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PubMed1984

Solubilization and affinity purification of the alpha 2-macroglobulin receptor from human fibroblasts.

Plasma membranes showing specific binding of alpha 2-macroglobulin (alpha 2M) complexes were isolated from normal human fibroblasts. The membrane preparation was solubilized with Triton X-100, and specific binding to solubilized receptor was demonstrated by precipitation of the alpha 2M X protease receptor complexes with polyethylene glycol. The solubilized receptor could be quantitatively adsorbed on immobilized alpha 2M complexes. Adsorbed receptor was then dissociated from the alpha 2M complexes with either EDTA, bacitracin, the monoclonal anti-receptor-recognition site antibody F2B2 , or low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-purified receptor preparation revealed polypeptides of 360, 130, and 83 kDa.

Antibodies, Monoclonal↗
PubMed1993

Affinity purification of histidine-tagged proteins.

Expression of recombinant proteins is a standard technique in molecular biology and a wide variety of prokaryotic as well as eukaryotic expression systems are currently in use. A limiting step is often the purification of the expressed recombinant protein, particularly if mammalian expression systems that yield low expression levels are employed. Here, we discuss the advantages and restrictions of tagging recombinant proteins with histidines and purifying them by Ni(2+)-NTA chromatography.

Animals↗
PubMed1985

Affinity purification and refined structural characterization of terminal deoxynucleotidyltransferase.

A total of 56 stable murine hybridoma monoclones that produce homogeneous antibodies against human or calf terminal deoxynucleotidyltransferase have been established. All of the antibodies exhibited specific binding to various Mr forms of terminal transferase and eight possessed neutralizing activity. Results are presented that permitted characterization of ten of these antibodies with respect to their immunoglobulin class, their recognition of calf or human terminal-transferase Mr species by immunoblotting techniques and their recognition of distinct antigenic sites. Terminal transferase was purified in a single step by using an immunoaffinity column constructed with a monoclonal antibody exhibiting a high binding affinity for the enzyme. Single monoclonal antibodies were also used to bind selectively to terminal-transferase antigen in tissue slices and individual cells.

Animals↗
PubMed1998

Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.

Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M(r) protein species (apparent M(r) 220,000 and 550,000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M(r) protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M(r) 54,597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M(r) estimation, fibrinogen binding and seroreactivity.

Amino Acid Sequence↗
PubMed1997

Overproduction and affinity purification of Saccharomyces cerevisiae replication factor C.

Yeast replication factor C (RF-C) is a heteropentamer encoded by the RFC1-5 genes. RF-C activity in yeast extracts was overproduced about 80-fold after induction of a strain containing all five genes on a single plasmid, with expression of each gene placed under control of the galactose-inducible GAL1-10 promoter. This strongly indicates that overexpression of the five known RFC genes is sufficient for overproduction of RF-C. Overexpression of all five genes was also necessary to achieve overproduction of RF-C as omission of any single gene from the plasmid gave uninduced, i.e. normal cellular levels of RF-C. The interaction between RF-C and proliferating cell nuclear antigen (PCNA) was studied with PCNA-agarose beads. Binding of RF-C to PCNA-agarose beads is negligible in buffers containing 0.3 M NaCl. However, addition of Mg-ATP to the binding buffer caused strong binding of RF-C to the beads even at 0.8 M NaCl. Binding of ATP, but not its hydrolysis, was required for the strong binding mode as nonhydrolyzable analogs were also effective. The existence of two distinct binding modes between PCNA and RF-C was used as the key step in a greatly improved procedure for the purification of RF-C. RF-C from the overproduction strain purified by this procedure was essentially homogeneous and had a severalfold higher specific activity than RF-C preparations that had previously been purified through multicolumn procedures.

Adenosine Triphosphate↗
PubMed1984

Production of monoclonal antibodies for affinity purification of bovine mullerian inhibiting substance activity.

