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PubMedDate not supplied

Affinity purification of the structural proteins of a fish Rhabdovirus by the use of monoclonal antibodies.

A simple procedure for optimization of affinity chromatography is described for parallel purification of four structural proteins of viral haemorrhagic septicaemia virus. Monoclonal antibodies to be used as affinity reagents were characterized initially with respect to elution conditions by enzyme-linked immunosorbent assay. Subsequently, immobilized antibodies were tested in a microscale nonradioactive direct immunoprecipitation assay, in order to establish optimal conditions for affinity purification of the viral proteins.

Animals↗

PubMed2004

Coated microwell plate-based affinity purification of antigens.

Antibody-based affinity capture of antigens is widely used in the isolation of antigens from complex mixtures. Antibody and the corresponding antigen are allowed to interact with each other to form immunocomplexes which are then typically captured by protein A or protein G immobilized on beaded support. Antigen capture performed using this method generally requires multiple centrifugation steps and careful pipetting to avoid loss of the bead-bound complexes. This traditional procedure is tedious and not easily reproducible, especially when working with multiple samples. To address these issues we have demonstrated that antigens can be captured with protein A/G, protein G, and high binding-capacity streptavidin 96-well strip-coated plates. The coated plate method of antigen purification is reproducible, within the same experiment and between experiments, due to the uniform binding capacity of the plates and wells. Here we report the use of coated microwell plates for antigen capture and for protein-protein interaction studies with the well-characterized BIR2-SMAC, transferrin receptor/ transferrin, and other systems.

Antibody Affinity↗

PubMed1998

Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis.

Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are synthesized starting from the apoprotein via several intermediates by the action of accessory proteins. The isolation and biochemical characterization of such intermediates is hampered by their low abundance and their lability. Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Sepharose matrix. The tag was fused to the large subunit of [NiFe] hydrogenase 3 (HycE) of Escherichia coli. No significant influence of the affinity peptide on maturation or activity of the protein was observed when the modified gene was integrated into the chromosome by homologous recombination. A tagged nickel-free precursor form of HycE bound quantitatively to a recombinant StrepTactin Sepharose column. More than 90% pure subunit could be obtained after elution with desthiobiotin. The procedure was shown to be more efficient than purification by immobilized metal affinity chromatography using a N-terminal His-tag. General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed.

Amino Acid Sequence↗

PubMed1994

Universal method for the facile production of glycolipid/lipid matrices for the affinity purification of binding ligands.

Glycolipid recognition is a common motif in cellular physiology and bacterial pathogenesis. Such protein/lipid interactions are most conveniently demonstrated by the thin-layer chromatogram overlay. We have designed a simple affinity matrix for the purification of such glycolipid (or lipid) binding ligands based on the same principle, i.e., glycolipid (or lipid) adsorbed onto silica. The versatility of the procedure is demonstrated by the purification of several anti-glycolipid antibodies and anti-phosphatidyl ethanolamine (anti-PE) and the affinity purification of the Escherichia coli-derived verotoxin which binds to globotriaosyl ceramide.

Affinity Labels↗

PubMed1990

CD4-affinity purification of recombinant and native HIV gp120 and comparison of the affinity constants for the receptor.

Soluble CD4-immunoglobulin chimeric proteins were covalently attached to CNBr-activated Sepharose. This affinity matrix was used to establish a powerful new method to isolate different species of the HIV external glycoprotein gp120 from cell-free culture supernatants. Recombinant gp120 was expressed in Baculovirus-infected insect cells and isolated from cell-free culture supernatants. The recombinant protein has an apparent molecular mass of 130 kDa. These two gp120 species were shown to be of identical molecular size after complete deglycosylation achieved by endoglycosidase treatment, and they bound to CD4-H gamma l with the same binding constant, that was reported for native forms of gp120 and CD4. Thus the different glycosylation of gp120 does not influence its affinity to CD4 and the gp120-CD4 complex can be reversibly dissociated.

