Showing papers on "Affinity chromatography published in 1996"

Strategies for Protein Purification and Characterization A Laboratory Course Manual

Abstract: Part 1 Purification of calmodulin. Experiments: activity assays - assay of calmodulin fractions preparation of a tissue extract bulk fractionation ion-exchange chromatography hydrophobic interaction chromatography characterization of calmodulin - calculation of recovery characterization of calmodulin - electrophoresis proteolytic digestion reverse-phase HPLC physical analysis of calmodulin chemical analysis of calmodulin. Preparation of reagents. Part 2 Purification of transcription factor AP-1 from HeLa cells. Experiments: preparation of a nuclear extract from HeLa cells gel filtration chromatography with Sephacryl S-300 HR sequence-specific DNA affinity chromatography DNase 1 footprinting gel-mobility-shift assay preparation of heparin-sepharose CL-2B preparation of reagents. Part 3 Purification of recombinant protein overproduced in escherichia coli. Experiments: breakage of E. coli cells and preparation of inclusion bodies solubilization, refolding, and ion-exchange chromatography of the inclusion body pellet (o32) polyethyleneimine precipitation and immunoaffinity chromatography of the soluble extract (core RNA polymerase o32 complex) quantitation and summary of preparation protein characterization. Protocol development trials - purification of o32 from a bacterial overexpresser preparation of reagents. Part 4 Solubilization and purification of the rat liver insulin receptor. Experiments: isolation of plasma membranes from rat liver solubilization of insulin receptor from membranes lectin affinity chromatography of solubilized receptors insulin affinity chromatography of partially purified receptors cross-linking of insulin receptors with [125I] insulin insulin-stimulated insulin receptor autophosphorylation analysis of insulin receptor glycosylation. Preparation of reagents.

RNA Aptamers That Bind l-Arginine with Sub-Micromolar Dissociation Constants and High Enantioselectivity

Abstract: A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 0 muM were used to select for tight binding arginine specific RNA aptamers. A modified in vitro selection scheme, based on affinity chromatography was applied to allow the enrichment of high affinity solution binders. The selection scheme included a negative selection with the non-cognate ligand citrulline, and a heat denaturation step prior to affinity elution with an excess of the cognate ligand arginine. After 20 cycles the majority of the pools bound specifically to the arginine matrix even after denaturation/renaturation in the presence of 20 mM of a non-cognate amino acid. When denatured and eluted in the presence of 20 mM arginine, the selected RNAs quantitatively washed off the column. These RNA aptamers were cloned and sequenced. Equilibrium dialysis performed with the most abundant clone among the selected sequence revealed Kd values of 330 nM for the RNA/arginine affinity, which is nearly a 200-fold improvement over the tightest binding arginine binding RNAs known to date. Arginine recognition by this RNA is highly enantioselectice: L- arginine is bound 12 000-fold better than D-arginine. Chemical modification analysis revealed that the secondary structure of the aptamer might contain a pseudoknot motif. Our tight binding arginine aptamers join a number of natural and in vitro selected RNAs which recognize arginine. The RNAs described here compare in their binding affinity with the tightest binding RNA aptamers for low molecular weight molecules isolated in other in vitro selection experiments.

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Abstract: Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).

Purification of a rat neurotensin receptor expressed in Escherichia coli.

Abstract: A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress.

Monoclonal Antibody to Thyroid Transcription Factor-1: Production, Characterization, and Usefulness in Tumor Diagnosis

Abstract: Thyroid transcription factor-1 (TTF-1), a member of the NKx2 family of homeodomain transcription factors, is expressed in epithelial cells of the thyroid gland and the lung. To produce monoclonal antibodies specific for TTF-1, the polypeptide was expressed in E. coli and purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fusion protein. Splenocytes from BALB/c mice immunized with recombinant TTF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas. Tissue culture supernatant was screened for anti TTF-1 activity by ELISA employing recombinant TTF-1 as antigen. Hybridomas producing high-affinity antibodies were subcloned by limiting dilution. Antibodies from tissue culture fluid from an IgG1 clone (8G7G3/1) that stained the nuclei of paraffin-embedded human thyroid tissues were precipitation-purified and further characterized. The antibody stained a single 40-kDa polypeptide in immunoblots of nuclear extracts or lystates of cell lines known to express TTF-1 mRNA. MAb 8...