Two monoclonal antibodies (IG8 and IG10) specific for Mullerian inhibiting substance (MIS) were obtained from the fusion between myeloma cell line SP2/0 and spleen cells from an A/J mouse immunized with partially purified MIS. The resulting hybridomas were screened by a solid-phase RIA and two lines were selected and cloned. Both MAbs IG8 and IG10 subsequently demonstrated specificity for MIS by their ability to inhibit biologically active MIS by precipitation with a second antibody, directly block MIS activity in the organ culture assay, and adsorb and elute active MIS when coupled to a solid support. SDS-polyacrylamide gel electrophoresis of affinity purified MIS demonstrated a major band at 140 kD in unreduced gels and two bands with approximate molecular weights of 70 and 74 KD following reduction. Protein bands were localized either directly by silver staining or on immunoblots developed with radiolabeled anti-MIS MA.

Animals↗
PubMed1986

Organ specific neoantigens reactive in the leukocyte adherence inhibition assay: affinity purification of human colon carcinoma antigen and its cross-reactive protein using monoclonal antibodies.

A monoclonal antibody (mAb) was prepared against a semipurified preparation of an organ specific neoantigen (OSN) reactive in the leukocyte adherence inhibition (LAI) assay. The mAb (LC20.1) induces a positive LAI response when incubated with leukocytes of normal individuals in the presence of OSN derived from either human colon or lung carcinoma cell lines. Absorption of crude OSN preparations from these cell lines on immobilized LC20.1 mAb eliminates all the LAI reactive material suggesting that the mAb recognizes a common determinant on OSN from both colon and lung carcinomas. The LC20 mAb was used to affinity purify the colon cancer OSN as well as a cross-reactive normal protein from the urine of colon cancer patients and healthy donors, respectively. The colon cancer OSN and normal cross-reactive protein display an apparent molecular weight of 29,000, have a similar linear tryptic peptide map, and are indistinguishable by isoelectric focusing analysis. Regardless of the molecular similarity, only the colon cancer OSN preparation could induce a positive LAI when incubated with leukocytes of colon cancer patients. Seven additional anti-colon cancer OSN mAbs were prepared against purified material. These mAbs can be divided into three groups, each of which recognizes a distinct antigenic determinant that is shared by the colon cancer neoantigen and its cross-reactive normal protein.

Animals↗
PubMed1989

Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transforming growth factor beta 2 affinity purification.

Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.

Animals↗
PubMed1990

Comparative studies of carbohydrate-binding proteins from Xenopus laevis skin and eggs. Sugar-binding specificities and affinity purification.

Salt and detergent extracts of acetone-dried powder of Xenopus laevis skin and eggs were fractionated on sugar-Sepharose columns, to which lactose, melibiose, galactose, rhamnose and mannose had been covalently linked, by successive elution with chelating reagent and specific sugars, resulting in separation of the different Ca2(+)-dependent and Ca2(+)-independent carbohydrate-binding proteins. The skin of X. laevis contains a salt-extractable Ca2(+)-dependent lactose-binding lectin of 30 kilodalton (kDa) and the eggs a similar lectin of 43 kDa, but they both lack Ca2(+)-dependent galactose-binding lectins. The 30 kDa lactose-binding lectin which agglutinates human A erythrocytes was isolated by successive affinity chromatography on two linked sugar-Sepharose columns, i.e., a galactose-Sepharose-lactose-Sepharose (GL) column system. Since the 30 kDa lectin was not recovered in the Ca2(+)-dependent lactose-binding protein fraction from the GL column system under the dithiothreitol (DDT)-free conditions, it was concluded that the lectin requires the presence of DTT and calcium for binding to the lactose-Sepharose column.

Animals↗
PubMed1995

Hepatitis C virus core protein: synthesis, affinity purification and immunoreactivity with infected human sera.

The genomic region encoding the core (C) protein (amino acids 1-162) of hepatitis C virus (HCV) was expressed in Escherichia coli as a recombinant (re-) protein with the maltose-binding protein (MBP) using the prokaryotic expression vector pMAL-CR1. The fusion protein (C::MBP) was identified as a approx. 62-kDa polypeptide by immunoblot analysis using antiserum to MBP and HCV-infected human sera. The size of C::MBP corresponded to the calculated combined molecular mass of the approx. 20-kDa HCV C protein and the approx. 42-kDa MBP. The approx. 62-kDa C::MBP was purified using amylose resin as a matrix in affinity chromatography, and showed specific reactivity with HCV-infected human sera. These results suggest that C::MBP may serve as a source of the core antigen for immunological studies on HCV infection.