Blotting, Western↗

PubMed1998

Affinity purification of Helicobacter pylori urease. Relevance to gastric mucin adherence by urease protein.

A simple, reproducible and high yield method of Helicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized with N-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction.

Animals↗

PubMedDate not supplied

Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag.

The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.

Canada↗

PubMed2000

Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia fructicola (Wint.) honey.

Trifluoromethyl ketones (TFK) are potent inhibitors of a variety of serine hydrolases. The TFK inhibitor, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone (MBTFP), was found to competitively inhibit cutinase activity (I50 = 9.4 x 10(-3)) from the fungal plant pathogen Monilinia fructicola and to serve as an effective affinity ligand for the purification of cutinases from culture filtrates. The TFK inhibitors, 3-n-octylthio-1,1,1-trifluoro-2-propanone (OTFP) and 3-n-pentylthio-1,1,1-trifluoro-2-propanone (PTFP), also inhibited cutinase activity with I50 values of 1.6 x 10(-6) and 2.3 x 10(-4) M, respectively. Buffer containing OTFP was the strongest eluant for cutinases of M. fructicola and provided the best purification factor and yield, although buffers containing OTFP, detergent, and salt were found to be effective for eluting cutinases bound to MBTFP-Sepharose. Buffer containing 0.5% Triton X-100 also selectively eluted cutinases from the affinity column. Two-dimensional electrophoretic analysis by SDS-PAGE and isoelectric focusing of the affinity-purified cutinase fraction indicated activity associated with proteins of pI 8.2 and molecular masses of approximately 18.6 and 20.8 kDa. These proteins hydrolyzed [3H]cutin and artificial substrates such as p-nitrophenylbutyrate and related esters, typical of other cutinases, but differ from previously characterized cutinases in molecular mass. The two low-molecular-weight proteins resolved by 2-D gel electrophoresis were subjected to in-gel digestion with Lys-C and the resulting peptide fragments were separated by Microbore-HPLC. The amino acid sequences of several internal peptide fragments had high homology with cutinase sequences from other fungi, particularly the plant pathogen Botrytis cinerea. Our study illustrates the potential of TFK ligands for the affinity purification of cutinases and indicates that the cutinases from M. fructicola have novel features warranting further study.

Acetone↗

PubMed2006

Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE.

A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1983

Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown.

By affinity chromatography of a crude reticulocyte extract on ubiquitin-Sepharose, three enzymes required for the conjugation of ubiquitin with proteins have been isolated. One is the ubiquitin-activating enzyme (E1), which is covalently linked to the affinity column in the presence of ATP and can be specifically eluted with AMP and pyrophosphate (Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) J. Biol. Chem. 257, 2537-2542). A second enzyme, designated E2, is bound to the ubiquitin column when E1 and ATP are present, and is eluted with a thiol compound at high concentration. The third enzyme, designated E3, is adsorbed to the affinity column by noncovalent interactions and can be eluted with high salt or increased pH. The presence of all three enzymes is absolutely required for the conjugation of 125I-ubiquitin with proteins. All three affinity-purified enzymes are also required for the breakdown of 125I-albumin to acid-soluble material in the presence of ubiquitin, ATP, and the unadsorbed fraction of the affinity column. The following observations indicate that the function of E2 is the transfer of activated ubiquitin to the site of conjugation in the form of an E2-ubiquitin thiol ester intermediate. (a) E2 is rapidly inactivated by iodoacetamide, but can be protected against inactivation by a prior incubation with E1, ATP, and ubiquitin. This suggests an E1-mediated transfer of activated ubiquitin to an iodoacetamide-sensitive thiol site of E2. (b) The requirements for the binding of E2 to the ubiquitin column and the mode of its elution, cited above, are consistent with the notion that a covalent linkage is formed between E2 and Sepharose-bound ubiquitin. (c) Upon the incubation of 125I-ubiquitin with E1 and ATP, followed by the addition of purified E2, activated ubiquitin is transferred from E1 to several low molecular weight forms of E2, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The linkage of ubiquitin to all these forms has the characteristics of a thiol ester bond. In a further incubation with E3 and a protein substrate for conjugation, activated ubiquitin was transferred from the different forms of E2-ubiquitin to stable ubiquitin-protein conjugates. Thus, E3 is involved in the last step of the ligase system.

