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PubMed2003

Affinity purification of ribosomes with a lethal G2655C mutation in 23 S rRNA that affects the translocation.

A method for preparation of Escherichia coli ribosomes carrying lethal mutations in 23 S rRNA was developed. The method is based on the site-directed incorporation of a streptavidin binding tag into functionally neutral sites of the 23 S rRNA and subsequent affinity chromatography. It was tested with ribosomes mutated at the 23 S rRNA position 2655 (the elongation factor (EF)-G binding site). Ribosomes carrying the lethal G2655C mutation were purified and studied in vitro. It was found in particular that this mutation confers strong inhibition of the translocation process but only moderately affects GTPase activity and binding of EF-G.

Base Sequence↗
PubMed1984

Affinity purification and subunit structures of beta-N-acetylhexosaminidases A and B from boar epididymis.

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

Animals↗
PubMed2006

Biotin-tag affinity purification of a centromeric nucleosome assembly complex.

Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, we used controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. Here, we highlight the benefits of our improved biotin-mediated purification method, and address the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres.

Animals↗
PubMed2003

Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification.

Human pituitary glutaminyl cyclase (hQC) was expressed in Drosophila S2 cells under the control of an inducible metallothionene promoter and fused to the Drosophila immunoglobulin-binding protein signal sequence to enable secretion into the culture media. Expression levels reached 50 microg/mL culture media after 7 days of induction. The enzyme was purified to homogeneity directly from culture media by affinity chromatography on Reactive Blue 4-agarose using a step pH elution. The identity of the expressed protein was confirmed by peptide mass mapping and Western blotting. Glutaminyl cyclase was expressed as a fully active 37 kDa enzyme with kcat/Km values of 14.3, 9.3, and 2.4 mM(-1)s(-1) for the substrates Gln-Gln, Gln-NH(2), and Gln-t-butyl ester, respectively. The two cysteines were disulfide bonded, and the lone predicted glycosylation site, asparagine 49, was shown by both enzymatic deglycosylation of the expressed enzyme and site-directed mutagenesis to be glycosylated.

Amino Acid Sequence↗
PubMed1989

Affinity purification of cellular retinoic acid-binding protein on 14-carboxy-13-cis-retinamide-sepharose 4B.

A biologically active bifunctional retinoid, ethyl 14-carboxyretinoate, has been synthesized and shown to bind cellular retinoic acid (RA)-binding protein (CRABP) via its free carboxy group. We describe herein the synthesis of 14-carboxy-13-cis-retinamide-Sepharose 4B, which is an affinity matrix bearing an all-trans-RA moiety, and thus was used to purify and characterize CRABP from chick-embryo skin. An amide bond was first formed between the free carboxy group of the retinoid and a primary amino group of aminohexyl-Sepharose 4B, by reaction with carbodi-imide, and the ester group of the resin-bound retinoid was then hydrolysed in an alkaline medium. Polyacrylamide-gel electrophoresis and f.p.l.c. Superose column-chromatographic analysis demonstrated that the affinity-purified CRABP (Mr 15,000) was close to electrophoretic homogeneity (greater than 90%) and specifically interacts with RA. By using affinity gel chromatography, conversion of holo-CRABP into apo-CRABP by treatment with p-hydroxymercuribenzoate and a possible involvement of a thiol group in RA binding to CRABP were established. This affinity procedure provides several advantages: (i) 14-carboxy-13-cis-retinamide-Sepharose exhibited high efficiency and selectivity for RA-binding protein (i.e. retinol- or fatty-acid-binding proteins did not bind); (ii) the presence of the amide linkage between the ligand and the matrix makes this affinity resin relatively stable to cytosolic enzymes; and (iii) other RA-binding proteins, e.g. nuclear receptor(s), may be purified.

Animals↗
PubMed1991

Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.

Animals↗
PubMed1984

Affinity purification of aminoacyl-tRNA.

