| 1. |
- Arvidsson, Pär, et al.
(författare)
-
Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns
- 2002
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 977:1, s. 27-38
-
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.
|
|
| 2. |
- Arvidsson, Pär, et al.
(författare)
-
Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent
- 2003
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 986:2, s. 275-290
-
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
|
|
| 3. |
- Dainiak, Maria, et al.
(författare)
-
Direct capture of product from fermentation broth using a cell-repelling ion exchanger.
- 2002
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 942:1-2, s. 123-131
-
Tidskriftsartikel (refereegranskat)abstract
- A new technique for treating anion exchangers has been proposed allowing direct capture of the fermentation product, shikimic acid directly from the cell-containing fermentation broth. A layer of hydrophilic polymer, poly(acrylic acid) (PAA) has been physically adsorbed on the anion exchanger followed by a covalent cross-linking of PAA. The PAA layer is penetrable for small molecules despite being negatively charged as PAA is, but the polymer layer repels large negatively charged structures like cell debris and cells preventing them from adsorption to the chromatographic matrix. The binding capacity for pure shikimic was about 81 mg/ml adsorbent for both cross-linked PAA-Amberlite and native Amberlite in the fluidized mode of column operation. Binding capacity dropped to 17 and 15 mg per ml adsorbent, respectively, when using filtrated fermentation broth and to about 10 mg/ml adsorbent for cross-linked PAA-Amberlite when using directly the fermentation broth containing cells. Native Amberlite cannot be used for the direct capture of shikimic acid due to the immediate clogging of the column and the collapse of the expanded bed. The cross-linked PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth.
|
|
| 4. |
- Bereli, Nilay, et al.
(författare)
-
Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels
- 2008
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1190:1-2, s. 18-26
-
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.
|
|
| 5. |
- Dainiak, Maria, et al.
(författare)
-
Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells
- 2006
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1123:2, s. 145-150
-
Tidskriftsartikel (refereegranskat)abstract
- Suitable conditions for separating cells using a chromatographic procedure were evaluated in parallel chromatography on minicolumns. A 96-hole minicolumn plate filled with cryogel monoliths (18.8 mm x 7.1 mm O) with immobilized concanavalin A was used. Chromatographic columns (113 mm x 7.1 mm O) were used for chromatographic resolution of a mixture of Saccharomyces cerevisiae and Escherichia coli cells. Separation of a cell mixture containing equal amounts of cells of both types performed in a column format under the determined optimal conditions, resulted in a quantitative capture of applied S. cerevisiae cells, while E. coli passed through the column. Bound S. cerevisiae cells were released by flow-induced detachment and by compression of the adsorbent in the presence of 0.3 M methyl U-D-manno-pyranoside. The flowthrough and the eluted fractions were analyzed by plate counting and by registering metabolic activity of S. cerevisiae cells in the eluted fractions after capturing on ConA-cryogel monoliths in a 96-minicolumn plate format. The flowthrough fraction contained E. coli cells with nearly 100% purity, whereas the fraction eluted by compression of the adsorbent contained viable S. cerevisiae cells with 95% purity. Thus, an efficient chromatographic separation of cells was achieved using affinity cryogel column.
|
|
| 6. |
- Dainiak, Maria, et al.
(författare)
-
Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels
- 2004
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1045:1-2, s. 93-98
-
Tidskriftsartikel (refereegranskat)abstract
- The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 mum) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)(6)-tagged single chain (sc) Fv antibody fragments, (His)(6)-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)(6)-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques. (C) 2004 Published by Elsevier B.V.
|
|
| 7. |
- Galaev, Igor, et al.
(författare)
-
High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format
- 2005
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1065:2, s. 169-175
-
Tidskriftsartikel (refereegranskat)abstract
- Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples. (C) 2005 Elsevier B.V. All rights reserved.
|
|
| 8. |
- Hanora, Amro, et al.
(författare)
-
Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format
- 2005
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1087:1-2, s. 38-44
-
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.
|
|
| 9. |
- Hedström, Martin, et al.
(författare)
-
Fast on-column protein digestion with subsequent peptide mapping using tandem mass spectrometry with information dependent acquisition
- 2005
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1080:2, s. 117-123
-
Tidskriftsartikel (refereegranskat)abstract
- A platform for rapid on-line protein digestion of protein mixtures for direct infusion to a mass spectrometer is presented. A mixture of protein A, staphylococcal enterotoxin B and cytochrome c was used as a model mixture injected on a gel filtration column and a trypsin reactor which were connected in series to a micro liquid chromatography (mu LC) system. The peptides in the column eluate were analyzed with ESI tandem mass spectrometry, utilizing information dependent acquisition (IDA). In one step, the proteins in the mixture (mu M concentrations) were concomitantly desalted, separated, digested and identified with an overall analysis time of less than 40 min. Protein sequence coverage of 78-95% for the involved substances was achieved. (c) 2005 Elsevier B.V. All rights reserved.
|
|
| 10. |
- Noppe, W, et al.
(författare)
-
Immobilised peptide displaying phages as affinity ligands purification of lactoferrin from defatted milk
- 2006
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1101:1-2, s. 79-85
-
Tidskriftsartikel (refereegranskat)abstract
- An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 mu m) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 mu m) phage particles as ligands without any risk of blocking the monolithic column. Bound lactoferrin was eluted using 1 M NaC1 with a purity of > 95%. The technique presents a good alternative to conventional immunoaffinity chromatography for purification of a protein of interest from complex samples due to (i) the robustness of the system in terms of recovery and ligand leakage and (ii) economical aspect in terms of low ligand cost.
|
|
