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PubMed1981

Purification of Phytochrome by Affinity Chromatography on Agarose-Immobilized Cibacron Blue 3GA.

The binding of phytochrome to Cibacron Blue 3GA was utilized to develop a new affinity purification procedure for phytochrome. Brushite-purified phytochrome from rye (Secale cereale c.v. Cougar) was bound to agarose-immobilized blue dye in 0.1 molar potassium phosphate (pH 7.8), contaminating proteins washed out with 0.5 molar KCl, and homogeneous phytochrome eluted with 10 millimolar flavin mononucleate. Ninety-five per cent of the phytochrome applied bound, and 60 to 65% was eluted, giving a 25 to 30% yield for the complete one-day procedure. Affinity-purified rye phytochrome was identical to conventionally purified phytochrome in its behavior on sodium dodecyl sulfate gels, in gel exclusion chromatography, in sedimentation in sucrose density gradients and in its spectral properties.

Journal Article↗
PubMed1988

Characterization of the RNA Required for Biosynthesis of delta-Aminolevulinic Acid from Glutamate : Purification by Anticodon-Based Affinity Chromatography and Determination That the UUC Glutamate Anticodon Is a General Requirement for Function in ALA Biosynthesis.

The heme and chlorophyll precursor delta-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3' terminus of yeast tRNA(Phe(GAA)) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNA(Glu(UUC)) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A(260) unit added was as much as 50 times greater with the affinity-purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA in plants and algae.

Journal Article↗
PubMed1977

Nucleotide phosphotransferase of Escherichia coli: purification by affinity chromatography.

Improved extraction and purification procedures permit the isolation from Escherichia coli W cells of much larger quantities and of more highly purified preparations of nucleotide phosphotransferase. Of various affinity resins tested for efficiency of purification, columns of agarose/5'-AMP (AGAMP), type 3, proved the best. In this way a 300- to 450-fold purification of the enzyme was achieved in a few steps. The enzyme, which, as reported before, transfers organically bound phosphate to the 2' or 3' hydroxyls of nucleosides and nucleotides, was tested in its behavior toward a series of ribonucleosidonucleotides, namely, CpC, ApA, CpA, and ApC. All were phosphate acceptors, but a detailed comparative study of adenosine and cytidine, 5'-AMP and 5'-CMP, and ApA and ApC revealed peculiar specificities in the relative distribution of the phosphorylated products.

Chromatography, Affinity↗
PubMed1985

Sepharose derivatives containing citric acid as affinity ligand. Purification of fumarase.

Six Sepharose derivatives, in which citrate was immobilized via methylene carbons, were prepared by coupling of the alpha- and beta-isomers of citrylpolymethylenediamine to Sepharose. The purification of fumarase from pig heart was dependent on the length of the spacer arm, but not on the isomeric configuration of the immobilized citrate. Gels having six methylene carbons had the largest adsorption capacity for the enzyme and therefore were the most suitable for use in affinity columns for its purification. Affinity chromatography with these gels was followed by hydrophobic interaction chromatography on an octamethylenediamine-Sepharose column.

Animals↗
PubMed2006

Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice.

Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

14-3-3 Proteins↗
PubMed2000

Essential role of the concentration of immobilized ligands in affinity chromatography: purification of guanidinobenzoatase on an ionized ligand.

Guanidinobenzoatase, a plasma protein with possible application as a 'tumor marker', has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by 'controlled' immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 microlmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 micromol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, x220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.

Adsorption↗
PubMed2006

Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes.

In recent years, MS has been widely used to study protein complex in eukaryotes. The identification of interacting proteins of a particular target protein may help defining protein-protein interaction and proteins of unknown functions. To isolate protein complexes, high-speed ultracentrifugation, sucrose density-gradient centrifugation, and coimmunoprecipitation have been widely used. However, the probability of getting nonspecific binding is comparatively high. Alternatively, by use of one- or two-step (tandem affinity purification) epitope-tag affinity purification, protein complexes can be isolated by affinity or immunoaffinity columns. These epitope-tags include protein A, hexahistidine (His), c-Myc, hemaglutinin (HA), calmodulin-binding protein, FLAG, maltose-binding protein, Strep, etc. The isolated protein complex can then be subjected to protease (i.e., trypsin) digestion followed by an MS analysis for protein identification. An example, the epitope-tag purification of the Arabidopsis cytosolic ribosomes, is addressed in this article to show the success of the application. Several representative protein complexes in eukaryotes been isolated and characterized by use of this approach are listed. In this review, the comparison among different tag systems, validation of interacting relationship, and choices of MS analysis method are addressed. The successful rate, advantages, limitations, and challenges of the epitope-tag purification are also discussed.

