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Showing papers on "Affinity chromatography published in 2005"


Journal ArticleDOI
TL;DR: It is found that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts.

469 citations


Journal ArticleDOI
TL;DR: A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)3 is presented here and is more selective, more cost effective and easily applicable to method optimization.
Abstract: A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.

283 citations


Journal ArticleDOI
TL;DR: The specific immobilization has been demonstrated for the cases of a genetically engineered horseradish peroxidase and ferredoxin-NADP(+) reductase, confirming the attachment of the fully functional proteins to the Co(II)-terminated nanointerface.
Abstract: This paper presents an efficient strategy for the specific immobilization of fully functional proteins onto the surface of nanoparticles. Thioctic acid-derivatized gold clusters are used as a scaffold for further stepwise modification, leading to a cobalt(II)-terminated ligand shell. A histidine tag introduced by genetic engineering into a protein is coordinated to this transition metal ion. The specific immobilization has been demonstrated for the cases of a genetically engineered horseradish peroxidase and ferredoxin-NADP+ reductase, confirming the attachment of the fully functional proteins to the Co(II)-terminated nanointerface. The absence of nonspecific protein adsorption and the specificity of the binding site have been verified using several analogues of the enzymes without the histidine tag.

264 citations


Journal ArticleDOI
TL;DR: A new method for the purification of recombinant proteins expressed in Escherichia coli using self-cleaving elastin-like polypeptide (ELP) fusion tags without the need for affinity chromatography or proteolytic tag removal is introduced.
Abstract: We introduce a new method for the purification of recombinant proteins expressed in Escherichia coli using self-cleaving elastin-like polypeptide (ELP) fusion tags without the need for affinity chromatography or proteolytic tag removal. Using this method we obtained high purity, activity and reasonable yields for ten diverse target proteins.

219 citations


Journal ArticleDOI
TL;DR: Empirope substitution improved purification in experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function.
Abstract: Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBP-calmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.

219 citations


Journal ArticleDOI
TL;DR: The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein‐protein interactions.
Abstract: We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin, and the human plasma protein loss in the affinity chromatography/strong cation exchange/reversed-phase liquid chromatography-tandem mass spectrometry methodology was investigated in detail. The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein-protein interactions.

196 citations


Journal ArticleDOI
TL;DR: Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum to indicate that the protein parent was fully sialylated at that specific glycosylation site.
Abstract: This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.

167 citations


Journal ArticleDOI
TL;DR: The purified S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride and showed random positional specificity on the thin-layer chromatography.

149 citations


BookDOI
19 Jul 2005
TL;DR: This chapter discusses Affinity Chromatography in Clinical and Pharmaceutical Analysis, as well as its applications in Biotechnology and Molecular Biology, and some of the techniques used in these applications.
Abstract: INTRODUCTION AND BASIC CONCEPTS An Introduction to Affinity Chromatography D.S. Hage and P.F. Ruhn Support Materials for Affinity Chromatography P. Gustavsson and P. Larsson Immobilization Methods for Affinity Chromatography H.S. Kim and D.S. Hage Application and Elution in Affinity Chromatography D.S. Hage, H. Xuan, and M.A. Nelson GENERAL AFFINITY LIGANDS AND METHODS Bioaffinity Chromatography D.S. Hage, M. Bian, R. Burks, E. Karle, C. Ohnmacht, and C. Wa Immunoaffinity Chromatography D.S. Hage and T.M. Phillips DNA Affinity Chromatography R.A. Moxley, S. Oak, H. Gadgil, and H.W. Jarrett Boronate Affinity Chromatography X. Liu and W.H. Scouten Dye-Ligand and Biomimetic Affinity Chromatography N.E. Labrou, K. Mazitsos, and Y.D. Clonis Immobilized Metal-Ion Affinity Chromatography D. Todorova and M.A. Vijayalakshmi PREPARATIVE APPLICATIONS General Considerations in Preparative Affinity Chromatography A. Subramanian Affinity Chromatography of Enzymes F. Friedberg and A.R. Rhoads Isolation of Recombinant Proteins by Affinity Chromatography A. Subramanian Affinity Chromatography in Antibody and Antigen Purification T.M. Phillips Affinity Chromatography of Regulatory and Signal-Transducing Proteins A.R. Rhoads and F. Friedberg Receptor-Affinity Chromatography P. Bailon, M. Nachman-Clewner, C. L. Spence, and G.K. Ehrlich ANALYTICAL AND SEMIPREPARATIVE APPLICATIONS Affinity Methods in Clinical and Pharmaceutical Analysis Carrie A.C. Wolfe, W. Clarke and D.S. Hage Affinity Chromatography in Biotechnology N. Jordan and I. Krull Environmental Analysis by Affinity Chromatography M.A. Nelson and D.S. Hage Affinity Chromatography in Molecular Biology L.A. Jurado, S.Oak, Himanshu Gadgil, R.A. Moxley, and H.W. Jarrett Affinity-Based Chiral Stationary Phases S. Patel, I.W. Wainer and W.J. Lough BIOPHYSICAL APPLICATIONS Quantitative Affinity Chromatography: Practical Aspects D.S. Hage and J. Chen Quantitative Affinity Chromatography: Recent Theoretical Developments D.J. Winzor Chromatographic Studies of Molecular Recognition and Solute Binding to Enzymes and Plasma Proteins S. Patel, I.W. Wainer and W.J. Lough Affinity-Based Optical Biosensors S.D. Long and D.G. Myszka RECENT DEVELOPMENTS Affinity Ligands in Capillary Electrophoresis Niels H. H. Heegaard and C. Schou Affinity Mass Spectrometry C.J. Briscoe, W. Clarke and D.S. Hage Microanalytical Methods Based on Affinity Chromatography T.M. Phillips Chromatographic Immunoassays A.C. Moser and D.S. Hage Molecularly Imprinted Polymers: Artificial Receptors for Affinity Separations K. Haupt

