Overexpression in Escherichia coli and affinity purification of chick kidney ferredoxin.
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Amino Acid Sequence↗
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Human lysosomal beta-glucosidase: purification by affinity chromatography.
Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase.
Chromatography, Affinity↗
Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling.
The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with [125I]ovine PRL [( 125I]oPRL). PRL receptors were solubilized from mammary microsomes with 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with 125I using the chloramine-T method. Covalent labeling of PRL receptors with [125I]oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of [125I]oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five [125I]oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit. From these observations, we conclude that this predominant 32,000 mol wt component is a major binding subunit of the PRL receptor molecule and does not aggregate with itself or with other subunits through S-S linkages.
Affinity Labels↗
Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries.
The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 micromol ml(-1), showed a maximum capacity of 9.1 mg Mab ml(-1) and a dynamic capacity of 3.9 mg Mab ml(-1). A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.
Antibodies, Monoclonal↗
The gene encoding the myeloid-related protein 14 (MRP14), a calcium-binding protein expressed in granulocytes and monocytes, contains a potent enhancer element in the first intron.
Myeloid-related proteins 8 and 14 (MRP8 and MRP14) are two Ca2+-binding proteins of the S-100 family highly abundant in myelomonocytic cells. The expression is not only dependent on the developmental status of the cell but also on the inflammatory situation in the tissue. In order to identify regulatory elements responsible for the high expression of MRP14 in myeloid cells, reporter gene constructs have been transfected into HL-60 cells, Mono Mac 6 cells, and L132 cells. We demonstrated that a DNA element in the first intron (positions 153-361) enhances the transcriptional activity of the homologous promoter and of the heterologous herpes simplex virus thymidine kinase promoter up to 37-fold. To further identify the functional site, the region between positions 153 and 192 was analyzed functionally using the thymidine kinase promoter. The region increased the expression in the same magnitude as the complete intron. This enhancer is highly conserved in the human and murine MRP genes, indicative of its involvement in the transcription of MRPs. Protein binding to the region is demonstrated using EMSA, DNA cross-linking, Southwestern blotting, and affinity purification. Affinity purification confirms that four proteins bind to the enhancer element.
Antigens, Differentiation↗
Comparison of affinity tags for protein purification.
Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.
Affinity Labels↗
Dye-ligand affinity adsorbents for enzyme purification.
Affinity chromatography is widely employed in laboratory and large-scale for the purification of biotherapeutics and diagnostics. Some of the most widely used ligands in affinity chromatography have been several reactive chlorotriazine dyes. In particular, immobilized anthraquinone dyes have found a plethora of applications in affinity chromatography because they are inexpensive, are resistant to chemical and biological degradation, are sterilizable and cleanable in situ, and are readily immobilized to generate affinity adsorbents which display high binding capacity for a broad spectrum of proteins. This article provides detailed protocols on the preparation of a dye-ligand affinity adsorbent. Also, detailed protocols for effective application of these media, emphasizing binding and elution conditions are presented.
Adsorption↗
CMRF44+ dendritic cells from peripheral blood stem cell harvests of patients with myeloma as potential cellular vectors for idiotype vaccination.
The optimal conditions required to harvest dendritic cells (DC) for immunotherapy were investigated in a series of preliminary investigations using peripheral blood stem cell (PBSC) harvests and blood from patients with myeloma. There was no difference in the number of DC (CMRF44+, CD19-, CD14-) in PBSC mobilized with G-CSF (mean 0.28%, n = 7) compared with GM-CSF (mean 0.24%, n = 6) and apheresis itself did not concentrate DC. In longitudinal studies (n = 10), the peak DC count (day 12 post PBSC harvest) did not correlate with the peak CD34+ cell count or white cell count. A simple affinity purification of DC resulted in a mean 63-fold purification. Affinity enriched suspensions from normal blood contained more DC (mean = 18.8%; n = 5) than those from patients with myeloma (mean = 9.9%; n = 13). The percentage of DC with a lymphoid phenotype (CD11c-, CDw123hi+) was significantly higher in G-CSF mobilized PBSC harvests (22.7%; n = 6) than in peripheral blood samples from patients with myeloma (7.0%; n = 13; p = 0.01). DC endocytosis was normal and did not change throughout the course of the disease. Neither DC numbers nor subsets changed significantly between days 1 and 3 of culture. Current mobilization procedures, optimized for PBSC, need to be altered when harvesting DC.
Antibodies, Monoclonal↗
Protein purification using a soluble affinity matrix: purification of estrogen receptor with estradiol-polylysine conjugate.