Adenosine Triphosphate↗

PubMed1995

Truncated human P450 2D6: expression in Escherichia coli, Ni(2+)-chelate affinity purification, and characterization of solubility and aggregation.

Cytochrome P450 2D6 is one of the clinically important drug-oxidizing enzymes in human liver. We constructed an Escherichia coli expression vector to obtain large amounts of this microsomal hemoprotein in a form suitable for purification and further structural analysis. The N-terminal 25 amino acids, which presumably serve as a membrane anchor, were replaced by codons for a [His]6 tag to increase solubility and to allow for rapid purification by Ni(2+)-chelate affinity chromatography. P450 2D6 apoprotein was synthesized under practically all growth conditions, whereas formation of heme-containing holoenzyme strictly depended on addition of the heme precursor delta-aminolevulinic acid to the E. coli culture. The truncated P450 was purified from the soluble cytosolic fraction to electrophoretic homogeneity (7 nmol of P450/mg protein) by affinity chromatography on Ni(2+)-nitrilotriacetate-agarose. The purified protein exhibited a CO-reduced difference spectrum with a delta max at 450 nm and no detectable P420. Kinetic analysis revealed a Km value for bufuralol 1'-hydroxylation similar to the Km of the native full-length enzyme purified from human liver microsomes. To characterize the purified truncated protein with respect to hydrodynamic properties, we performed sedimentation velocity and sedimentation equilibrium analysis. These studies demonstrated that approximately 50% of the protein was in a highly aggregated state. Another 30% consisted of a single protein species with an approximated molecular weight of 200,000 and the residual 20% represented at least two other species with lower molecular weights. To prevent formation of such unexpectedly high aggregation states, purification was also performed in the presence of "nonaethyleneglycol monododecyl ether" (C12E9), a nonionic, chemically defined detergent often used in attempts to crystallize membrane proteins. Over 80% of this preparation was found to consist of a single protein species with a M(r) of 62,000 indicating a monomeric state. These data demonstrate that (i) N-terminal truncation of P450 2D6 does not principally interfere with heme incorporation and function of the enzyme, (ii) deletion of the hydrophobic N-terminus leads to a protein product which can be recovered largely from the soluble E. coli fraction, and (iii) this soluble truncated protein is highly aggregated unless detergents are added to maintain it in a monomeric state.

Amino Acid Sequence↗

PubMed1998

Affinity-purification and characterization of caveolins from the brain: differential expression of caveolin-1, -2, and -3 in brain endothelial and astroglial cell types.

Caveolins 1, 2 and 3 are the principal protein components of caveolae organelles. It has been proposed that caveolae play a vital role in a number of essential cellular functions including signal transduction, lipid metabolism, cellular growth control and apoptotic cell death. Thus, a major focus of caveolae-related research has been the identification of novel caveolins, caveolae-associated proteins and caveolin-interacting proteins. However, virtually nothing is known about the expression of caveolins in brain tissue. Here, we report the purification and characterization of caveolins from brain tissue under non-denaturing conditions. As a final step in the purification, we employed immuno-affinity chromatography using rabbit polyclonal anti-caveolin IgG and specific elution at alkaline pH. The final purified brain caveolin fractions contained three bands with molecular masses of 52 kDa, 24 kDa and 22 kDa as visualized by silver staining. Sequencing by ion trap mass spectrometry directly identified the major 24-kDa component of this hetero-oligomeric complex as caveolin 1. Further immunocyto- and histochemical analyses demonstrated that caveolin 1 was primarily expressed in brain endothelial cells. Caveolins 2 and 3 were also detected in purified caveolin fractions and brain cells. The cellular distribution of caveolin 2 was similar to that of caveolin 1. In striking contrast, caveolin 3 was predominantly expressed in brain astroglial cells. This finding was surprising as our previous studies have suggested that the expression of caveolin 3 is confined to striated (cardiac and skeletal) and smooth muscle cells. Electron-microscopic analysis revealed that astrocytes possess numerous caveolar invaginations of the plasma membrane. Our results provide the first biochemical and histochemical evidence that caveolins 1, 2 and 3 are expressed in brain endothelial and astroglial cells.

