Showing papers on "Affinity chromatography published in 2004"
TL;DR: This work has used the ability of different lectins to recognize specific glycosylation motifs to develop a specific affinity system that can achieve a comprehensive capture of serum glycoproteins through a multi-lectin affinity column.
295 citations
TL;DR: Protein A affinity chromatography is often employed as a capture step to meet the purity, yield, and throughput requirements for pharmaceutical antibody purification, but a trade-off exists between step performance and price.
265 citations
TL;DR: It is shown that several subunits of RNA polymerases I, II, and III, members of the transcription repression and chromatin remodeling machineries previously not known to be sumoylated, are modified by SUMO-1.
259 citations
TL;DR: A novel method for identifying transient protein‐protein interactions using in vivo cross‐linking and MS‐based protein identification is described and the RasGAP‐related protein IQGAP1 is identified to be a novel interaction partner of M‐RasQ71L.
Abstract: The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.
246 citations
TL;DR: The usefulness of arginine as an efficient eluent for Protein-A chromatography is demonstrated and it is demonstrated that the eluted antibodies were mostly monomeric whether eluted with citrate or Arginine.
209 citations
TL;DR: A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system) that allows for the efficient purification of natural protein complexes and their identification by mass spectrometry.
Abstract: A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). B...
207 citations
TL;DR: A new purification procedure involving affinity chromatography on N-acetylcysteine-derivatized Sepharose on the basis of the selectivity of L-ficolin is developed, which shows that it is not easily defined as a lectin.
192 citations
TL;DR: Microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments, and though further macroscaleAssays are needed, these microscale assay showed that reuniting Mytilus galloprovincialis foot protein type 5 with hexahistidine affinity ligand has significanthesive ability.
Abstract: Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments
187 citations
TL;DR: Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection.
177 citations
TL;DR: It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative effect on protein solubility, but the effect is target protein specific and a solubilizing fusion tag was able to partly counteract this negative effect.
Abstract: We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative effect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins. abbreviations:
BSA – bovine serum albumin; DBD – DNA binding domain; DCW – dry cell weight; EDTA – ethylenediaminetetraacetic acid; GFP – green fluorescent protein; IMAC – immobilized metal ion affinity chromatography; IPTG – isopropyl-β-d-thiogalactopyranoside; LB – Luria-Bertani; MES – 2-(N-morpholino) ethane sulfonic acid; OD – optical density; ORF – open reading frame; PCR – polymerase chain reaction; SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis.
174 citations
TL;DR: This work has used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6, revealing the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism.
TL;DR: In this paper, a placenta-derived galectin-13 (PP13) was cloned from human term placentia and a specific monoclonal antibody to PP13 was developed, which showed its conserved structural and functional homology to members of the Galectin family.
Abstract: Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin-13. Similar to human eosinophil Charcot-Leyden crystal protein/galectin-10 but not other galectins, its weak lysophospholipase activity was confirmed by 31P-NMR. In this study, recombinant PP13/galectin-13 was expressed and specific monoclonal antibody to PP13 was developed. Endogenous lysophospholipase activity of both the purified and also the recombinant protein was verified. Sugar binding assays revealed that N-acetyl-lactosamine, mannose and N-acetyl-glucosamine residues widely expressed in human placenta had the strongest binding affinity to both the purified and recombinant PP13/galectin-13, which also effectively agglutinated erythrocytes. The protein was found to be a homodimer of 16 kDa subunits linked together by disulphide bonds, a phenomenon differing from the noncovalent dimerization of previously known prototype galectins. Furthermore, reducing agents were shown to decrease its sugar binding activity and abolish its haemagglutination. Phosphorylation sites were computed on PP13/galectin-13, and phosphorylation of the purified protein was confirmed. Using affinity chromatography, PAGE, MALDI-TOF MS and post source decay, annexin II and beta/gamma actin were identified as proteins specifically bound to PP13/galectin-13 in placenta and fetal hepatic cells. Perinuclear staining of the syncytiotrophoblasts showed its expression in these cells, while strong labelling of the syncytiotrophoblasts' brush border membrane confirmed its galectin-like externalization to the cell surface. Knowing its colocalization and specific binding to annexin II, PP13/galectin-13 was assumed to be secreted to the outer cell surface by ectocytosis, in microvesicles containing actin and annexin II. With regard to our functional and immunomorphological results, PP13/galectin-13 may have special haemostatic and immunobiological functions at the lining of the common feto-maternal blood-spaces or developmental role in the placenta.
TL;DR: An affinity selection-mass spectrometry method for protein-ligand affinity ranking and the classification of ligands by binding site, readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.
Abstract: To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein−ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein−ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affin...
TL;DR: AraA gene encoding l-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence and had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures.
Abstract: The araA gene encoding l-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni2+ affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90°C and pH 7.5 under the assay conditions used. Its apparent Km values for l-arabinose and d-galactose were 31 and 60 mM, respectively; the apparent Vmax values (at 90°C) were 41.3 U/mg (l-arabinose) and 8.9 U/mg (d-galactose), and the catalytic efficiencies (kcat/Km) of the enzyme were 74.8 mM−1 · min−1 (l-arabinose) and 8.5 mM−1 · min−1 (d-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn2+ and/or Co2+ than in the absence of these ions. The enzyme carried out the isomerization of d-galactose to d-tagatose with a conversion yield of 56% for 6 h at 80°C.
TL;DR: Improvements have been made to these processes including sophisticated changes to the mobile phase composition and the recycling of aggregates to improve yield and in the use of immobilized metal affinity chromatography.