Affinity Labels↗
PubMed2005

Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.

We describe the application of tandem affinity purification-mass spectrometry (TAP-MS) to the study of protein complexes in Drosophila. We have constructed vectors for inducible expression of TAP-tagged fusion proteins in Drosophila cultured cells and in vivo. Using these vectors, we tagged, as a paradigm, several components of the Notch signaling pathway, isolated protein complexes containing the baits and associated proteins from cells and embryos, and identified the subunits by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several known interactions involving Notch pathway elements were confirmed, and many novel potential interactions were uncovered. For some of the novel associations, we validated the interaction genetically and biochemically. We conclude that TAP, in combination with MS, can be used as an effective method for the studies of the Drosophila proteome.

Affinity Labels↗
PubMed2025

Proximity Proteomics to Profile Ebola Virus Protein Interactome in Its Functional Context.

Proximity labeling-based proteomics (proximity proteomics) has emerged as a popular and versatile approach to illuminate the molecular interactions between viruses and their hosts. In this approach, a proximity labeling enzyme tag is fused to a bait protein and labels neighboring proteins with a chemical handle such as biotin, allowing for downstream affinity purification. Compared to another widely used technique, affinity purification coupled mass spectrometry, proximity proteomics enables the detection of low affinity or transient interactors that might have important functions in the viral life cycle. Further, proximity proteomics can identify interactors of a labile bait protein, of which affinity purification is technically challenging. Here, we describe a proximity proteomic protocol to identify cellular interactors of the Ebola virus polymerase. A similar strategy is readily applicable to elucidate the virus-host interactions for Marburg virus.

Ebolavirus↗
PubMed1998

Affinity chromatographic purification of antibodies to a biotinylated fusion protein expressed in Escherichia coli.

HOP1, a protein component of the synaptonemal complex in Saccharomyces cerevisiae which is believed to play an important role in meiotic synapsis, was expressed in Escherichia coli as a fusion protein incorporating a "tag" polypeptide which is biotinylated naturally in the bacteria. The HOP1 fusion protein was produced in an insoluble form within the bacteria; once solubilized using a denaturing agent, the protein was purified by avidin monomer affinity chromatography. The recombinant protein was used to immunize rabbits and produce polyclonal antibodies. Procedures for affinity purification of antibodies using the recombinant protein attached to the avidin column and a magnetic method for concentration of antibodies are described. Antibody elution conditions in these procedures do not affect the affinity of the column for the recombinant protein, which can be recovered afterward. Affinity-purified antibodies show high binding capacity to HOP1 recombinant protein in immunoblotting experiments, but reduced background compared with crude antiserum or purified IgG fraction. The affinity-purified antibodies recognize a major band around 70 kDa in Western blots of yeast protein extracts following meiotic induction.

Animals↗
PubMed2002

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein.

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.

Chitin↗
PubMed1982

Affinity chromatography purification of Pseudomonas aeruginosa exotoxin A on specifically linked NAD agarose.

The specific binding of P. aeruginosa exotoxin A to NAD was exploited for the rapid purification of the toxin. Affinity chromatography on a column of agarose-N6-(aminohexyl)carbamoylmethyl-NAD resulted in an enzymatically, biologically, and immunologically active purified toxin preparation. Other NAD-agarose resins were not efficient substrates for toxin purification.

ADP Ribose Transferases↗
PubMed1997

COS-1 cell expression and one-step affinity protein purification and activity of epitope-tagged human erythropoietin and of site-directed mutants.