Protein a mimetic peptide ligand for affinity purification of antibodies

Abstract: A peptide mimicking protein A for its ability to recognize the Fc immunoglobulin portion has been identified through screening of a synthetic multimeric peptide library. Screening of the multimeric library, composed of randomized synthetic tripeptide tetramers, has been carried out using a very simple assay, measuring the library ability to interfere with the interaction between protein A and biotinylated immunoglobulins, monitored on solid phase using an enzyme-linked immunosorbent assay format. The tetrameric tripeptide identified after three screening cycles was produced in larger amounts and then immobilized in high yield on preactivated solid support for the preparation of affinity columns, which proved useful for a very convenient one-step purification of antibodies directly from crude sera. Antibody purity after affinity purification was close to 95 per cent, as determined by densitometric scanning of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of purified fractions, and up to 2 mg of antibody could be purified from 1 ml of peptide-derivatized affinity support. The ligand was stable to treatment with a vast array of sanitation agents, such as ethanol and 0.1 M sodium hydroxide, and to repeated use, thus making the ligand applicability extremely attractive for the purification of monoclonal antibodies for therapeutic use. Column binding selectivity was similar to that of protein A-affinity columns, since immunoglobulin G from several sources (rabbit, goat, sheep, mouse) was conveniently purified, with no detection of leaked ligand fragments in the purified preparations.

Engineering a de novo designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins.

Abstract: Using the techniques of genetic engineering and the principles of protein de novo design, we have developed a unique affinity matrix protein tag system as a rapid, convenient and sensitive method to detect, purify and characterize newly expressed recombinant peptides or proteins from cell extracts. The method utilizes two de novo-designed linear peptide sequences that can selectively dimerize to form the stable protein motif, the two-stranded alpha-helical coiled-coil. In this method, a recombinant bacterial expression vector pRLDE has been engineered so that one of the dimerization strands (E-coil) is expressed as a C-terminal fusion tag on newly expressed peptides or proteins, while the other (K-coil) is either biotin-labeled for detection in a Western blot-type format or immobilized on an insoluble silica support for selective dimerization affinity chromatography. Recombinantly expressed peptides from Escherichia coli containing the dimerization tag have been produced, detected and purified using this method. The recombinant peptides were easily and clearly identified using the biotin-labeled coil, while the single-step affinity purification results indicated the purity of the affinity purified expressed peptides to be > 95%, as assessed by reversed-phase chromatography. The stability of the dimerization domain also allows for the purified peptide to be left attached to the matrix, thus creating a new peptide-bound column that can be used to study peptide-protein or peptide-ligand interactions. Therefore this system offers a new alternative to existing peptide or protein fusion tags and demonstrates the utility of a de novo-designed system.

Method for purifying viruses by chromatography

Abstract: A method for purifying viruses from a cell line culture by chromatography, comprising an anion exchange chromatography step followed by a cation exchange chromatography step and optionally a metal-binding affinity chromatography step. The method is particularly suitable for producing viruses for use in vaccines.

A novel support with artificially created recognition for the selective removal of proteins and for affinity chromatography

Abstract: Acrylamide and N,N′-methylenebisacrylamide were copolymerized in the presence of a protein to form a gel which was pressed through a sieve. The gel particles obtained were packed into a chromatographic tube. The experimental conditions for the polymerization are such that the pores of the gel particles are large enough to permit the protein to diffuse out of the particles, so that the entrapped protein can be removed from the bed by washing with an aqueous solution. However the interaction with the matrix is so strong that the protein can be desorbed only by a buffer containing 0.5 M sodium chloride or by a 10% solution of acetic acid containing 10% SDS. When a sample containing the protein present during the polymerization was applied to the column along with other proteins this protein was the only one adsorbed. The technique worked selectively with hemoglobin, cytochrome C and transferrin.

