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At least 91 records · Page 5Linked to original sources
PubMed2004

Rational affinity purification of native Streptomyces family 10 xylanase.

Xylanase SoXyn10A from Streptomyces olivaceoviridis E-86 comprises a family 10 catalytic module linked to a family 13 carbohydrate-binding module (SoCBM13). The SoCBM13 has a beta-trefoil structure, with binding sites in each subdomain (alpha, beta and gamma). Subdomain alpha, but not subdomains beta and gamma, binds tightly to lactose. It was, therefore, thought that immobilized lactose could be used for the affinity purification of SoXyn10A. Lactosyl-Sepharose was prepared and tested as an affinity matrix. SoXyn10A produced from the cloned xyn10A gene by Escherichia coli, and native SoXyn10A in culture supernatants from S. olivaceoviridis, were purified to homogeneity in a single step by affinity chromatography using this matrix. This simple purification of SoXyn10A makes the enzyme an attractive candidate for applications requiring xylanase. The CBM also has the potential for use as an affinity tag for the purification of other proteins.

Blotting, Western↗
PubMed1992

Affinity purification and characterization of CIF, an insect immunoresponsive factor with NF-kappa B-like properties.

1. A rapid DNA affinity purification procedure was worked out for the purification of the Cecropia Immunoresponsive Factor (CIF) from the pupae of Hyalophora cecropia. 2. CIF consists of a single polypeptide chain of 65 kDa and is present as a homodimer under native conditions. 3. CIF binds to the kappa B-like sequences upstream of the H. cecropia immune genes with the following order of affinity: attacin kappa B greater than lysozyme kappa B greater than cecropin A kappa B greater than cecropin B kappa B. 4. The purified CIF also strongly binds to the kappa B sequences from both the immunoglobulin kappa light chain gene and the MHC class I gene. 5. The DNA binding of CIF can be inhibited by antisera directed against NF-kappa B-related proteins. 6. The cytoplasmic factor Cl, co-purified from the affinity column, contains two polypeptide chains, one of which has the same molecular weight as CIF.

Animals↗
PubMed1991

Affinity purification of Trypanosoma brucei small nuclear ribonucleoproteins reveals common and specific protein components.

We have developed a procedure for the affinity purification of small nuclear ribonucleoproteins (snRNPs) of Trypanosoma brucei (U2 and U4/U6 snRNPs), which are essential for trans splicing. Each of these snRNPs can be specifically and efficiently selected from T. brucei extracts through biotinylated antisense 2'-O-methylated RNA oligonucleotides immobilized on streptavidin-agarose. Protein analysis revealed a set of five low molecular weight polypeptides common to the U2 and U4/U6 snRNPs and the spliced leader RNP. In addition, several U2 and U4/U6 snRNP-specific protein components were identified. Using monoclonal antibodies against human snRNP proteins, we could not detect any significant cross-reaction with the trypanosomal U2 snRNP proteins. Thus, the trypanosomal snRNPs exhibit principal differences from the higher eukaryotic snRNPs not only in their RNA but also in their protein components.

Animals↗
PubMedDate not supplied

Immunization and affinity purification of antibodies using resin-immobilized lysine-branched synthetic peptides.

A new method has been developed to raise antibodies against synthetic peptides. A multiple antigenic peptide system (MAP) containing a branched oligolysine was synthesized on a beaded polystyrene polyoxyethylene graft copolymer resin, which acts as a synthetic hapten carrier for use in immunization. The peptides, already attached to the carrier, can be used directly after final deprotection without any further purification steps. The utility of this peptide-carrier conjugate is highlighted by its additional application for affinity purification of antibodies generated.

Amino Acid Sequence↗
PubMed1993

Affinity purification of molecular chaperones of the yeast Hansenula polymorpha using immobilized denatured alcohol oxidase.

We used peroxisomal alcohol oxidase (AO) for the affinity purification of molecular chaperones from yeasts. Methodical studies showed that up to 0.8 mg of purified bacterial GroEL was able to bind per ml of immobilized denatured AO column material. Using crude extracts of Hansenula polymorpha or Saccharomyces cerevisiae, several proteins were specifically eluted with Mg-ATP which were recognized by antibodies against hsp60 or hsp70. One H. polymorpha 70 kDa protein was strongly induced during growth at elevated temperatures, whereas a second 70 kDa protein as well as a 60 kDa protein showed strong protein sequence homology to mitochondrial SSC1 and hsp60, respectively, from S. cerevisiae.