A procedure for separating Escherichia coli aminoacyl-tRNA from unacylated tRNA or components of the aminoacylation reaction, thereby achieving an aminoacyl-tRNA product with a very high specific activity, is described. The method utilizes the specific recognition of aminoacyl-tRNA for E. coli protein synthesis elongation factor Tu which has been immobilized on an affinity matrix. The application of the affinity procedure as a means of purifying a single aminoacyl-tRNA from an unfractionated mixture of tRNAs is also discussed.

Chemical Phenomena↗
PubMed1996

Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina.

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.

Amino Acid Sequence↗
PubMed1993

Receptor affinity purification of a lipid-binding adhesin from Helicobacter pylori.

Our previous work has shown that Helicobacter pylori specifically recognizes gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro. This binding specificity is shared by exoenzyme S from Pseudomonas aeruginosa, and monoclonal antibodies against this adhesin prevent the attachment of H. pylori to its lipid receptors. We now report the use of a novel, versatile affinity matrix to purify a 63-kDa exoenzyme S-like adhesin from H. pylori which is responsible for the lipid-binding specificity of this organism.

Adhesins, Bacterial↗
PubMed1989

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid.

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

Animals↗
PubMed1988

Affinity purification and chemical analysis of the interleukin-1 receptor.

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.

Chromatography, Affinity↗
PubMed2002

Moenomycin-mediated affinity purification of penicillin-binding protein 1b.

The antibiotic moenomycin A inhibits the biosynthesis of peptidoglycan, the main structural polymer of the bacterial cell wall. The inhibition is based on a reversible binding of the antibiotic to one of the substrate binding sites at enzymes such as the penicillin binding protein 1b (PBP 1b). This binding has been employed to isolate PBP 1b by affinity chromatography. Suitable ligands have been prepared from moenomycin A and coupled both to affinity supports and to surface plasmon resonance sensor surfaces. The reactions that take place upon immobilization of the ligands to the affinity support and the sensor surface, respectively, have been studied in detail. With the help of surface plasmon resonance the optimal conditions for binding of PBP 1b to moenomycin-derivated ligands have been established. For the first time the selective binding of the moenomycin sugar moiety to the enzyme has been demonstrated.

Bacterial Proteins↗
PubMed1987

Dog mastocytoma tryptase: affinity purification, characterization, and amino-terminal sequence.

A tryptic protease with the characteristics of a mast cell tryptase was purified from dog mastocytoma cells propagated in nude mice. Partial amino acid sequence of the mastocytoma tryptase revealed unexpected differences in comparison with other mast cell and leukocyte granule protease sequences. Extraction from mastocytoma homogenates at high ionic strength, followed by gel filtration and benzamidine affinity chromatography yielded a product with several closely spaced bands (Mr 30,000-32,000) on gel electrophoresis and a single N-terminal sequence. Nondenaturing analytical gel filtration revealed an apparent Mr of 132,000, suggesting noncovalent association as a tetramer. Studies with peptide p-nitroanilides indicated pronounced substrate preferences, with P1 arginine preferred to lysine. Benzoyl-L-Lys-Gly-Arg-p-nitroanilide was the best of the substrates screened. Inhibition by diisopropyl fluorophosphate and tosyllysine chloromethyl ketone indicated that the enzyme is a serine protease. Like the tryptases of human mast cells, mastocytoma tryptic protease was inhibited by NaCl, resistant to inactivation by alpha 1-proteinase inhibitor and plasma, and stabilized by heparin. Comparison of the N-terminal 24 residues of mastocytoma tryptase revealed 80% identity with the more limited sequence reported for human lung tryptase, and surprisingly, closer homology to serine proteases of digestion and clotting than to other leukocyte granule proteases sequenced to date, including mast cell chymase. The N-terminal isoleucine is the homolog of trypsinogen Ile-16 which becomes the new N-terminus upon cleavage of the activation peptide. Thus, the tryptase N-terminus is related to the catalytic domain of activated serine proteases, and lacks the N-terminal regulatory domains found in most clotting and complement serine proteases. These findings provide further evidence that tryptases are unique serine proteases and that they may be less closely related in evolution and function than are other leukocyte granule proteases described to date.