140 citations


Journal ArticleDOI
TL;DR: The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification, and in some cases, it is even possible to identify the site of oxidation.
Abstract: It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of a wide variety of routes by which proteins are oxidized is the generation of carbonyl groups. This paper reports a method for the isolation of oxidized proteins, which involves (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. The selectivity of the method was validated by adding in vitro oxidized biotinylated BSA to a yeast lysate and showing that the predominant protein recovered was BSA. This method was applied to the question of whether large doses of 2-nitropropane produce oxidized proteins. A study of rat liver homogenates showed that animals dosed with 2-nitropropane produced 17 times more oxidized protein than controls in 6 h. Tryptic digestion of these oxidized proteins followed by reversed-phase chromatography and tandem mass spectrometry led to the identification of 14 peptides and their parent proteins. Nine of the 14 identified peptides were found to carry 1 or 2 oxidation sites and 5 of the 9 peptides were biotinylated. The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification. In some cases, it is even possible to identify the site of oxidation.

140 citations


Journal ArticleDOI
01 Feb 2005-Peptides
TL;DR: An antifungal peptide with a molecular mass of 9 kDa was isolated from fresh fruiting bodies of the mushroom Agrocybe cylindracea and exhibited weaker mitogenic activity than Con A on isolated murine splenocytes, but was devoid of antiproliferative activity when tested at 110 microM.

Journal ArticleDOI
TL;DR: Arginine was effective in eluting monoclonal antibodies IgG1 and IgG4 and effective in fractionation of pAbs using antigen-conjugated affinity columns, and GdnHCl was also effective under similar conditions, but the eluted material showed more aggregation than did the protein eluted by arginine.

Journal ArticleDOI
TL;DR: This study successfully purified a PP2A holoenzyme protein complex from a cultured mammalian cancer cell line using the Strep‐tag affinity chromatography method, enabling fast and simple one‐step purification, coupled with competitive elution under physiological conditions.
Abstract: Identification of protein complexes is the key to understanding cellular functions. In this study, we present a novel method for the identification of multiprotein complexes from mammalian cells. By using the Strep-tag affinity chromatography method, enabling fast and simple one-step purification, coupled with competitive elution under physiological conditions, we successfully purified a PP2A holoenzyme protein complex from a cultured mammalian cancer cell line. We identified, by mass spectrometry, both known and novel interacting proteins for PP2A, and demonstrate that the purified PP2A complex is functional. The benefits and potential applications of the Strep-tag method for protein complex purification are discussed.