Alcohol Oxidoreductases↗
PubMed1989

Immuno-affinity purification of specific antibodies against vasoactive intestinal polypeptide (VIP) on VIP (1-10)-linked polydimethylacrylamide resin.

A novel method for immuno-affinity purification of specific antibodies against amino-terminal (N-terminal) porcine vasoactive intestinal polypeptide (p-VIP) was developed. The antiserum VP (No. 6203) elicited by p-VIP-immunoglobulin G (IgG) conjugate was heterogenous and reacted not only with the N-terminal fragments of p-VIP but also with carboxy-terminal (C-terminal) fragments. To obtain specific antibodies against the N-terminal fragment of p-VIP, antiserum VP was purified by column chromatography on N-terminal decapeptide (p-VIP 1-10)-linked polydimethylacrylamide resin. The antibody thus obtained was highly specific to the N-terminal sequence of p-VIP and hardly reacted with the C-terminal fragments of VIP in enzyme-linked immunosorbent assay.

Antibodies↗
PubMed2005

Intracellular production of a soluble and functional monomeric streptavidin in Escherichia coli and its application for affinity purification of biotinylated proteins.

Monomeric forms of avidin and streptavidin [(strept)avidin] have many potential applications. However, generation of monomeric (strept)avidin in sufficient quantity is a major limiting factor. We report the successful intracellular production of an improved version of monomeric streptavidin (M4) in a soluble and functional state at a level of approximately 70 mg/L of an Escherichia coli shake flask culture. It could be affinity purified in one step using biotin agarose with 70% recovery. BIAcore biosensor analysis using biotinylated bovine serum albumin confirmed its desirable kinetic properties. Two biotinylated proteins with different degrees of biotinylation (5.5 and 1 biotin per protein) pre-mixed with cellular extracts from Bacillus subtilis were used to examine the use of M4-agarose in affinity purification of protein. Both biotinylated proteins could be purified in high purity with 75-80% recovery. With the mild elution and matrix regeneration conditions, the M4-agarose had been reused four times without any detectable loss of binding capability. The relatively high-level overproduction and easy purification of M4, excellent kinetic properties with biotinylated proteins and mild procedure for protein purification make vital advancements in cost-effective preparation of monomeric streptavidin affinity matrix with desirable properties for purification of biotinylated molecules.

Bacillus subtilis↗
PubMed1999

One-step affinity purification of the G protein betagamma subunits from bovine brain using a histidine-tagged G protein alpha subunit.

An efficient one-step affinity purification of bovine brain G protein betagamma subunits (betagamma's) is described. The betagamma's, in a detergent extract of brain membranes, are first dissociated from the alpha subunits (alpha's), reassociated with decahistidine-tagged alphail produced in bacteria, and then adsorbed onto Ni2+-nitrilotriacetic acid-agarose via the histidine tag. This mild adsorption retained the high activity of the ligand alpha's, in contrast to the commonly used chemical crosslinking methods. A wash step with a buffer containing chaotropic ions (SCN-) completely removed contaminating proteins that were refractory to washes with high concentrations of detergents, after which the highly purified betagamma's were eluted with a buffer containing Al3+, Mg2+, and F- ions. The obtained betagamma's were found to be fully functional, as assessed by their ability to support pertussis toxin-catalyzed ADP-ribosylation of alphail. Since the combination of the mild adsorption via the histidine tag and the wash with chaotropic ions can be easily applied to purifying betagamma's from various animal tissues, this new chromatographic method is expected to facilitate the purification of other membrane proteins that bind to Galpha and/or Galphabetagamma.

Adsorption↗
PubMed2003

Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides.

Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues followed by addition of an affinity tag has recently been pursued as a strategy for enriching phosphorylated species from complex mixtures. Here we report the use of an introduced thiol tag as the ligand for affinity purification via disulfide exchange with an activated thiol resin and the development of a protocol to improve the sensitivity considerably over previous reports (i.e., to subpicomole levels.) During our experiments, we observed a side reaction in which water was eliminated from unmodified serine residues. This side reaction resulted in the introduction of the affinity tag into unphosphorylated proteins, confounding attempts to specifically purify phosphoproteins from mixtures. Unchecked, this side reaction will also prevent application of the beta-elimination strategy to phosphopeptide samples where the phosphorylated species are minor components (i.e., most current phosphoproteomics applications). Quantitation of the side reaction products using three synthetic unphosphorylated peptides showed varying conversion efficiencies; at maximum, 1.7% of unphosphorylated peptide was converted to the affinity-tagged form. Inclusion of EDTA into the reaction reduced the side reaction but also greatly reduced the conversion efficiency of one of the phosphoserine residues of ovalbumin, suggesting a role for trace metal ions in the beta-elimination chemistry. Despite the presence of the side reaction, the affinity strategy was shown to be effective at enriching phosphopeptides from fairly complex peptide mixtures. The strategy was applied to the analysis of in vitro phosphorylation of bovine synapsin I by Ca(2+)/calmodulin-dependent kinase II, resulting in the identification of four phosphorylation sites, two of which have not been previously reported.