Biological properties of recombinant chicken interferon-γ

Abstract: Supernatants of the chicken T cell line 855 contain antiviral and macrophage-activating factor activity and strongly activate transcription of the guanylate-binding protein (GBP) gene in chicken cells. To characterize the cytokine responsible for the GBP-inducing activity, we chose a cDNA expression cloning strategy in COS cells. Sequencing a positive clone revealed that it encodes chicken interferon-γ (ChIFN-γ). Histidine-tagged ChIFN-γ was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. ChIFN-γ from COS cells and E. coli both potently induced GBP RNA synthesis but were rather poor antiviral agents. In macrophages, recombinant ChIFN-γ strongly stimulated secretion of nitric oxide and enhanced expression of major histocompatibility complex class II antigen. A rabbit antiserum to E. coli-derived ChIFN-γ effectively neutralized the macrophage-activating factor activity secreted by concanavalin A-induced spleen cells and various T cell lines, suggesting that IFN-γ is the major macrophage-activating factor of the chicken.

Improvements on the purification of mannan-binding lectin and demonstration of its Ca(2+)-independent association with a C1s-like serine protease.

Abstract: Mannan-binding lectin (MBL), previously called 'mannan-binding protein' or MBP, is a plasma C-type lectin which, upon binding to carbohydrate structures on micro-organisms, activates the classical pathway of complement. Purification of MBL relies on its Ca(2+)-dependent affinity for carbohydrate, but existing methods are susceptible to contamination by anti-carbohydrate antibodies. In the present study a sequential-sugar-elution method has been developed which can achieve a preparation of virtually pure MBL and its associated serine protease (MBL-associated serine protease, MASP) by two steps of affinity chromatography. In further separation of MASP from MBL, it was found that activated MASP was associated with MBL independent of Ca2+. Since MBL was found to bind to underivatized Sepharose 4B, the MBL-MASP complex was purified using Sepharose 4B and protease inhibitors were included to purify the complex with MASP in its proenzyme form. Analysis of thus-purified MBL-MASP complex by gel filtration on a Sephacryl S-300 column at pH 7.8 showed that the proenzyme MASP was also associated with MBL independently of Ca2+, but that the complex could be disrupted at a low pH (5.0). Therefore the mechanism of MBL-MASP-mediated complement activation appears to be significantly different from the C1-mediated classical pathway.

Induction of Muscle Protein Degradation and Weight Loss by a Tumor Product

Abstract: Splenocytes from mice bearing a cachexia-inducing tumor (MAC16) have been fused with mouse myeloma cells to produce hybridomas, which have been cloned to produce antibody reactive to a material which copurified with a lipid-mobilizing factor isolated from the same tumor. The monoclonal antibody has been used to investigate factors potentially involved in the development of cachexia. The major protein detectable by immunoprecipitation of a partially purified lipid-mobilizing factor was Mr 69,000, whereas Western blotting showed two bands of Mr 69,000 and Mr 24,000. Although the monoclonal antibody did not neutralize lipidmobilizing activity in an in vitro assay, it did neutralize a serum factor capable of protein degradation in isolated gastrocnemius muscle. Affinity purification of MAC16 tumor homogenates using the monoclonal antibody yielded two immunoreactive bands of Mr 69,000 and Mr 24,000, which were further fractionated on a hydrophobic column (C8). This material was capable of inducing tyrosine release from isolated gastrocnemius muscle, and the effect could be blocked with the monoclonal antibody. The two immunoreactive bands from the hydrophobic column were capable of inducing weight loss in mice, whereas nonimmunoreactive fractions had no effect on body weight. The Mr 24,000 species had a unique amino acid sequence, whereas the Mr 69,000 species gave the same sequence as the Mr 24,000 material, together with that for albumin. The Mr 24,000 species contained carbohydrate, and lectin blotting showed a strong reaction with wheat germ and Erythrina crystagalli agglutinins. This suggests that the material is a glycoprotein or proteoglycan that shows strong binding affinity for albumin, possibly through the carbohydrate residues.

Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells

Abstract: The FLAG peptide, AspTyrLysAspAspAspAspLys, has been used as an epitope tag in a variety of cell types. The modification of the cytomegalovirus (CMV) promoter containing vector, pCMV5, to create two transient expression vectors designed for secretion and intracellular expression of FLAG-fusion proteins in mammalian cells is described. As a functional test, the bacterial alkaline phosphatase gene was cloned into both vectors, and anti-FLAG monoclonal antibodies were used for detection of FLAG epitope-tagged bacterial alkaline phosphatase in mammalian cells. In addition, secreted bacterial alkaline phosphatase was purified from the extracellular medium by anti-FLAG affinity chromatography.

Human protoporphyrinogen oxidase : expression, purification, and characterization of the cloned enzyme

Abstract: Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.

Immobilized membrane vesicle or proteoliposome affinity chromatography. Frontal analysis of interactions of cytochalasin B and D-glucose with the human red cell glucose transporter.

Abstract: Human red cell membrane vesicles stripped of peripheral proteins and proteoliposomes with reconstituted red cell glucose transporter (Glut1) were sterically immobilized in gel beads by freeze-thawi...

Characterization of a ligand for receptor protein-tyrosine kinase HTK expressed in immature hematopoietic cells.

Abstract: HTK is a receptor tyrosine kinase that belongs to the Eph subfamily. An extensive screening using BIAcore system revealed that a colon cancer cell line, C-1, expressed the ligand for HTK. From the conditioned medium of C-1 cells, a soluble form of ligand was purified by receptor affinity chromatography, and the isolation of full-length cDNA revealed that this ligand is identical to the human HTK ligand (HTKL) previously reported. HTK receptor tyrosine phosphorylation was induced by membrane-bound or clustered soluble HTKL but not by unclustered soluble HTKL, indicating that HTKL requires cell-to-cell interaction for receptor activation. Binding analysis demonstrated that HTKL binds to HTK with a much higher affinity (Kd: 1.23 nM) than the other transmembrane-type ligand for Eph family, LERK-2/ELKL (Kd: 135 nM). The expression of HTK in cord blood cells was upregulated after the culture in the presence of stem cell factor. Clustered soluble HTKL stimulated the proliferation of sorted HTK+ cord blood cells and a hematopoietic cell line, UT-7/EPO from which HTK was isolated. These findings suggest the involvement of HTK-HTKL system in the proliferation of HTK+ hematopoietic progenitor cells in the hematopoietic environment.

Identification and characterization of an active soluble form of human CD38 in normal and pathological fluids.

Abstract: Human CD38 is a transmembrane glycoprotein involved in lymphocyte activation and adhesion to endothelium. The ectocellular domain of the molecule possesses properties of a bifunctional enzyme catalyzing both the synthesis from NAD+ and the hydrolysis of the calcium-releasing metabolite cyclic ADP-ribose (cADPR). Surface expression of CD38 (mCD38) is rapidly and almost completely down-modulated upon ligation by specific mAb in cells from different lineages. The data presented here also show that, in addition to the existence of a mCD38, a soluble form of CD38 (sCD38) is detectable in the cell culture supernatant of allo-activated T lymphocytes and of several tumor cell lines. sCD38 is also present in vivo and is assayable in normal (fetal serum and amniotic fluid) and pathological (serum and ascites from patients with multiple myeloma, and serum from patients with AIDS) biological fluids. Immunoaffinity chromatography, SDS-PAGE and Western blot analyses with mAb and polyclonal antibodies, along with metabolic labeling, yield a body of data concerning the structure of sCD38, which displays a M(r) of 39 kDa. Native sCD38 maintains the ability to inhibit the binding activity of different anti-CD38 mAb and still catalyzes the synthesis and the hydrolysis of cADPR at the same ratio observed with mCD38. Furthermore, cross-linking experiments indicate that the purified soluble molecule binds a 120 kDa molecule expressed by monocytoid cells and identified as a candidate ligand for human mCD38.