Amino Acid Sequence↗

PubMed1992

Brain casein kinase 2: affinity purification procedure using immobilized polyethylenimine.

A simplified procedure for casein kinase 2 purification from bovine brain is described. The purification procedure consists of two affinity chromatography steps, using heparin and polyethylenimine immobilized on a synthetic matrix (Toyopearl 650M). The adsorption and elution conditions for each column were optimized, resulting in a simple elution protocol for each column. A stable, highly purified casein kinase 2 preparation was obtained in 4 h using this procedure. Polyethylenimine was shown to stimulate the casein kinase 2 activity using exogeneous substrates (casein, calmodulin, MAP2, and tau) but not the enzyme's autophosphorylation activity. The polyethylenimine stimulation could be overcome by applying a mass excess of the casein kinase 2 inhibitor, heparin.

Animals↗

PubMed1988

Medium-scale ligand-affinity purification of two soluble forms of human interleukin-2 receptor.

Recombinant technology has facilitated the production of two soluble forms of human 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 IL-2Rs, 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 and the receptor-affinity purification of human recombinant interleukin-2.

Amino Acids↗

PubMed1992

Affinity purification of proteins using expanded beds.

The use of expanded beds of affinity adsorbents for the purification of proteins from feedstocks containing whole or broken cells is described. It is demonstrated that such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. A stable, expanded bed can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Circulation and mixing of the adsorbent particles is minimal and liquid flow through the expanded bed shows characteristics similar to those of plug flow. Frontal analysis performed with the highly selective affinity system involving the adsorption of human polyclonal immunoglobulin G onto Protein A Sepharose Fast Flow indicate that the adsorption performance of the expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. The adsorption performance of the expanded bed was not diminished when adsorption was carried out in the presence of intact yeast cells. Batch adsorption experiments also indicated that the adsorption characteristics of the affinity system were not greatly altered in the presence of cells in contrast to results from a less selective ion-exchange system. An expanded bed of Cibacron Blue Sepharose Fast Flow was used to purify phosphofructokinase from feedstock of disrupted yeast prepared by high pressure homogenisation without the need for prior removal of particulate material. The potential for the use of expanded beds in large scale purification systems is discussed.

Adsorption↗

PubMed2001

Affinity purification of DNA-binding proteins.

The focus of this review is on DNA affinity chromatography, which is the most powerful tool for purification of DNA binding proteins. The use of nonspecific-, sequence specific- and single stranded-DNA affinity columns in purification of various DNA binding proteins is discussed. The purification strategies for transcription factors, restriction enzymes, telomerases, DNA and RNA polymerase and DNA binding antibodies are described. Different applications of DNA affinity chromatography are presented.

Animals↗

PubMed1998

A synthetic ligand for IgA affinity purification.

We reported previously that TG19318, a synthetic ligand deduced from the screening of combinatorial libraries, displays specific and selective recognition properties for immunoglobulins of the G class and can be used conveniently for affinity chromatography purification of monoclonal and polyclonal antibodies. In this study we have extended the ligand characterization, examining its ability to bind IgA from cell culture supernatants and from IgG-deprived serum. Affinity columns prepared by immobilizing TG19318 on Sepharose allowed convenient one-step purification of monoclonal IgA directly from crude feedstocks, in high yield and with full recovery of immunoreactivity. Optimal column adsorption occurred with phosphate buffer at neutral pH, while elution of adsorbed IgA could be accomplished by a buffer pH change to acidic or basic conditions. Column capacity was close to 7 mg IgA/ml support.

Adsorption↗

PubMed1987

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex.

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.

Acetylation↗