Animals↗
PubMed1990

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta.

We describe a simple method for the affinity purification of specific RNA-binding proteins. DNA sequences corresponding to the protein-binding site of the RNA are subcloned into an in vitro transcription vector between the T7 viral promoter and a poly(A) track. A polyadenylated RNA transcript is bound to poly(U)-Sepharose and subsequently incubated with a cellular extract prepurified on heparin-agarose. Specifically adsorbed proteins are recovered in high yield and purity from the affinity matrix by high salt elution. Using this method we isolated the iron regulatory factor (IRF), a cytoplasmic protein which binds to specific palindromic elements in the 5' and 3' untranslated sequences of ferritin and transferrin receptor mRNA, respectively. Activation and binding of this regulatory factor correlates with increased transferrin receptor mRNA stability and inhibition of ferritin translation. The purified factor from human placenta migrates as a monomer in gel chromatography, but is present in equimolar amounts of two proteins with molecular weights of 95 and 100 kDa when analysed by SDS/PAGE. The two proteins are highly related as judged by the identity of their isoelectric points and their specificity to form RNA-protein complexes.

Base Sequence↗
PubMed1995

Affinity purification of rat cortical and chicken forebrain synaptosomes using a biotinylated derivative of omega-CgTx GVIA.

We describe a magnetophoretic method for the affinity purification of synaptosomes expressing omega-CgTx GVIA-sensitive, N-type voltage-sensitive calcium channels (VSCCs). The method utilizes a biotinylated derivative of omega-CgTx GVIA which retains its ability to displace [125I] omega-CgTx GVIA from its binding sites on rat synaptic membranes. When coupled to streptavidin coated magnetizable beads, the hexanoyl spacer between omega-CgTx GVIA and the biotin:streptavidin bead complex is sufficiently long to allow flexibility of the toxin to bind to its receptor on synaptosomes. We have used this ligand successfully to isolate deaggregated synaptosomes from the parent fractions of chicken forebrain and rat cortex. In the chicken synaptosome parent fraction, omega-CgTx GVIA (1 nM-1 microM) produced a concentration-dependent block of the KCl-induced intracellular free Ca2+, [Ca2+]i, elevation with an IC50 of 28 nM. After affinity magnetophoresis no increase in [Ca2+]i elevation was observed in either the bound or unbound fractions. In the rat synaptosome parent fraction, the KCl-induced increase in free intracellular Ca2+ ([Ca2+]i) elevation was partially blocked by omega-CgTx GVIA (17 +/- 2% / 1 microM) and to a greater extent by omega-Aga IVA (55 +/- / 1 microM): a combination of the two toxins was additive (72 +/- 4% / 1 microM). The block obtained by omega-CgTx GVIA (1 microM) in the unbound fraction was reduced to 3 +/- 2%, whereas that by omega-Aga IVA (1 microM) increased to 82 +/- 3%. The block obtained by a combination of both toxins (83 +/- 2%) was the same as that with omega-Aga IVA alone (82 +/- 3%). No increase in free [Ca2+]i elevation was observed in the bound fraction although single synaptosome-like structures, displaying synaptophysin immunoreactivity, were detected on the beads. We conclude that omega-CgTx GVIA-sensitive N-type calcium channels are present on all chicken forebrain synaptosomes but only a subset of rat cortical synaptosomes.

Animals↗
PubMed2000

Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand.

