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| 2. |
- Bansal, Vibha, et al.
(författare)
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Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography
- 2006
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 19:4, s. 332-339
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Tidskriftsartikel (refereegranskat)abstract
- An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(11)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different ellution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography. Copyright (c) 2006 John Wiley & Sons, Ltd.
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| 3. |
- Barrios del Pino, Yvelise, et al.
(författare)
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Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor
- 2004
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 17:4, s. 332-338
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Tidskriftsartikel (refereegranskat)abstract
- lThe antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen-binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by Immoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen-specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour-associated antigen mucin-1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen-specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity. Copyright (C) 2004 John Wiley Sons, Ltd.
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| 4. |
- Becker, Kristian, et al.
(författare)
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Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences.
- 2009
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 22:2, s. 104-109
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Tidskriftsartikel (refereegranskat)abstract
- Hydrophobic interaction chromatography (HIC) has been developed as a powerful technique for separating and purifying proteins. In this study, we have characterized the ability of new multimodal pH-HIC media to resolve proteins with only small differences in their primary structures. This was done by determining the retention times of different green fluorescent protein (GFP) mutants prepared from Escherichia coli extracts. The mutants, modified with single or double hydrophobic amino acid substitutions in two positions, N212 and T230, could be resolved successfully, up to 2.1 column volumes in retention difference for single substitutions and 2.6 column volumes for double substitutions, at two pH and on two media with varying ligand density. The retention times also correlated well with calculated theoretical retentions (R(2) = 0.91) using a hydrophobic descriptor. This medium can therefore be very useful in a final polishing step during purification and the protein library prepared represents a good screening set in validating and characterizing new future media due to the accessible, but yet, extremely small differences in protein structure. Copyright (c) 2008 John Wiley & Sons, Ltd.
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| 6. |
- Björkelund, Hanna, et al.
(författare)
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Avoiding false negative results in specificity analysis of protein-protein interactions
- 2011
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 24:1, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed.
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| 7. |
- Bywater, Robert P.
(författare)
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Location and nature of the residues important for ligand recognition in G-protein coupled receptors
- 2005
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 18:1, s. 60-72
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Forskningsöversikt (refereegranskat)abstract
- The overall structure of the biogenic amine subclass of the G-protein-coupled receptors, and of their ligand binding sites, is discussed with the aim of highlighting the major structural features of these receptors that are responsible for ligand recognition. A comparison is made between biogenic amine receptors, peptide receptors of the rhodopsin class, and the secretin receptors which all have peptide ligands. The question of where the peptide ligands bind, whether at extracellular sites or within the transmembrane helix bundle, is discussed. The suitability of the rhodopsin crystal structure as a template for construction of homology models is discussed and it is concluded that there are many reasons why a caution should be issued against using it uncritically.
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| 8. |
- Chen, Zhiyong, et al.
(författare)
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Controlling size and uniformity of molecularly imprinted nanoparticles using auxiliary template.
- 2012
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 25:6, s. 370-376
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Tidskriftsartikel (refereegranskat)abstract
- Molecularly imprinted nanomaterials are gaining substantial importance. As a simple and efficient synthetic method, precipitation polymerization has been used to prepare uniform molecularly imprinted microspheres for numerous template compounds. Despite of its general applicability, the difficulty of obtaining uniform particles for some difficult templates by precipitation polymerization has been reported. In this work, we attempted to produce uniform atrazine-imprinted nanoparticles using propranolol as an auxiliary template under standard precipitation polymerization condition. When propranolol was added in the prepolymerization mixture for atrazine imprinting, it displayed a significant effect on particle size and size distribution of atrazine-imprinted polymers. The molecular binding characteristics of the molecularly imprinted polymer (MIP) nanoparticles were found to be dependent on the relative ratios of the two templates. Under an optimal template propranolol-atrazine ratio of 1:3 mol/mol, very uniform imprinted nanoparticles (d(H) = 106 nm) with a polydispersity index of 0.07 were obtained. The loading of the auxiliary template (propranolol) could be reduced to as low as 5% without sacrificing the uniformity of the MIP nanoparticles. The uniform MIP nanoparticles could be easily encapsulated into polyethylene terephthalate nanofibers using a simple electrospinning technique. The composite nanofibers containing the MIP nanoparticles maintained specific molecular binding capability for both atrazine and propranolol. Copyright © 2012 John Wiley & Sons, Ltd.
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| 9. |
- Ellmark, Peter, et al.
(författare)
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A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement
- 2004
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 17:4, s. 316-322
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Tidskriftsartikel (refereegranskat)abstract
- The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component. Copyright (C) 2004 John Wiley Sons, Ltd.
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