Amino Acid Sequence↗
PubMed1992

Immunochemical and biochemical characterization of purified canine interferon-gamma. Production of a monoclonal antibody, affinity purification, and its effect on mixed lymphocyte culture and mixed lymphocyte kidney culture reactions.

We have advanced the hypothesis that the primary autolymphoproliferative response of dog T cells in mixed lymphocyte kidney cultures (MLKC) results from their recognition of tissue-specific (kidney-associated) antigen(s) presented in conjunction with class II MHC antigens. Lymphocyte culture-derived supernatants had been found previously to upregulate class II antigen expression on kidney cells and enhance T cell activation. In the present study we have isolated and characterized dog IFN-gamma, a class II-inducing substance that is secreted in the culture supernatant of activated T lymphocytes. Dog IFN-gamma was induced with A-23187 and PMA and purified stepwise using controlled-pore glass, Mono Q anion exchange chromatography, and Superose 6-gel filtration on FPLC. The purification resulted in two molecules of 42 Kd and 31 Kd molecular weights. An IgG1 monoclonal antibody was engendered to these molecules. With this mAb reagent, in immunochemical experiments, we have developed a sensitive ELISA and a method for purifying dog IFN-gamma by affinity chromatography. Species specificity studies indicated that purified dog IFN-gamma reacted with a polyclonal rabbit antihuman IFN-gamma, but not with a mAb to human IFN-gamma. However, the antidog IFN-gamma mAb that was generated also reacted with recombinant human IFN-gamma. In in vitro biological studies, the purified IFN-gamma (two mol. wt. species) upregulated the expression of canine class II MHC molecules on dog tubular epithelial cells and the dog kidney epithelial cell line (MDCK). The antidog IFN-gamma mAb blocked T cell proliferative response to kidney cell and, by inference, the interaction between endogenously released IFN-gamma in vitro with its cell surface receptor, thus inhibiting the induced upregulation of class II. Interestingly, although antidog IFN-gamma markedly blocked the MLKC (10 micrograms mAb/well), there was no effect on the allogeneic MLC. This observation indicates that the cytokine IFN-gamma may be a uniquely key substance amplifying the immune response of T cells to tissue-associated antigens on surrogate antigen-presenting cells that require induced upregulation of class II MHC antigen expression (MLKC), in contrast to reactions in which these antigens are already constitutively expressed on the antigen-presenting cells (mixed lymphocyte culture).

Animals↗
PubMed1995

Affinity purification of the ryanodine receptor/calcium release channel from fast twitch skeletal muscle based on its tight association with FKBP12.

The ryanodine receptor (RyR)/calcium release channel isolated from skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) is tightly associated with FK506 binding protein of 12.0 kDa (FKBP12) (Jayaraman et al., (1992) J.Biol.Chem. 267, 9474-9477). In this study, we describe a new method of affinity chromatography for purifying the RyR from skeletal muscle SR based on: 1) its tight association with FKBP12; and 2) the finding that bound FKBP on the RyR can be exchanged with soluble FKBP12 (Timerman et al., (1995) J.Biol.Chem. 270, 2451-2459). Soluble glutathione S-transferase/FKBP12 (GST/FKBP12) fusion protein was first exchanged with bound FKBP12 on the RyR of TC. The TC were then solubilized with CHAPS and the complex of RyR.GST/FKBP12 was specifically adsorbed by glutathione Sepharose 4B and then eluted with glutathione. The RyR, purified by this method, has similar characteristics by SDS-PAGE, radioligand binding and immuno-reactivity as the RyR purified by multiple sequential column chromatography.

Animals↗