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At least 109 records · Page 6Linked to original sources
PubMed1989

Immuno-affinity purification of specific antibodies against human gastrin releasing peptide (h-GRP) by the h-GRP(1-8)-linked polydimethylacrylamide resin.

A new method for immuno-affinity purification of specific antibodies against human gastrin releasing peptide(h-GRP) was developed. The antiserum GP(No. 6201) elicited by h-GRP-BSA conjugate was heterogeneous and reacted not only with h-GRP and its fragments but also partially with other structurally related peptides, such as other GRPs (porcine, canine, and chicken), bombesin, and neuromedin-C. To obtain specific antibodies against human GRP, antiserum GP was purified by column chromatography on the amino-terminal octapeptide h-GRP(1-8)-linked polydimethylacrylamide resin. The antibody thus obtained was highly specific to amino-terminal sequence of h-GRP and hardly reacted with other GRPs (porcine, canine and chicken), bombesin, and even carboxy-terminal h-GRP fragments in ELISA.

Acrylamides↗
PubMed1998

A convenient method for affinity purification of maltose binding protein fusions.

Maltose binding protein of Escherichia coli is frequently employed as a fusion partner for the biosynthesis of polypeptides and proteins, largely because of the advantage provided by affinity purification of such a fusion. A mixture of cellulose and starch is shown to be a simple and effective alternative to the use of other, more complicated media which are often used for affinity chromatography of maltose binding protein fusions.

ATP-Binding Cassette Transporters↗
PubMed1991

Affinity purification of antibodies from sera using polyvinylidenedifluoride (PVDF) membranes as coupling matrices for antigens presented by autoantibodies to triosephosphate isomerase.

Polyvinylidenedifluoride (PVDF) membranes covered with antigen can be used for affinity purification of antibodies from sera. These antibodies serve as an appropriate substitute for monoclonal antibodies when performing cytological and analytical experiments with the respective antigen. The usefulness of this method is demonstrated with the purification of IgM autoantibodies produced against human triosephosphate isomerase in acute Epstein-Barr virus infection. Compared with the original serum, the specific activity of the purified antibodies was up to 1400-fold higher, and the recovery of the anti-triosephosphate isomerase activity between 25 and 33%.

Animals↗
PubMed2000

Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps.

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.

Chromatography, Affinity↗
PubMed1999

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.

Animals↗
PubMed1988

Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels.

Improved affinity chromatography procedures for the purification of cytosolic epoxide hydrolase are described. An earlier affinity purification method using immobilized 7-methoxycitronellyl thiol (MCT) sporadically produced final enzyme preparations containing major impurities. To eliminate these impurities, we tested alternate ligands, spacer arms, and ligand concentrations. A series of alkyl and aryl thiols coupled to epoxy-activated Sepharose were found to exhibit markedly different binding characteristics as compared with commercially available alkyl- and aryl-Sepharose gels. Using one of these new matrices, benzylthio-Sepharose, cytosolic epoxide hydrolase from mouse liver was purified over 100-fold, appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was obtained with 60-90% recovery of enzyme activity. The impurities previously observed with the MCT-Sepharose procedure were reduced or eliminated by using an MCT ligand concentration of 5 microequivalents per gram or less. MCT-Sepharose and benzylthio-Sepharose provide rapid and convenient one-step procedures for obtaining purified cytosolic epoxide hydrolase from numerous species and tissues.

Adult↗
PubMed1996

Affinity purification of phospholipase A2 on immobilized artificial membranes containing and lacking the glycerol backbone.

Immobilized artificial membranes (IAMs) are chromatography surfaces containing monolayers of phospholipid ligands. etherIAM.PCC10/C3 contains the glycerol backbone whereas delta GIAM.PCC10/C3 lacks the glycerol backbone. Affinity purification of PLA2 on these IAM surfaces demonstrated that the surface structural differences were not important for phospholipase A2 (PLA2) binding. This suggests that the chromatographically important binding event involves the PLA2 surface and the monolayer of polar choline headgroups on the IAM surface. After sample loading, short-chain alkylsulfonates were used as low eluotropic strength detergents to remove contaminating proteins, and PLA2 were eluted with CH3CN (30%). Octyllysophosphatidylcholine (0.5%) can replace CH3CN to elute PLA2 from IAM surfaces. The PLA2 purity after IAM chromatography depends on the protein loading; analytical-scale loadings (0.8 mg protein/g IAM) resulted in a PLA2 purity of ca. 70% based on densitometric scans of proteins in polyacrylamide gels after electrophoresis. Preparative loadings of 3.21 mg protein/g IAM resulted in 48% PLA2 purity. Purification of PLA2 to electrophoretic homogeneity was achieved using an IAM column followed by a strong anion-exchange column. These results suggests that IAMs may be used to develop purification methods for PLA2 enzymes obtained from diverse biological specimens.