A new strategy for protein purification using a soluble affinity matrix is described. The method was used for purification of estrogen receptor. Cytosols from rat uteri and human fibroid uterine tissue, after fractionation by ammonium sulfate, were treated with estradiol-polylysine conjugate. The highly basic affinity complex was separated from other proteins by DEAE-Sephacel chromatography. After dissociation of the eluted complex with excess estradiol, the receptor was recovered by CM-Sephadex chromatography. A 2000-fold purification of the rat uterine estrogen receptor was obtained with an activity recovery of 35%.
Animals↗
Human platelet factor 4: Purification and characterization by affinity chromatography. Purification of human platelet factor 4.
Platelet factor 4 is a low molecular weight protein contained in the storage granules of platelets and released during aggregation with a variety of aggregating agents. In vitro, it is a potent antiheparin. This property has been used for a rapid, simple purification procedure using affinity chromatography on heparin epsilon-aminocaproic aced Sepharose. Supernatants collected from outdated platelet concentrates, or platelet extracts prepared from washed, outdate platlets themselves, are first precipitated with 50% ammonium sulfate. The supernatant is dialyzed and applied to the affinity column. Contaminating proteins are washed from the column with 0.5 M NaCl in 0.005 M sodium barbital buffer, pH 7.4 and the column is then eluted with a gradient of 0.5 to 3.0 M NaCl in 0.005 M sodium barbital buffer, pH 7.4. When prepared from platelet extracts, a single protein peak with high platelet factor 4 activity is eluted at 0.9 to 1.0 M NaCl. The peak fractions demonstrate a single band on Na dodecyl-SO4-polyacrylamide gel electrophoresis. The molecular weight as determined by Na dodecyl-SO4 gel electrophoresis was 11,600 +/- 330, and was 40,000 by gel filtration.
Amino Acids↗
Biomimetic dyes as affinity chromatography tools in enzyme purification.
Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins. Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation, the model of the protein and also a number of suitable synthetic ligands which mimic natural biological ligands of the protein. There are several examples of enzyme purifications (trypsin, urokinase, kallikrein, alkaline phosphatase, malate dehydrogenase, formate dehydrogenase, oxaloacetate decarboxylase and lactate dehydrogenase) where synthetic biomimetic dyes have been used successfully as affinity chromatography tools.
Chromatography, Affinity↗
The use of affinity chromatography for the purification of affinity labeled peptides from staphylococcal nuclease.
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Journal Article↗
Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.
Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.
Adsorption↗
Novel ligands for the affinity-chromatographic purification of antibodies.
Affinity chromatography represents one of the most powerful fractionation techniques for the large-scale purification of biotechnological products. Despite its potential, the use of this methodology is limited by the availability of specific ligands for each target. Combinatorial chemistry and molecular modeling, often combined, have become interesting and innovative methods for generating novel ligands, tailored to specific biotechnological needs. One of the greatest area of application has been the discovery of novel ligands for the purification of antibodies, which represent an emerging but very important class of innovative therapeutic agents for the treatment of a vast array of diseases. Naturally available affinity ligands, such as Protein A or G for IgG purification or lectins for IgA and IgM purification, which are obtained from microorganisms or genetically modified bacteria through complex and expensive procedures, are not well suited for large-scale purification and require moreover time-consuming analytical controls to check for the presence of contaminants which may affect the safety of the purified antibody for clinical purposes. Recent results suggest that the application of combinatorial technologies and molecular modeling for the discovery of synthetic ligands may open new avenues for the development of more efficient, less expensive and--more importantly--safer procedures for antibody purification at the industrial level.
Antibodies↗
Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation.
Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5+/-3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.
Chromatography, Affinity↗
Purification of plasmids by triplex affinity interaction.
Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.
DNA, Bacterial↗
Purification of antibodies by affinity chromatography.
This review focusses on affinity purification of immunoglobulins, a methodology which is a powerful tool to obtain pure and intact antibodies. Affinity techniques allow antibody purification both in a single step chromatographic procedure as well as in complex purification protocols depending on the intention to use the target antibody. The purification strategies for antibodies by interaction with affinity ligands such as antibodies and Fe receptors or low molecular weight compounds are described.
Animals↗
Immobilised peptide displaying phages as affinity ligands. Purification of lactoferrin from defatted milk.
An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 microm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 microm) phage particles as ligands without any risk of blocking the monolithic column. Bound lactoferrin was eluted using 1M NaCl with a purity of >95%. The technique presents a good alternative to conventional immunoaffinity chromatography for purification of a protein of interest from complex samples due to (i) the robustness of the system in terms of recovery and ligand leakage and (ii) economical aspect in terms of low ligand cost.
Acrylamides↗