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Results for “Chromatography, Affinity”

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PubMed1986

Purification of guanosine triphosphate cyclohydrolase I from Escherichia coli. The use of competitive inhibitors versus substrate as ligands in affinity chromatography.

Different affinity chromatography ligands have been compared for the purification of guanosine triphosphate (GTP) cyclohydrolase I, an enzyme that catalyses the transformation of GTP into formate and dihydroneopterin triphosphate, the first metabolite in the biosynthetic pathway of the pterins. When this enzyme is purified by affinity chromatography on GTP-Sepharose a major fraction of the activity is lost and the yield of enzyme decreases as the amount of enzyme applied to the column decreases. The use of nucleotide competitive inhibitors (UTP and ATP) as ligands in the affinity column has shown that the extent of inactivation of the enzyme is related to the affinity of the enzyme for the ligand. Further, the extent of inactivation was reduced by reducing the length of the columns when using the same volume of GTP-Sepharose. Dihydrofolate-Sepharose gave consistently higher yields of GTP cyclohydrolase I regardless of the amount of enzyme applied, but several other proteins were also obtained. For a high purification of GTP cyclohydrolase I the best yield may be obtained with UTP as the affinity ligand and with the shortest length possible of the affinity column, and the purity of enzyme is comparable with that obtained with GTP-Sepharose.

Aminohydrolases↗

PubMed1994

Purification of humanized murine and murine monoclonal antibodies using immobilized metal-affinity chromatography.

An affinity purification technique has been developed using mild elution conditions for the isolation of humanized murine and murine IgG1. This technique is based on the innate affinity of IgG1 for metal and utilizes immobilized metal-affinity chromatography. Antibody bound to the metal-affinity column is eluted with a descending pH gradient and IgG1 elutes around pH 6.0. Humanized murine IgG1 isolated from cell culture media using this procedure is approximately 90% pure and does not contain any free light chain, light-chain dimer, or contaminating serum albumin. In addition, murine IgG1 has been isolated from ascites fluid by direct application to the metal-affinity column. Murine IgG1 is recovered essentially free from albumin in approximately 60% purity, the principal contaminant being transferrin. This method facilitates further purification which is easily achieved by cation-exchange chromatography. The near complete removal of albumin makes metal-affinity chromatography an alternative to salt precipitation of antibody from ascites fluid. Recoveries of antibody from the column are high, typically greater than 90%. We have identified the location of the metal affinity in the IgG1 as being near the carboxy terminus of the heavy chain. A histidine-rich sequence is present in this region which offers the possibility of genetically engineering this sequence to form an even higher affinity metal binding site for potential application in antibody imaging and therapeutics.

Amino Acid Sequence↗

PubMed1990

The preparation of an enzyme associated with aflatoxin biosynthesis by affinity chromatography.

An affinity matrix for the purification of norsolorinic acid dehydrogenase, an enzyme involved in aflatoxin biosynthesis, was prepared by coupling norsolorinic acid to an agarose gel. This matrix was found to be ineffective in isolating active enzyme, and was therefore modified by methylation, using diazomethane. The methylated matrix produced a one-step purification of the enzyme from a crude homogenate, resulting in a 138-fold purification. The active isolate was found to contain one major and two minor bands upon nondenaturing electrophoresis, and all the norsolorinic acid dehydrogenase activity was associated with the major band. It was concluded that the matrix exhibited true affinity for the enzyme, and that affinity chromatography was a valuable approach to isolating other secondary metabolic enzymes involved in the biosynthesis of the aflatoxins.

Aflatoxins↗

PubMed1991

Production of plasma selectively depleted in fibrinogen by affinity chromatography.

An affinity chromatography column was prepared using a polyclonal anti-fibrinogen antibody bound to Sephacryl S 1000. Normal human plasma was depleted of fibrinogen by passage through this column. The fibrinogen free plasma corrected the prolonged clotting times of plasmas deficient in factors V, VII, VIII and IX. Clottable fibrinogen was recovered from the column. An immunodepleted fibrinogen free plasma could fulfil a useful role in internal and external quality control alone, or in combination with other test materials; it could also be used in research.

Antibodies↗

PubMed1977

Extraction and purification of specific antibody against Pseudomonas aeruginosa by immune affinity chromatography.

Immune affinity chromatography has been used to extract and purify specific immune antibody against P. aeruginosa immunotype 4 from hyperimmune gamma glubulin containing antibody against each of seven major immunotypes of P. aeruginosa. A column with a 4 ml. bed volume was construced which had a "working capacity" of approximately 1,680 agglutination units. With water used as the eluting agent, 100 percent yields of functionally active antibody were obtained when amounts equal to or less than the working capacity were loaded onto the column. The technique appears to be suitable for large scale extraction of specific immune antibody for therapeutic trials or investigative use.

Animals↗

PubMed1987

Determination of glycated hemoglobin by affinity chromatography.