Journal ArticleDOI
TL;DR: These findings provide conclusive evidence for the existence of an inactive proenzyme to this plasminogen activator and thus demonstrate and additional step in a cascade-like reaction leading to extracellular proteolysis.
Abstract: We have previously reported the purification of a plasminogen-activating serine pro1 ease with an approximate Mr of 48000 from sarcoma-virus-transformed murine cells. We now report that under serum-free conditions the enzyme is released from the cells in an inactive form. After affinity chromatography with 4-aminobenzamidine-cellulose, ion-exchange chromatography and gel filtration, the proenzyme could be obtained from culture fluid as a pure, homogeneous protein as evaluated by polyacrylamide gel electrophoresis with sodium dodecylsulphate. Proenzyme was quantitatively converted to active enzyme by incubation with catalytic amounts of plasmin. Analysis by polyacrylainide gel electrophoresis with sodium dodecylsulphate under reducing and non-reducing conditions showed that the inactive form consisted of a single polypeptide chain with an Mr of approximately 48000, while the active form consisted of two chains with Mr values of approximately 18000 and 29000, held together by one or more disulphide bridges. The active-site reagent diisopropylfluorophosphate in radiolabelled form was incorporated into the 29000-Mr chain of thc active enzyme, but not into the inactive form. These findings provide conclusive evidence for the existence of an inactive proenzyme to this. plasminogen activator and thus demonstrate an additional step in a cascade-like reaction leading to extrac ellular proteolysis. Regulatory as well as methodological implications of this finding are discussed.

Journal ArticleDOI
TL;DR: Inhibition analysis indicates that racemization and beta-elimination activities of mSR reside at the same active site and has a broader specificity for l-amino acids with a suitable leaving group at the beta-carbon and optimal spatial orientation of the alpha-carboxyl and leaving groups.
Abstract: Mouse serine racemase (mSR) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the biosynthesis of the N-methyl-d-aspartate receptor coagonist d-serine in the brain. Furthermore, mSR catalyzes beta-elimination of serine and l-serine-O-sulfate into pyruvate. The biological significance of this beta-elimination activity and the factors influencing mSR substrate and reaction specificity, which are crucial for prospective inhibitor design, are poorly understood. Using a bacterial expression system and ATP-agarose affinity chromatography, we have generated a pure and active recombinant mSR and investigated its substrate and reaction specificity in vitro by analyzing a systematic series of compounds derived from l-Ser and l-serine-O-sulfate. The analysis revealed several competitive inhibitors of serine racemization including glycine (K(I) = 1.63 mM), several dicarboxylic acids including malonate (K(I) = 0.077 mM), and l-erythro-3-hydroxyaspartate (K(I) = 0.049 mM). The latter compound represents the most effective inhibitor of SR reported to date. A simple inversion of the beta-carbon configuration of the compound yields an excellent beta-elimination substrate l-threo-3-hydroxyaspartate. Inhibition analysis indicates that racemization and beta-elimination activities of mSR reside at the same active site. While the racemization activity is specific to serine, the beta-elimination activity has a broader specificity for l-amino acids with a suitable leaving group at the beta-carbon and optimal spatial orientation of the alpha-carboxyl and leaving groups. The possible implications of our observations for inhibitor design, regulation of activity, and function of mSR are discussed.

Journal ArticleDOI
TL;DR: This represents a successful crystallization of a mammalian membrane protein derived from a heterologous expression system, and it opens the way for the study of mutant forms of SERCA1a.
Abstract: The Ca2+-ATPase SERCA1a (sarcoplasmic–endoplasmic reticulum Ca2+-ATPase isoform 1a) from rabbit has been overexpressed in Saccharomyces cerevisiae. This membrane protein was purified by avidin agarose affinity chromatography based on natural biotinylation in the expression host, followed by HPLC gel filtration. Both the functional and structural properties of the overexpressed protein validate the method. Thus, calcium-dependent ATPase activity and calcium transport are essentially intact after reconstitution in proteoliposomes. Moreover, the recombinant protein crystallizes in a form that is isomorphous to the native SERCA1a protein from rabbit, and the diffraction properties are similar. This represents a successful crystallization of a mammalian membrane protein derived from a heterologous expression system, and it opens the way for the study of mutant forms of SERCA1a.

Journal ArticleDOI
TL;DR: The results indicated that the multi-lectin affinity column (M-LAC) is sensitive to changes in the content of sialic acid and fucosyl residues present in serum glycoproteins, and has the potential to be used to screen serum proteins for glycosylation changes due to disease.

Journal ArticleDOI
TL;DR: A chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase using Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates is described.
Abstract: Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second nonenzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the alkylated phosphorothioate epitope recognize these labeled substrates, but not alkylation products of other cellular nucleophiles. This strategy is demonstrated with Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates.

Journal ArticleDOI
TL;DR: Serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph, as identified in the proteins eluted from the immunoaffinity column using serpins antibody.