ATP-Binding Cassette Transporters↗
PubMed1992

Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Trehalase of Dictyostelium discoideum: inhibition by amino-containing analogs of trehalose and affinity purification.

The inhibitory effects of three nitrogen containing analogs of trehalose, validamycin A, MDL 25,637 and castanospermine, on Dictyostelium discoideum trehalase were examined. Prior to this study, the effects of glycohydrolase inhibitors on D discoideum trehalase have not been reported. Validamycin A, MDL 25,637 and castanospermine were found to be potent, reversible, competitive inhibitors of D discoideum vegetative trehalase in vitro with IC50 values of 1 x 10(-9) M, 2 x 10(-8) M and 1.25 x 10(-4) M, respectively. Validamycin A and MDL 25,637 also exhibited time-dependent inhibition of D discoideum trehalase, whereby the potencies of these two inhibitors were observed to increase when pre-incubated with the enzyme for up to 60 min. The competitive natures of validamycin A and MDL 25,637 were also altered during pre-incubation with enzyme such that the compounds behaved as mixed inhibitors under these conditions. Taken together, these results suggest that the inhibitory action of validamycin A and MDL 25,637 on trehalase is of a slow-binding nature. A trehalase-specific affinity resin was synthesized by covalently coupling validamycin A to Sepharose 6B. This resin was used to purify D discoideum trehalase to near homogeneity in a two-step procedure. SDS-PAGE of affinity-purified trehalase, and silver staining or in situ staining for trehalase activity, revealed a major protein species of 42 kDa, exhibiting trehalase activity, and two minor protein species of approximately 45 and 49 kDa. Since validamycin A demonstrates strict binding specificity for trehalase, validamycin A-Sepharose has potential and novel applications in rapid, large scale, purification of trehalases from a variety of species origins.

Animals↗
PubMed2006

Monosize poly(glycidyl methacrylate) beads for dye-affinity purification of lysozyme.

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for purification of lysozyme from chicken egg white. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by a dispersion polymerization technique. The content of epoxy groups on the surface of the poly(GMA) sample determined by the HCl-pyridine method (3.8 mmol/g). Cibacron Blue F3GA loading was 1.73 mmol/g. The monosize beads were characterized by elemental analysis, FTIR and SEM. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature and ionic strength). Maximum lysozyme adsorption amount of poly(GMA) and poly(GMA)-Cibacron Blue F3GA beads were 1.6 and 591.7 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, poly(GMA)-Cibacron Blue F3GA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg-white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the eluted lysozyme was analyzed by SDS-PAGE and found to be 88% with recovery about 79%. The specific activity of the eluted lysozyme was high as 43,600 U/mg.

Adsorption↗
PubMed1989

Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide.

Tumor cells resistant to chloroethylnitrosourea (CENU) therapy contain high levels of O6-alkylguanine DNA-alkyltransferase (GATase), a DNA repair enzyme that aborts DNA interstrand cross-linking by removing CENU-induced O6-alkylguanine adducts. Because the transferase binds covalently to CENU-treated oligonucleotides, we reacted partially purified GATase from cultured human lymphoblasts with a BCNU-treated, 35S-5'-end-labeled, synthetic oligonucleotide designed to have a polyadenylated 3' terminus. Immunoprobing Western blots of this reaction mixture with GATase-specific monoclonal antibody indicated that 25-30% of the transferase became complexed. We purified this complex by affinity chromatography with oligo(dT) cellulose, recovering homogenous material that appeared as a discrete 35-kDa Coomassie blue or silver-stained band after SDS-polyacrylamide gel electrophoresis. Autoradiography and Western immunoblotting confirmed that this band contained both the radiolabeled oligonucleotide and the GATase protein.

Base Sequence↗
PubMed1994

Affinity purification of latex antigens.