An affinity chromatography microcolumn assay for glycated hemoglobin was compared with electrophoretic and microcolumn ion exchange methods. The affinity method has an imprecision of less than 4% for non-diabetic and less than 2% for diabetic specimens. The method is neither affected by carbamylated or acetylated hemoglobin nor by high glucose concentrations. Method comparisons showed discordant results due to interferences in electrophoretic and ion exchange methods in 3%-6.5% of all cases. Nonlinear relationships between affinity methods on one hand and electrophoretic as well as ion exchange methods on the other indicate that the affinity method measures not only beta-terminal glycated hemoglobin but all glycated hemoglobins, providing enhanced sensitivity and diagnostic value.

Blood Glucose↗

PubMed1999

Affinity chromatography of human estrogen receptor-alpha expressed in Saccharomyces cerevisiae. Combination of heparin- and 17beta-estradiol-affinity chromatography.

Estrogen receptor-alpha is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein. Human estrogen receptor-alpha has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor. Pilot scale quantities of receptor have been produced by a yeast strain transformed with expression plasmid YEpE13 [Graumann et al., J. Steroid Biochem. Mol. Biol. 57 (1996) 293] in a 14 l stirred tank reactor. The yeast extract contained 2-4 pmol of receptor protein per mg total protein. A purification scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17beta-estradiol 17-hemisuccinate. Heparin-affinity chromatography was very efficient to remove host cell protein. Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution. The receptor could be purified 5-10-fold in ligand-free state. In contrast to previous reports, we did not find a difference of the binding affinity of liganded and unliganded receptor for heparin immobilized onto Sepharose. The unoccupied receptor could be further purified 100-fold with ligand-affinity chromatography using 17beta-estradiol 17-hemisuccinate-bovine serum albumin-Sepharose. The receptor could be kept in its native state, although saturated with 17beta-estradiol. The purification sequence allows an efficient production of receptor. Further improvement of productivity can be only accomplished by increasing the expression level.

Base Sequence↗

PubMed1978

Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.

Arginine↗

PubMed1988

Quantitative analysis of fibrin-binding affinity of fibrinolytic components by frontal affinity chromatography.

Binding affinity of fibrinolytic factors to insolubilized lysine and fibrin was quantitatively measured by frontal affinity chromatography using lysine-Toyopearl and fibrin-Sepharose column. The highest binding affinity was found with recombinant tissue-type plasminogen activator (t-PA), followed by lysyl-plasminogen and glutamyl-plasminogen (Glu-PLg) with intermediate affinity, but very low affinity by single chain UK-type plasminogen activator, high molecular weight UK and low molecular weight UK. At the coexistence of EACA, fibrin-binding affinity of Glu-PLg was greatly reduced, but those of UK's were substantially unchanged. It was concluded that high fibrin-binding affinity of t-PA and plasminogens were largely related to the lysine-binding affinity of these enzymes, but that of UK's would be related to the other binding affinity.

Arginine↗

PubMed1975

Statistical theory of chromatography: new outlooks for affinity chromatography.

We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods.

Analysis of Variance↗

PubMed2004

Applications of affinity chromatography in proteomics.

Affinity chromatography is a powerful protein separation method that is based on the specific interaction between immobilized ligands and target proteins. Peptides can also be separated effectively by affinity chromatography through the use of peptide-specific ligands. Both two-dimensional electrophoresis (2-DE)- and non-2-DE-based proteomic approaches benefit from the application of affinity chromatography. Before protein separation by 2-DE, affinity separation is used primarily for preconcentration and pretreatment of samples. Those applications entail the removal of one protein or a class of proteins that might interfere with 2-DE resolution, the concentration of low-abundance proteins to enable them to be visualized in the gel, and the classification of total protein into two or more groups for further separation by gel electrophoresis. Non-2-DE-based approaches have extensively employed affinity chromatography to reduce the complexity of protein and peptide mixtures. Prior to mass spectrometry (MS), preconcentration and capture of specific proteins or peptides to enhance sensitivity can be accomplished by using affinity adsorption. Affinity purification of protein complexes followed by identification of proteins by MS serves as a powerful tool for generating a map of protein-protein interactions and cellular locations of complexes. Affinity chromatography of peptide mixtures, coupled with mass spectrometry, provides a tool for the study of protein posttranslational modification (PTM) sites and quantitative proteomics. Quantitation of proteomes is possible via the use of isotope-coded affinity tags and isolation of proteolytic peptides by affinity chromatography. An emerging area of proteomics technology development is miniaturization. Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. More applications of affinity-based purification can be expected, including increasing the resolution in 2-DE, improving the sensitivity of MS quantification, and incorporating purification as part of multidimensional liquid chromatography experiments.

Chromatography, Affinity↗

PubMed1997

D-Glucose, forskolin and cytochalasin B affinities for the glucose transporter Glut1. Study of pH and reconstitution effects by biomembrane affinity chromatography.

