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PubMed1997

Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata).

Adenosine Monophosphate↗
PubMed2000

Polyreactivity as an acquired artefact, rather than a physiologic property, of antibodies: evidence that monoreactive antibodies may gain the ability to bind to multiple antigens after exposure to low pH.

Evidence is presented that monoreactive antibodies exposed to low pH may acquire the ability to bind to multiple antigens. M11, a murine, monoclonal, IgM(K) anti-goat IgG (GIgG) was purified from a hybridoma supernatant by elution at low pH from an anti-mu-Sepharose 4B affinity column. By measuring the specific antiGIgG activities and the affinity constants for the interactions of M11, pre- and post-affinity-purification, with GIgG, M11 was shown to be monoreactive before purification. Quite unexpectedly, however, the affinity-purified M11 reacted extensively with size-fractionated liver proteins when tested in an immunoblot, clearly indicating that it was polyreactive. It was concluded that the exposure to low pH had altered the M11 binding-site so as to allow it to bind to many different proteins. This phenomena provides an alternative basis for interpreting the polyreactivity observed following affinity-purification.

Animals↗
PubMed2003

Purification of neutral protease by dye affinity chromatography.

Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25 degrees C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8 degrees C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25 degrees C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.

Affinity Labels↗
PubMed1986

Computer controlled affinity chromatographical purification of soluble Plasmodium falciparum antigens from supernatants of in vitro cultures.

Affinity chromatographic procedures are difficult to scale up from the analytical to the preparative level when the ligand used for purification is a limiting factor. A versatile, computer-controlled affinity chromatographic system is described which permits automatic repetition of the purification process and sophisticated control functions based on the ultra-violet absorbance of fluid passing through the affinity column. The system has been used for automation and scaling up of the purification of Plasmodium falciparum exoantigens.

Antigens, Protozoan↗
PubMed1991

Studies on the 52 kDa antigen of Salmonella typhi: physicochemical stability, purification by affinity chromatography and immunochemical specificity.

The 52 kDa specific protein antigen of Salmonella typhi, as identified by monoclonal antibodies (Ekpo et al. 1990) has been studied with respect to its physicochemical stability, purification by affinity chromatography and immunochemical specificity. It was found that the 52 kDa protein was degraded into smaller antigenic fragments of MW 30-51 kDa when treated with acetone, ethanol, sodium thiocyanate, 0.3M sodium chloride and Veronal and Tris buffers. The exact chemical nature of the degradation of the protein under these conditions is not known but digestion by conventional proteases and dissociation of the non-covalent subunit type have been ruled out. It is proposed that the degradation may be the result of yet unidentified enzyme(s) which become activated by various physical or chemical treatments. Affinity chromatography using a specific monoclonal antibody has been carried out in an attempt to purify the 52 kDa protein. The binding of S. typhi protein to the column was saturable at 65.6 microgram protein/ml gel. The amount of S. typhi protein adsorbed on the column was 0.51% of the total sonicated cell protein. SDS-PAGE of the immunoadsorbent purified protein revealed bands at Mr 15-58 kDa, indicating that the protein obtained had been severely degraded. However, Western blot of the purified protein stained with a specific monoclonal antibody and with rabbit polyclonal antibody against S. typhi showed striking similarity, indicating that the protein obtained was close to immunochemical purity. The 52 kDa protein purified by affinity adsorbent was used as an antigen for the detection of specific IgM in sera of patients. It was shown that sera of patients infected with S. typhi as well as those infected with other bacteria, contained specific IgM against the 52 kDa protein. Thus, it appears that the 52 kDa protein contains species specific as well as cross-reacting epitopes. The possible development of specific diagnosis of S. typhi based on the present experimental results in discussed.

Antibodies, Monoclonal↗
PubMed2002

Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins.

The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.

Amino Acid Sequence↗
PubMed2003

Protein purification by affinity precipitation.

Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale.