Due to the peculiar composition of the egg yolk and the lack of specific affinity ligands, Y immunoglobulins are normally purified using complex and time consuming procedures involving a combination of precipitation and chromatographic steps first to extract and capture and then to polish IgY. In this study, we have examined the applicability for IgY affinity purification of TG19318, a synthetic ligand for immunoglobulin, obtained from the screening of combinatorial libraries, and already characterized for its capability to purify immunoglobulins of class G, M, E and A. Soluble proteins were separated from the lipidic fraction of egg yolk by the water dilution method and loaded on to TG19318 affinity columns prepared by immobilizing the ligand on the commercially available support Emphaze. In a single chromatographic step TG19318 affinity columns led to an efficient capture of IgY directly from crude samples, and with a purity degree higher than 90%, as determined by densitometric scanning of SDS-PAGE analysis of bound fractions, and with full recovery of antibody activity, as determined by ELISA assay. Higher recovery and purity of IgY was obtained by using loading buffers at pH close to 6.5. Column capacity, determined by applying 4x excess IgY to 1 ml bed volume column, and eluting the retained immunoglobulins, was close to 65 mg of IgY per ml of resin. Chemical and chromatographic stability of TG19318/Emphaze was tested before and after various treatments. The derivatized matrix was found to be very stable, in terms of ligand leakage and maintenance of IgY binding capacity, under conditions of normal column usage, cleaning and storage.

Animals↗
PubMed1990

Immobilization of alpha-human atrial natriuretic peptide on insoluble supports and affinity purification of specific antibodies from a polyclonal goat anti-alpha-human atrial natriuretic peptide serum.

alpha-Human atrial natriuretic peptide (alpha-hANP) was covalently coupled via single attachment onto two different insoluble matrices. Controlled-pore glass-alpha-hANP matrices were well suited for the purification of monospecific antibodies, whereas Enzacryl AA-alpha-hANP did not withstand the inevitable chemical and physical stresses during affinity purification.

Animals↗
PubMed1993

The use of phenyl-Sepharose for the affinity purification of proteinases.

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media. Purification factors in the range of 2.9-60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts.

Bacteria↗
PubMed1997

Affinity purification of RNA: sequence-specific capture by nonionic morpholino probes.

Nucleic acid isolation for amplification-based diagnostics requires techniques that do not co-purify inhibitors of DNA polymerases. Also, other requirements for an ideal sample preparation technology include ease of use, capability for automation, high recovery and the use of nontoxic reagents. Affinity purification techniques provide high purification factor with minimal sample processing. Hybridization is the affinity interaction specific to nucleic acids and thus provides a uniquely advantageous method for purifying DNA or RNA for subsequent manipulation. Nonionic (morpholino) probes (Neu-Probes, AntiVirals, Corvallis, OR, USA) have several unique hybridization properties, including resistance to nucleases and the ability to hybridize independently of salt concentration. Therefore, such probes provide advantages over DNA probes for sample preparation by hybridization capture. Three formats for hybridization-based purification of human immunodeficiency virus (HIV) RNA were evaluated using RNA transcripts spiked into crude lysates of normal human plasma. Indirect capture used streptavidin-coated microparticles to capture hybrids of biotinylated capture probes and HIV RNA. Direct capture used particles precoated with probes. In addition, a novel method for acceleration of sequence-specific hybridization was developed and shown to give consistently high recoveries.

DNA-Directed DNA Polymerase↗
PubMed1990

Affinity purification of HIV-1 and HIV-2 proteases from recombinant E. coli strains using pepstatin-agarose.

A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E. coli constructs that were developed for the expression of these enzymes. HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates. A Mono S ionic exchange step following pepstatin-agarose chromatography was sufficient to bring the HIV-1 protease to homogeneity. Purification of either enzyme can be completed in several days yielding homogeneous preparations suitable for crystallization and other physical characterization.

Chromatography, Affinity↗
PubMed1993

Affinity purification of beta-amylases originating from plant using cyclomaltohexaose-immobilized Sepharose 6B in the presence of ammonium sulfate.

This paper reports a novel method of affinity purification of soybean and barley beta-amylase on cyclomaltohexaose-immobilized Sepharose. Until now, it has been shown that sweet potato beta-amylase can be purified using the above absorbent but beta-amylases from soybean and barley seeds cannot. We found that soybean and barley beta-amylase becomes adsorbed specifically on the above absorbent if it is in solution with 1 to 2 M ammonium sulfate, and the adsorbed enzyme can be easily eluted with a buffer containing no ammonium sulfate. Employing this procedure, soybean beta-amylase was demonstrated to be purified about 10-fold to homogeneity as judged from analysis of both a sodium dodecyl sulfate-gel electrophoresis and the specific activity, using a crude enzyme preparation (sp act 95 U/mg) as a starting material. The specific activity of this highly purified enzyme (950 U/mg) was almost the same as that of crystallized soybean beta-amylase at 37 degrees C.

Ammonium Sulfate↗