
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Ingår i: Colloids and Surfaces B: Biointerfaces. - : Elsevier BV. - 1873-4367 .- 0927-7765. ; 68:1, s. 33-38
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Tidskriftsartikel (refereegranskat)abstract
- Recent years molecular imprinting has received considerable attention as an excellent and simple approach to recognize small molecules and bioactive substances. The aim of this study is to prepare the bilirubin-imprinted supermacroporous cryogels which can be used for the adsorption of bilirubin from human plasma. N-methacryloyl(L)-tyrosinemethylester (MAT) was chosen as the preorganization monomer. In the first step, bilirubin was complexed with MAT and the bilirubin-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-tyrosine methylester) [BR-MIP] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After that, the template molecules (i.e., bilirubin) were removed from the polymeric structure using sodium carbonate and sodium hydroxide. The maximum bilirubin adsorption amount was 3.6 mg/g polymer. The relative selectivity coefficients of the BR-MIP cryogel for bilirubin/cholesterol and bilirubin/testosterone mixtures were 7.3 and 3.2 times greater than non-imprinted poly(HEMA-MAT) [NIP] cryogel, respectively. The BR-MIP cryogel could be used many times without decreasing bilirubin adsorption amount significantly. Therefore, as a reusable carrier possessing high selectivity, BR-MIP cryogel has a potential candidate as a clinical hemoperfusion material. (C) 2008 Elsevier B.V. All rights reserved.
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| 8. |
- Baydemir, Goezde, et al.
(författare)
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Supermacroporous poly(hydroxyethyl methacrylate) based cryogel with embedded bilirubin imprinted particles
- 2009
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Ingår i: Reactive & Functional Polymers. - : Elsevier BV. - 1873-166X .- 1381-5148. ; 69:1, s. 36-42
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Tidskriftsartikel (refereegranskat)abstract
- Molecular imprinted polymers are artificial, template-made materials with the ability to recognize and to specifically bind the target molecule. The aim of this study is to prepare supermacroporous cryogel with embedded bilirubin-imprinted particles which can be used for the selective removal of bilirubin from human plasma. N-methacryloyl-(L)-tyrosinemethylester (MAT) was chosen as the pre-organization monomer. In the first step, bilirubin was complexed with MAT and the bilirubin-imprinted poly(hydroxyethyl methacrylate-N-methacryloly-(L)-tyrosine methyl-ester) [MIP] monolith was produced by bulk polymerization. MIP monolith was smashed and the particles ground and sieved through 100 pm sieves. In the second step. the supermacroporous poly(hydroxyethyl methacrylate) (PHEMA) cryogel with embedded MIP particles [PHEMA/MIP composite cryogel] was produced by free radical polymerization initiated by N.N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After that, the template (i.e., bilirubin) molecules were removed using sodium carbonate and sodium hydroxide. Compared with the PHEMA cryogel (0.2 mg/g polymer), the bilirubin adsorption capacity of the PHEMA/MIP composite cryogel (10.3 mg/g polymer) was improved significantly due to the embedded MIP particles into the polymeric matrix. The relative selectivity coefficients of PHEMA/MIP composite cryogel for bilirubin/cholesterol and bilirubin/testosterone were 8.6 and 4.1 times greater than the PHEMA cryogel, respectively. The PHEMA/MIP composite cryogel could be used many times without decreasing the bilirubin adsorption capacity significantly. (c) 2008 Elsevier Ltd. All rights reserved.
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| 9. |
- Bereli, Nilay, et al.
(författare)
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Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels
- 2008
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1190:1-2, s. 18-26
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Tidskriftsartikel (refereegranskat)abstract
- Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. The aim of this study is to prepare lysozyme-imprinted supermacroporous cryogels which can be used for the purification of lysozyme (Lyz) from egg white. N-Methacryloyl-(L)-histiclinemethylester (MAH) was chosen as the metal-coordinating monomer. In the first step, Cu2+ was complexed with MAH and the lysozyme-imprinted poly(HEMA-MAH) [Lyz-MIP] cryogel were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) in an ice bath. After that, the template (i.e., lysozyme) was removed using 0.05 M phosphate buffer containing 1 M NaCl (pH 8.0). The maximum lysozyme adsorption capacity was 22.9 mg/g polymer. The relative selectivity coefficients of Lyz-MIP cryogel for lysozyme/bovine serum albumin and lysozyme/cytochrome c were 4.6 and 3.2 times greater than non-imprinted poly(HEMA-MAH) (NIP) cryogel, respectively. Purification of lysozyme from egg white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 94% with recovery about 86%. The Lyz-MIP cryogel could be used many times without decreasing the adsorption capacity significantly.
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| 10. |
- Berillo, Dmitry, et al.
(författare)
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Formation of macroporous self-assembled hydrogels through cryogelation of Fmoc-Phe-Phe.
- 2012
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 1095-7103 .- 0021-9797. ; 368:1, s. 226-230
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Tidskriftsartikel (refereegranskat)abstract
- In this study, it was found that macroporous hydrogels were formed when self-assembly of fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Phe-Phe) peptides was induced using glucono-δ-lactone (GdL) in apparently frozen samples. Formed cryogels exhibited a heterogeneous structure with pore walls of densely packed fibres of assembled dipeptides and pores in the range 10-100μm. Hydrogels formed from the same composition above the freezing point exhibited a homogenous structure without any apparent porosity. The formed gels were characterised using microscopy techniques, CD-spectroscopy and stress sweeps. The cryogels exhibited less mechanical strength than the hydrogels that might be due to the heterogeneous structure of the former. It appeared that the self-assembled peptide both in the cryo- and hydrogel maintained the β-sheet structure commonly attributed to these.
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