Chromatography, Affinity↗
PubMed1981

Tumor and placental histaminase, I. affinity chromatography purification and characterization of the placental enzyme.

Histaminase (diamine oxidase) is an enzyme produced at very high levels by the decidua of the placenta and is found to be associated with a number of human cancers. A procedure for the affinity chromatography purification of this enzyme is described. In this procedure, cadaverine-AH-sepharose was used to bind the enzyme in the placental extract. After extensive washing of the column with 2.5% Triton X-100 in 1 M NaCl, the enzyme was released from the column by 0.1 N chromotropic acid. This purification, essentially a one step procedure, provided 1800-fold purification, and yielded mg quantities of histaminase, homogeneous by SDS-gel electrophoresis and immunodiffusion tests. The procedure usually recovered more than 40% of the enzyme applied and the specific activity of the final enzyme preparation was around 5000 units/mg protein. SDS-gel electrophoresis of the enzyme in different concentrations of acrylamide indicated that the subunit molecular weight of histaminase was about 90,000. Isoelectric focusing of the enzyme in polyacrylamide gel revealed 5 major enzyme components. Results of amino acid analyses indicated that the enzyme had a low content of sulfur-containing amino acids and a relatively high content of dicarboxylic amino acids. The availability of this purification will be useful for the development of immunological methods for detections and quantitation of this enzyme in specimens from cancer patients.

Amine Oxidase (Copper-Containing)↗
PubMed1985

Affinity chromatographic purification of antibody subsets depends on the mode of cross-linking of antigen to the affinity matrix.

Affinity columns for the separation of rabbit antibodies (Abs) to the purified nicotinic acetylcholine receptor (AcChR) from Torpedo californica electroplax were constructed in two ways: (1) by direct cross-linking of purified AcChR to cyanogen bromide activated Sepharose 4B, and (2) by first cross-linking a purified curarimimetic neurotoxin to cyanogen bromide activated Sepharose 4B, subsequently saturating with AcChR, and finally cross-linking the toxin to noncovalently bound receptors with bisimidate cross-linkers such as dimethyl suberimidate (DMS). Ab(AcChR) from pooled rabbit antisera were chromatographed in near stoichiometric proportions to the AcChR bound to the column to allow equilibrium selection of antibodies directed against sites which were not sterically hindered. The concept that columns of the second type subfractionate Ab(AcChR) was tested by analyzing Ab(AcChR) from column fractions with an ELISA and radioimmunoassay (RIA) procedure. The ELISA was constructed to that the exposed face of the AcChR was the same as that expected for columns of the second type, i.e. for the internal or cytoplasmic face. Enhanced ratios of ELISA to RIA measures of Ab(AcChR) reflected substantial purification of cytoplasmic face antibodies in DMS-cross linked columns. Total Ab(AcChR) was measured by RIA. Both types of columns gave substantial purification of tightly bound antibodies, i.e. those which were eluted with 3M potassium thiocyanate. Thirty-fifty percent of the IgG eluted was found in these fractions, which contained 2-6% of the total eluted protein. Approximately half of the total IgG present in these fractions represented specific IgG against AcChR. Both types of columns could be reutilized giving similar results; however, their efficiency was diminished.

Animals↗
PubMed1991

Characterization of a murine IgG3 monoclonal antibody against Chlamydia-specific lipopolysaccharide and its purification by affinity chromatography on IgG Fc-receptors from Streptococcus dysgalactiae.

Purification of a murine IgG3 monoclonal antibody (Mab 22) directed against an epitope of Chlamydia-specific lipopolysaccharide by affinity chromatography on recently described solid phase IgG Fc-receptors from Streptococcus dysgalactiae is reported. SDS-PAGE studies revealed the purity of the eluted antibody. The purified Mab 22 was characterized by determination of class, subclass and light chain-type, and by dot tests and immunoblot analysis.

Animals↗
PubMed1976

Chorismate mutase/prephenate dehydratase from Escherichia coli K12. 1. The effect of NaCl and its use in a new purification involving affinity chromatography on sepharosyl-phenylalanine.