The affinities of D-glucose and the transport inhibitors, forskolin and cytochalasin B (CB), for Glut1 were studied by frontal affinity chromatography at pH 5-10 on sterically immobilized proteoliposomes with reconstituted human red cell glucose transporter Glut1. The affinity of D-glucose for Glut1 became slightly weaker as the pH was increased. The inhibitor affinities decreased and became immeasurably weak above pH 9. At pH 7.4, the dissociation constants were 44 mM for glucose, 1.8 microM for forskolin and 72 nM for CB. The affinities of these solutes for Glut1 in red cell membrane vesicles and particularly for Glut1 in red cells were higher, as shown by chromatographic analyses.

Adult↗

PubMed1990

Purification of the IL-2 receptor (TAC) by ligand-affinity chromatography and utilization of the immobilized receptor for receptor-affinity chromatography (RAC) purification of IL-2, mutant IL-2, and IL-2 fusion proteins.

Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug-screening assays. The purified IL-2R subsequently has been immobilized on silica gel and employed for the purification of recombinant IL-2. Receptor-affinity-chromatography-purified IL-2 contains only a highly active monomeric form of the lymphokine, in contrast to immunoaffinity chromatography where several molecular forms of IL-2 with varying degrees of biologic activity are recovered. Receptor-affinity chromatography has been successfully applied to the purification of several mutant IL-2 as well as an IL-2-Pseudomonas exotoxin (IL2-PE40) fusion protein that is a 54.5-kDa chimeric protein in which the cell recognition domain is replaced by IL-2. The IL-2-PE40 is a potential cytotoxic agent for cells bearing the IL-2 receptor.

ADP Ribose Transferases↗

PubMed1998

Fast assay and mini-purification of protein by high performance membrane affinity chromatography.

Several affinity ligands of protein A, human immunoglobulin G, iminodiacetate (IDA)-Cu2+, and cibacron blue F3GA were coupled to GMA-modified cellulose membrane matrices. We prepared the columns based on the membrane supports in high performance membrane affinity chromatography (HPMAC). Next we evaluated nonspecific adsorption of proteins on the membrane media. The rapid assay of proteins on the affinity columns based on membrane supports was achieved. It was observed that the affinity columns based on membrane supports provided good reproducibility, high separation efficiency, and low pressure drop of the column. The columns with the membrane media could also be used for purification of proteins.

Adsorption↗

PubMed1997

Glucose affinity for the glucose transporter Glut1 in native or reconstituted lipid bilayers. Temperature-dependence study by biomembrane affinity chromatography.

The affinity of D-glucose and the transport inhibitor cytochalasin B (CB) for the glucose transporter Glut1 was studied at 5-42 degrees C by quantitative frontal affinity chromatography on sterically immobilized human red cell membrane vesicles, and on proteoliposomes containing reconstituted red cell membrane proteins. Glut1 in the vesicles showed the highest glucose affinity; the dissociation constant Kd(glc) was nearly constant (16 +/- 3 mM) from 15 degrees C to 37 degrees C. For Glut1 in proteoliposomes Kd(glc) decreased from 56 mM at 5 degrees C to 26 mM at 42 degrees C. The CB-Glut1 affinity was strongest around 20 degrees C and was mostly higher with the vesicles, Kd (CB) being 49 nM at 19 degrees C. The entropy and entropy and enthalpy changes for the interactions were calculated.

Adult↗

PubMed1979

[Heterogeneity of alpha-L-fucosidase from human kidney during affinity chromatography].

During affinity chromatography on N-(epsilon-aminocaproyl)-beta-L-fucopyranosylaminosepharose of the enzyme preparation of alpha-L-fucosidase from human kidney the elution profile of the enzyme revealed two components, which can be designated as alpha-L-fucosidases A and B. In the absence of sodium aside in the buffer mixtures used one of the components (fucosidase A) was retained by an affinity adsorbent, while the other one (fucosidase B) was adsorbed under the same conditions; the latter component was eluted with a solution containing the enzyme inhibitor--L-fucose. Data from the enzyme rechromatography suggest an equilibrium of the fucosidases A and B, which differ in their affinities for the affinity sorbent.

Chromatography, Affinity↗

PubMed2001

Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions.

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.

Adsorption↗

PubMed2006

High-throughput analysis of lectin-oligosaccharide interactions by automated frontal affinity chromatography.

Frontal affinity chromatography (FAC) is a quantitative method that enables sensitive and reproducible measurements of interactions between lectins and oligosaccharides. The method is suitable even for the measurement of low-affinity interactions and is based on a simple procedure and a clear principle. To achieve high-throughput and efficient analysis, an automated FAC system was developed. The system designated FAC-1 consists of two isocratic pumps, an autosampler, and a couple of miniature columns (bed volume, 31.4 microl) connected in parallel to either a fluorescence or an ultraviolet detector. By use of this parallel-column system, the time required for each analysis was reduced substantially. Under the established conditions, fewer than 10 hrs are required for 100 interaction analyses, consuming as little as 1 pmol pyridylaminated oligosaccharide for each analysis. This strategy for FAC should contribute to the construction of a lectin-oligosaccharide interaction database essential for future glycomics. Overall features and practical protocols for interaction analyses using FAC-1 are described.

Animals↗