Chromatography, Affinity↗
PubMed2005

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification.

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

Bornaviridae↗
PubMed1991

Bovine N-acetyl-beta-D-glucosaminidase: affinity purification and characterization of its active site with nitrogen containing analogs of N-acetylglucosamine.

Two N-acetylglucosaminidases were isolated from bovine kidney with a three step procedure featuring affinity purification on 2-acetamido-1,2,5-trideoxy-1,5-iminoglucitol (2-acetamido-1,2-dideoxynojirimycin, II). The major isoenzyme, Hex A, is an alpha, beta hetero-dimer (57 and 52 kDa) with isoelectric points from pH 5.3 to 6.6 and comprised about 80% of the total activity. Its kinetic properties with respect to discrimination between N-acetylglucosaminide, N-acetylgalactosaminide and the corresponding 6-sulfate ester were similar to human hexosaminidase A. The minor isoenzyme, Hex B, a homodimer, isoelectric points 7.0 to 7.4, was similar to Hex A but was without detectable activity with methylumbelliferyl-N-acetyl-beta-glucosaminide-6-sulfate. Inhibition studies with Hex A were carried out with 2-acetamido-2,5-dideoxy-1,5-imino-D-glucopyranose (2-acetamido-2-deoxynojirimycin, (1), the corresponding 1,5-lactam (III), with II and its N,N-dimethyl derivative, and with 2-acetamido-2-deoxy-D-glucono-1,5-lactone (IV). In comparison with N-acetylglucosamine (Ki 1.9 mM) Hex A was inhibited 10(6)-fold better by I, 2600-fold better by II, 2900-fold better by III, and 55,000-fold better by IV. A slow approach to the inhibition equilibrium was observed with I and IV. For IV and Hex A it is the first example of a slow inhibition of a glycoside hydrolase by the corresponding glycono-1,5-lactone. The pH-dependence of Ki for the permanently cationic N,N-dimethyl II (15.4 microM (pH 3.5) to 0.47 microM (pH 7.0)) indicated that formation of the enzyme inhibitor complex is governed by deprotonation of a group with pKa 5.0. The results are discussed with respect to structural features and water accessibility of the active site.

Acetylglucosamine↗
PubMed1986

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

1-Deoxynojirimycin↗
PubMed1987

Glycoproteins of human parainfluenza virus type 3: affinity purification, antigenic characterization and reconstitution into lipid vesicles.

Monoclonal antibodies to the envelope glycoproteins, HN and F, of human parainfluenza virus type 3 were coupled to a Sepharose 4B matrix and used for affinity purification of the viral glycoproteins. The purity of the glycoproteins was demonstrated by SDS-PAGE followed by fluorography or silver staining. The antigenicity of the glycoproteins was determined by immunization of rabbits; polyclonal rabbit antisera demonstrated inhibition of functional activities of the virus glycoproteins. The F glycoprotein, when reconstituted into lipid vesicles, showed distinct spike-like projections similar to those of intact virions.

Animals↗
PubMed2006

A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4.

With the completion of numerous genome projects, new high-throughput methods are required to ascribe gene function and interactions. A method proven successful in yeast for protein interaction studies is tandem affinity purification (TAP) of native protein complexes followed by MS. Here, we show that TAP, using Protein A and CBP tags, is not generally suitable for the purification and identification of proteins from tissues. A head-to-head comparison of tags shows that two others, FLAG and His, provide protein yields from Drosophila tissues that are an order of magnitude higher than Protein A and CBP. FLAG-His purification worked sufficiently well so that two cofactors of the Drosophila nuclear receptor protein dHNF4 could be purified from whole animals. These proteins, Hsc70 and Hsp83, are important chaperones and cofactors of other nuclear receptor proteins. However, this is the first time that they have been shown to interact with a non-steroid binding nuclear receptor. We show that the two proteins increase the ability of dHNF4 to bind DNA in vitro and to function in vivo. The tags and approaches developed here will help facilitate the routine purification of proteins from complex cells, tissues and whole organisms.