A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl-phenylalanine, has been developed. The method utilizes the effect of NaCl on the binding properties of the enzyme. NaCl inhibits both the mutase and dehydratase activities of the enzyme. In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme. In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites. NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine.

Binding Sites↗
PubMed1972

Purification by affinity chromatography of the molecular forms of acetylcholinesterase present in fresh electric-organ tissue of electric eel.

An acetylcholinesterase inhibitor-Sepharose conjugate was prepared by coupling a derivative of the powerful acetylcholinesterase inhibitor, N-methylacridinium, to CNBr-activated Sepharose. Use of this conjugate permitted direct purification, by affinity chromatography, of the two molecular forms of acetylcholinesterase, 14 and 18 S, present in fresh electric organ tissue. The purified 14S and 18S acetylcholinesterases retained the capacity to aggregate at low ionic strength displayed by crude extracts of the enzyme. The major polypeptide components of the 14S and 18S enzymes, as revealed by acrylamide gel electrophoresis, closely resemble those observed in the 11S form of acetylcholinesterase, previously purified after tryptic digestion of electric-organ tissue.

Acetylcholinesterase↗
PubMed1992

Metabolism in rat and affinity chromatographic purification of bone sialoprotein secreted by metabolically labelled rat osteoblastic cells (UMR-106).

An osteoblastic, established cell line UMR-106 was shown to synthesize high levels of the bone-specific, bone sialoprotein (BSP). BSP could be radiolabelled to high specific activity by adding 3H-glucosamine and 35S-sulfate to the UMR-106 cultures and was isolated to high purity using ion-exchange and affinity chromatography on immobilized serotonin. The radiolabelled BSP, partially purified by ion-exchange chromatography, was injected intravenously into a rat in order to study its tissue distribution and urinary clearance. About 43% of the total recovered radioactivity was excreted in the urine within 75 h and the remainder was widely distributed, with the liver, kidney, heart and pelt showing the highest concentrations. The use of established cell lines for the synthesis of radiolabelled glycoconjugates, in conjunction with rapid purification on affinity matrix, provides a useful approach for studying the metabolism of glycoconjugates in whole animals.

Animals↗
PubMed2006

Gateway-compatible vectors for plant functional genomics and proteomics.

Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.

Amino Acid Sequence↗
PubMed1993

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Ammonium Sulfate↗
PubMed1981

Use of triazine dyes in the affinity chromatographic purification of alkaline phosphatase from calf intestine.

Dye-ligand chromatography was examined as a method for the purification of alkaline phosphatase (EC.3.1.3.1). Forty six dye-Matrex Gels were assessed for their ability to bind alkaline phosphatase. Most dye adsorbents bound significant quantities of the enzyme. Three dye columns were examined in more detail for their selectivity using gradients of potassium chloride to desorb enzyme protein. Purification of alkaline phosphatase using Cibacron blue 3GA-Sepharose 6B chromatography was enhanced by using affinity elution. The best purifications (290-fold) were obtained using pulsed elution with the substrate alpha-naphthyl phosphate although the inhibitor, inorganic phosphate, was also useful (128-fold purification).

Alkaline Phosphatase↗
PubMed2006

Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin.

Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-beta3 (alphaIIbbeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

Animals↗
PubMed1976

NADP+-specific isocitrate dehydrogenase of Excherichia coli. III. Two-step purification employing affinity chromatography.

The NADP+-specific isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) of Excherichia coli has been purified to electrophoretic homogeneity by a two-step purification procedure employing affinity chromatography. The overall yield of enzyme was 30% with specific activity 125 mumol/min per ng protein. Electrophoretic homogeneity of the isocitrate dehydrogenase was deterimed in analytical polyacrylamide gels in a Tris/acetate/EDTA buffer system at pH 7.5 and in a citrate/phosphate buffer system at pH 6.0.

Chromatography, Affinity↗