TL;DR: The fluorescence spectra, analytical size exclusion chromatography, and the specific activity of the purified protein were observed to be similar to the native protein which demonstrated that the protein had folded properly and was present in its active tetramer form in the culture supernatant.
TL;DR: In order to enable detection of improvements regarding the alkaline resistance of the Z domain, a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework is used.
Abstract: Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.
TL;DR: The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.
Abstract: Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS The analysis resulted in the identification of 83 different gene products in the untreated plasma Removal of the high-abundance proteins resulted in the visualization of new protein signals In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies
TL;DR: This work reports the specific removal of 98% of albumin and 80% of immunoglobulin heavy chain from human plasma by affinity chromatography, and the subsequent improvement in the number of spots detected and their resolution following two‐dimensional gel electrophoresis.
Abstract: In studies of the plasma proteome, the high abundance of proteins such as albumin and immunoglobulin impedes the investigation of lower abundance proteins that may be more suitable as biomarkers of disease. We report the specific removal of 98% of albumin and 80% of immunoglobulin heavy chain from human plasma by affinity chromatography, and the subsequent improvement in the number of spots detected and their resolution following two-dimensional gel electrophoresis.
TL;DR: An alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture of rare earth elements.
TL;DR: Results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.
Abstract: Immunological strategies for the detection of N(epsilon)-(carboxymethyl)lysine (CML), one of the major antigenic structures of advanced glycation end products (AGE), are widely applied to demonstrate the contribution of CML to the pathogeneses of diabetic complications and atherosclerosis. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly through the Embden-Meyerhof and polyol pathways, reacts with proteins to form MG-derived AGE structures such as N(epsilon)-(carboxyethyl)lysine (CEL). In order to accurately measure the CML contents of the proteins by means of an immunochemical method, we prepared CML-specific antibodies since conventionally prepared polyclonal anti-CML antibody and monoclonal anti-CML antibody (6D12) cross-reacted with CEL. To prepare polyclonal CML-specific antibody, CML-keyhole limpet hemocyanin (CML-KLH) were immunized with rabbit and CEL-reactive antibody was removed by CEL-conjugated affinity chromatography. Monoclonal antibody specific for CML (CMS-10) was obtained by immunization with CML-KLH, followed by successive screening according to CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both polyclonal CML-specific antibody and CMS-10 significantly reacted with CML-proteins but not with CEL-proteins. It is likely therefore that these antibodies can recognize the difference of one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with BSA modified with several aldehydes and its reactivity was highly correlated with the CML content, which was determined by high performance liquid chromatography, whereas 6D12 showed a low correlation. These results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.
TL;DR: Comparison of a CYP102A2 homology model with the CYP 102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences inThe catalytic properties of these two enzymes.
Abstract: The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelate affinity chromatography (IMAC) and characterised. CYP102A2 is a 119-kDa self-sufficient monooxygenase, consisting of an FMN/FAD-containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from B. megaterium (59%) and CYP102A3 from B. subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 449 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ω-3, ω-2 and ω-1 hydroxylated fatty acids. Activity was highest towards oleic acid (KM=17.36±1.4 μM, kcat=2,244±72 min−1) and linoleic acid (KM=12.25±1.8 μM, kcat=1,950±84 min−1). Comparison of a CYP102A2 homology model with the CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in the catalytic properties of these two enzymes.
TL;DR: This is the first study to biochemically describe PfHsp70 and establishes a foundation for future studies on its chaperone properties.
TL;DR: A laccase with a novel N-terminal sequence, a low molecular mass smaller than those of previously reported laccases, a pH optimum of 4, and a temperature optimum at 70 degrees C was isolated from fresh fruiting bodies of the mushroom Tricholoma giganteum.
TL;DR: Recombinant domain III of dengue virus type-2 envelope is expressed and purified without the aid of a carrier protein in Escherichia coli, and could effectively protect cells in culture against d Dengue virus types-2 infection by blocking the virus from binding to host cells.
TL;DR: The peptidoglycan recognition protein, PGRP, known as an intracellular component of neutrophils, has been isolated from camel milk by acid precipitation followed by heparin-sepharose affinity chromatography of the supernatant.
TL;DR: The integrated system reduces the time required for the lectin affinity chromatography reaction to approximately 3%, thus, the overall analysis time from 4 h to 400 s, which results in improvement of performance, as compared to the conventional method.
Abstract: Lectin affinity chromatography was miniaturized into a microfluidic format, which results in improvement of performance, as compared to the conventional method. A lectin affinity monolith column was prepared in the microchannel of a microfluidic chip. The porous monolith was fabricated by UV-initiated polymerization of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) in the presence of porogeneities, followed by immobilization of pisum sativum agglutinin (PSA) on the monolith matrix. Using electroosmosis as the driven force, lectin affinity chromatographies of three kinds of glycoprotein, turkey ovalbumin (TO), chicken ovalbumin (CO), and ovomucoid (OM), were carried out on the microfluidic system. All the glycoproteins were successfully separated into several fractions with different affinities toward the immobilized PSA. The integrated system reduces the time required for the lectin affinity chromatography reaction to ∼3%, thus, the overall analysis time from 4 h to 400 s. Only 300 pg of g...
TL;DR: It appears that MCLs have unusual carbohydrate specificities for N-acetyl-d-galactosamine, which contains both mucin-type carbohydrate chains and highly branched mannans from marine halophilic bacteria.
Patent•
01 Mar 2004
TL;DR: In this article, a method for selectively removing leaked protein A from antibody purified by means of protein A affinity chromatography is described, which can be used to identify the source of leaks.
Abstract: A novel method for selectively removing leaked protein A from antibody purified by means of protein A affinity chromatography is disclosed.
TL;DR: The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state.