Affinity Labels↗
PubMed1993

Affinity purification of antibodies specific for Asn-linked glycans containing alpha 1-->3 fucose or beta 1-->2 xylose.

Antisera raised against the plant glycoproteins beta-fructosidase and horseradish peroxidase can be fractionated on an affinity column of honeybee venom phospholipase A2 to produce serum fractions that are specific for either the alpha 1-->3 fucose or beta 1-->2 xylose epitopes commonly found on the Asn-linked glycans of plant glycoproteins. This affinity purification strategy relies on the absence of beta 1-->2 xylose from the glycan of the venom protein. Such antibody preparations can be used for the detection of these sugar epitopes on glycoproteins.

Animals↗
PubMed2003

Identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system.

Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4. We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture. We have identified HSP70 as a specific interacting protein of SMAD3. We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.

Amino Acid Sequence↗
PubMed1989

Affinity purification of endothia protease with a novel renin inhibitor SQ 32,970.

A novel tripeptidic renin inhibitor is described, SQ 32,970, that will potently inhibit endothia protease. This inhibitor can be coupled to Sepharose and will allow the affinity-purification of endothia protease in one step to greater than 95% purity as measured by SDS PAGE. The purified endothia protease cleaves the Lys-Pro-Ala-Glu-Phe-Nph-Arg-Leu substrate at the Phe-Nph bond with a Kcat/Km of 7445 (s-1 mM-1) at pH 3.1 and 4057 (s-1 mM-1) at pH 6.0. Affinity purified endothia protease can be crystallized in the pH range in which it is enzymatically active and can be inhibited by renin inhibitors.

Ascomycota↗
PubMed1998

Affinity purification of recombinant cholera toxin B subunit oligomer expressed in Bacillus brevis for potential human use as a mucosal adjuvant.

For use as a mucosal adjuvant for human vaccines, a simple method has been developed for the affinity purification of recombinant cholera toxin B subunit which had been expressed in a safe host, Bacillus brevis. Recombinant cholera toxin B subunit, adsorbed quantitatively to a D-galactose-agarose column, was eluted with an 0.1-0.4 M D-galactose gradient with a yield of > 90%. The cholera toxin B subunit preparation was similar to the native cholera toxin B subunit with respect to GM1 binding ability, remarkable stability of the pentamer, and the dissociation-reassociation property by shifting pHs. Cross-linking experiments with glutaraldehyde demonstrated that the pentameric form was predominant; tetrameric, trimeric, dimeric and monomeric forms were detected to a lesser extent, and additionally 10- and 15-mers were observed depending on the concentration of the cholera toxin B subunit.

Adjuvants, Immunologic↗
PubMed2006

Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragments.

Endotoxins are frequent contaminants of recombinant proteins produced in Escherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg(-1) is, however, one of the most problematic steps during protein purification from E. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced in E. coli using a non-ionic detergent. Endotoxin content was as low as 5 to 9 EU mg(-1) with a recovery of antibody fragments of over 90%. Non-ionic detergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinant antibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.

Antibodies, Monoclonal↗
PubMed1997

Affinity purification of a human RNA polymerase II complex using monoclonal antibodies against transcription factor IIF.

Five different monoclonal antibodies that immunoreact with RAP74, the large subunit of general transcription factor (TF) IIF, were produced and characterized. Using one of these antibodies, an affinity purification procedure was devised to isolate a human RNA polymerase II complex. This procedure is fast, simple, and reproducible and does not require extensive purification. The RNA polymerase II complex isolated using this procedure contains SRB (suppressor of RNA polymerase B) polypeptides, transcription factors IIE and IIF, limiting amounts of TFIIH, and the TATA-binding protein, but was devoid of TFIIB.

Amino Acid Sequence↗