Latex extracts are complex mixtures of antigenic peptides. We attempted to raise monoclonal antibodies to latex and to use these antibodies to purify latex antigens. A monoclonal antibody, CRI-C, was raised by standard techniques. Peptides of nonammoniated latex (NAL) and ammoniated latex were electrophoretically separated and transferred for immunoblots. CRI-C was covalently attached to an agarose column. NAL was passed over the column, and purified antigen was then eluted. The eluate was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and RAST inhibition with sera from health care workers and children with spina bifida. CRI-C recognized a single band in ammoniated latex immunoblots and several distinct bands in NAL. The affinity-purified antigen of CRI-C (C-Ag) had multiple bands of less than 20 kd and was 3.9 times more potent in RAST inhibition than NAL when sera from patients with spina bifida were used. However, when health care workers' sera were used, there was no significant difference in the inhibitory potency of NAL and C-Ag. CRI-C appears to recognize a distinct and important epitope in the IgE immune response to latex of patients with spina bifida.

Animals↗
PubMed1998

Affinity purification of HC-Pro of potyviruses with Ni2+-NTA resin.

The HC-Pro of zucchini yellow mosiac virus (ZYMV) was found to bind to Ni2+-NTA resin with or without His-tagging. The binding stringency was similar to that observed in proteins with a zinc finger motif like the HC-Pro. Using this characteristic we developed an efficient and rapid method (2-3 h) for purification of the HC-Pro of several potyviruses. A dominant protein of about 150 kDa was extracted and identified as the HC-Pro of ZYMV by means of immunoblotting. About 150 microg of HC-Pro were partially purified from the soluble fraction of 1 g of leaves. High titers of HC-Pro protein were obtained from plants infected with four potyviruses [ZYMV, watermelon mosaic virus II (WMVII), papaya ringspot virus (PRSV) and turnip mosaic virus (TuMV)]. The HC-Pros of potato virus Y (PVY) and tobacco vein mottling virus (TVMV) did not bind to the Ni2+-NTA resin. The ZYMV-HC-Pro purified by the Ni2+-NTA resin could bind in vitro to ZYMV virions blotted onto a membrane. All the HC-Pros which had been successfully purified by the Ni2+-NTA resin were bound in vitro to membrane-blotted ZYMV coat protein. However, only the HC-Pros of ZYMV and WMVII were able to mediate aphid transmission of purified ZYMV virions. The purification procedure described herein is efficient and convenient, and enables HC-Pro for a number of potyviruses to be obtained in larger amounts and at higher purity than possible by means of most existing methods, based on ultracentrifugation.

Animals↗
PubMed1983

Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification.

Hybridomas producing a monoclonal IgG1 antibody to a human plasminogen-activating enzyme with an apparent mol. wt. of 66,000 (66 K, HPA66) from human melanoma cells were obtained by fusion of NSI-Ag 4/1 mouse myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of the enzyme. Screening for clones of hybridomas producing antibodies to HPA66 was performed with the impure enzyme preparation. A preliminary screening included enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting; the final identification was based on inhibition of the enzymatic activity of HPA66 which was complete at high antibody concentrations. No inhibition of three other human and murine plasminogen activators or of plasmin was observed. Employing a one-step affinity procedure with the antibody coupled to Sepharose, HPA66 was purified approximately 200-fold from conditioned medium from the melanoma cells with a yield of 79%. The purified HPA66 was homogeneous as evaluated by SDS-PAGE. Electrophoresis under reducing conditions indicated that it consisted of one polypeptide chain. The binding constant between the antibody and 125I-labelled HPA66 was approximately 2.5 x 10(9) l/mol. The antibody did not bind to a variety of other plasminogen activators, including 52-K and 36-K human enzymes and 48-K and 75-K murine enzymes. Previously, a monoclonal antibody against another enzyme was derived by the sole use of enzyme inhibition for screening. The present study represents a modification of this procedure that can be used when antibody-unrelated inhibitors of the enzyme are present in hybridoma culture fluid.

Animals↗
PubMed1983

Monoclonal antibodies to human interferon-gamma: production, affinity purification and radioimmunoassay.

Human interferon-gamma (IFN-gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN-gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN-gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN-gamma. Moreover, IFN-gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN-gamma subtypes are immunologically cross-reactive. Immune precipitation of partially purified 125I-labelled IFN-gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN-gamma gave a 50-fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid-phase radioimmunoassay, allowing the detection of IFN-gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h.

Animals↗