Recombinant human erythropoietin (rhEPO) is an important glycoprotein hormone which has been successfully used in the treatment of anaemia. To facilitate the rapid evaluation of wild-type and mutant forms of rhEPO in structure-function studies, we have developed an expression system in which the recombinant hormone is tagged at the C-terminus with a c-myc peptide. One-step affinity purification of culture supernatants on an anti-myc antibody column yielded proteins which were greater than 50% pure with a specific activity of 300,000 U/mg, in agreement with the value of wild-type protein. We conclude that the additional myc-peptide does not affect receptor binding. The expression system was used to study three mutants in which the N-glycosylation sites were changed to cysteines (Asn24Cys, Asn38Cys and Asn83Cys). Specific activities of these cysteine mutants were significant, but reduced (60%, 22% and 70%, respectively), compared to wild-type. The reduction in specific activity may be due to reduced stability of the mutant proteins.

Amino Acid Sequence↗
PubMed2003

Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes.

Multi-protein complexes are emerging as important entities of biological activity inside cells that serve to create functional diversity by contextual combination of gene products and, at the same time, organize the large number of different proteins into functional units. Many a time, when studying protein complexes rather than individual proteins, the biological insight gained has been fundamental, particularly in cases in which proteins with no previous functional annotation could be placed into a functional context derived from their 'molecular environment'. In this minireview, we summarize the current state of the art for the retrieval of multiprotein complexes by affinity purification and their analysis by mass spectrometry. The advances in technology made over the past few years now enable the study of protein complexes on a proteomic scale and it can be anticipated that the knowledge gathered from such projects will fuel drug target discovery and validation pipelines and that the technology is also going to prove valuable in the emerging field of systems biology.

Animals↗
PubMed1978

Affinity chromatographic purification of beta-glucosidase of Candida gulliermondii.

A beta-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethyleneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacrylamide disc electrophoresis of the purified enzyme gave a single band. A km of 1.25 x 10(-4)M was obtained using p-nitrophenyl beta-D-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at temperature of 37 degrees C. Enzyme activity was completely inhibited by Hg++, Pb++, and Zn++ ions. The molecular weight of the enzyme is 48,000 as estimated by sucrose density gradient centrifugation.

Candida↗
PubMed2006

LBT/PTD dual tagged vector for purification, cellular protein delivery and visualization in living cells.

Cellular protein delivery is an emerging technique, by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an E. coli expression vector suited for protein cellular delivery experiments. The plasmid is designed to generate a C-terminal fusion with the 12 amino acid HIV-Tat peptide as a protein transduction domain (PTD), whereas the protein N-terminus is fused to an 17-residue peptide lanthanide-binding tag (LBT). LBT is used for both purification by affinity chromatography and fluorescent detection with Tb(3+) as a coordinating metal. We have employed the TA-cloning site between the two tags, LBT and PTD, according to the PRESAT-vector methodology [N. Goda, T. Tenno, H. Takasu, H. Hiroaki, M. Shirakawa, The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics, Protein Sci. 13 (2004) 652-658], which facilitates unidirectional cloning of any PCR-amplified DNA fragments corresponding to the protein of interest. A simple three-step protocol consisting of affinity purification of LBT/PTD dual-tagged proteins has also been developed, in which the proteins are purified by heparin-, then immobilized Ni(2+)-, and then heparin-affinity chromatography, in this order. The purified protein is ready for protein delivery experiment, and the delivered protein is visible by fluorescent microscopy. Our LBT/PTD dual-tagged PRESAT-vector provides a powerful research tool for exploring cellular functions of proteins in the post-genomic era.

Amino Acid Sequence↗
PubMed2005

Selective ligand purification using high-performance affinity beads.

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.

Animals↗
PubMed1998

One-step purification of cathepsin D by affinity chromatography using immobilized propeptide sequences.

In vivo, active cathepsin D proteinase is generated by removal of a 44-residue propeptide at its N-terminus. Here we report that mature cathepsin D and pseudocathepsin D (a partially activated form of cathepsin D with 25 amino acid residues removed from the propeptide) bind to the immobilized propeptide, while procathepsin D does not. The N-terminal 25 amino acid residues of the propeptide are sufficient for this binding. Based on this observation, a simple one-step procedure was developed to purify mature cathepsin D from whole cell extracts to near homogeneity. This method has the advantage over existing affinity-purification systems that active forms of the proteinase can be separated from inactive precursors and other aspartic proteinases. Furthermore, this technique was effective for pepsin as well, suggesting it may have general utility for all activated aspartic proteinases and perhaps other families of proteinases.

Amino Acid Sequence↗