Journal ArticleDOI
TL;DR: A novel application of SPR biosensor technology to screen solubilization conditions automatically and to assess receptor activity directly is described and the benefits of using the biosensor as a tool for isolating functional membrane receptors and for analyzing ligand/receptor interactions are illustrated.

Journal ArticleDOI
TL;DR: The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennar glycans, and the relative amount of biantENNary glycan versus tri-and- tetraanavalin glycans was different at separate glycosylation sites within the same glycoprotein.
Abstract: This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein.

Journal ArticleDOI
TL;DR: A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate.
Abstract: A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin−intein tag, and the target protein is then isolated from the elastin−intein fusion by an additional phase transition cycle.

Journal ArticleDOI
TL;DR: Thermodynamic analysis of the activity data showed that the activation energies are dependent on the substrate, and entropy changes appear to be the main determinant contributing to substrate specificity.

Journal ArticleDOI
TL;DR: It appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa, which was able to breakdown intact glucosinolates in a crude extract of broccoli.

Journal ArticleDOI
TL;DR: Results clearly demonstrated that NDH-1S is associated with NDh-1M in vivo, and the BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH
Abstract: NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Journal ArticleDOI
TL;DR: A simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.
Abstract: Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.

Journal ArticleDOI
TL;DR: The enzyme specificity was revealed to be that of a true mammalian collagenase splitting the native type I tropocollagen molecule into three-quarter and one-quarter fragments according to dodecyl sulfate/polyacrylamide gel electrophoresis.
Abstract: Latent and active human polymorphonuclear leukocyte collagenase could be purified to apparent homogenei by a rapid and reproducible method involving affinity chromatography on concanavalin-A–Sepharose, collage Sepharose and activated thiol-Sepharose 4-B, gel filtration on Sephacryl S-300 and ion-exchange chromatograpl on DEAE-Sephacel. The latent and active collagenase were shown to be single entities with apparent Mr 91000 and 6400 respectively, as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without mercapto ethanol. Dodecyl sulfate gel electrophoresis of the latent enzyme in the presence of mercaptoethanol showed two bands with apparent Mr of 64000 and 24000 indicating the cleavage of a disulfide bond. Activation of the native latent collagenase could be achieved by_ disulfide-containing compounds. A concomitant decrease in molecular weight of approximately 25000 was observed on activation of the latent enzyme. The activators proved ineffective after alkylation of the latent enzyme with iodoacetamide. This lends support to the proposed thiol/disulfide interchange mechanism in the activation reaction [Macartney, H. W. and Tschesche, H (1980) FEBS Lett. 119, 327–332]. The active collagenase could be completely inhibited by an inhibitor dissociated from the latent enzyme, i.e. a complex of enzyme and inhibitor. Regulation of the collagenase activity could be achieved by the redox potential of the glutathione cycle [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190]. The latent and active enzymes were shown to be acidic proteins with isoelectric points at pH 5.5 and 6.4 respectively. Amino acid analyses of both enzymes have been obtained and indicate a difference of about 222–223 amino acid residues between the latent and active collagenases. This corresponds to a difference of approximately Mr25000 between the active and the latent enzyme, the latter being a mixed disulfide of the enzyme and its inhibitor. The optimum pH for collagenolytic activity was observed near neutrality. The enzyme specificity was revealed to be that of a true mammalian collagenase splitting the native type I tropocollagen molecule into three-quarter and one-quarter fragments according to dodecyl sulfate/polyacrylamide gel electrophoresis. No other contaminating proteolytic activity was observed in the purified latent and active collagenase preparations. The active enzyme was found to be stable for over one year when stored at - 70°C. The collagenase was capable of reducing the specific viscosity of a native type I collagen solution by approximately 40%. The amount of latent collagenase present in the leukocytes was estimated to 0.65 mg/1010 cells. The overall yield obtained after the purification to homogeneity was 75–80% of the theoretical value.

Journal ArticleDOI
TL;DR: Human peripheral‐type cannabinoid receptor was expressed in Escherichia coli as a fusion with the maltose‐binding protein, thioredoxin, and a deca‐histidine tag to confirm functional activity and structural integrity of the receptor in bacterial protoplast membranes.
Abstract: Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%–90% homogeneity by affinity chromatography followed by ion-exchange chromatography. By high-resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques.

Journal ArticleDOI
TL;DR: Ligand 8/7, which emerged as the lead from a de novo designed combinatorial library of ligands, inhibits the interaction of PpL with IgG and Fab by competitive ELISA and shows negligible binding to Fc.