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Results for “Affinity purification”

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At least 127 records · Page 7Linked to original sources
PubMed2001

Protein adsorption on histidyl-aminohexyl-Sepharose 4B. II. Application to the negative one-step affinity purification of human beta2-microglobulin and immunoglobulin G.

The adsorption of two human proteins, beta2-microglobulin and Immunoglobulin G, from uremic patient's blood ultrafiltrate and plasma, respectively, was investigated on the histidyl-aminohexyl-Sepharose 4B adsorbent. Both target proteins could be adsorbed on the gel through a low affinity for immobilized histidine ligand. However, a fine adjustment of the operating conditions (ionic strength, buffer, pH) prevented their adsorption and thus allowed their "negative affinity" purification (purity estimated by silver nitrate SDS-PAGE) by the removal of the contaminating proteins. This simple and efficient method provides purification under gentle chromatographic conditions and a further characterization of both molecules.

Adsorption↗
PubMed2002

Monoclonal antibody affinity purification of a 78 kDa membrane protein of Leishmania donovani of Indian origin and its role in host-parasite interaction.

Monoclonal antibodies were raised against pathogenic promastigotes of Leishmania donovani of Indian origin. Among these, one was used for immuno-affinity purification of a 78 kDa membrane protein present in both the amastigote and promastigote forms of the parasite. Results of immunoblot experiments with the anti-78 kDa antibody revealed that the protein was present only in parasites belonging to the L. donovani complex. The expression of the protein was observed to be the same during different phases of growth of the promastigotes. Therefore, the 78 kDa protein is neither stage-specific nor differentially regulated. Surface iodination and subcellular fractionation of the promastigotes indicated that the protein was localized on the cell surface. The 78 kDa protein was found to inhibit the binding of promastigotes to macrophages significantly, suggesting that it may play a role in the process of infection. Thus, here we report the purification of a surface protein of L. donovani of Indian origin, which may play an important role in the process of infection.

Animals↗
PubMed2005

Fluorous affinity purification of oligonucleotides.

[structure: see text] Nucleoside phosphoramidites bearing a fluorous dimethoxytrityl (FDMT) group were used to synthesize fluorous-tagged oligonucleotides, which were subjected to solid-phase extraction using a pH-stable fluorinated adsorbent. On-column detritylation afforded the purified oligonucleotides. The fluorous affinity purification method offers one-pass loading without ammonia removal, high selectivity for the removal of failure sequences, high recoveries (typically 70-100%), and the ability to purify long oligonucleotides (e.g., 50-100-mers).

Affinity Labels↗
PubMed2006

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.

To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.

Adenosine Triphosphatases↗
PubMed1997

Affinity purification and kinetic analysis of mutant forms of yeast NAD+-specific isocitrate dehydrogenase.

Polyhistidine tags were added to the carboxyl termini of the two homologous subunits of yeast NAD+-specific isocitrate dehydrogenase (IDH). The tag in either the IDH1 or IDH2 subunit permits one-step affinity purification from yeast cellular extracts of catalytically active and allosterically responsive holoenzyme. This expression system was used to investigate subunit-specific contributions of residues with putative functions in adenine nucleotide binding. The primary effect of simultaneous replacement of the adjacent Asp-279 and Ile-280 residues in IDH1 with alanines is a dramatic loss of activation by AMP. In contrast, alanine replacement of the homologous Asp-286 and Ile-287 residues in IDH2 does not alter the allosteric response to AMP, but produces a 160-fold reduction in Vmax due to a 70-fold increase in the S0.5 value for NAD+. These results suggest that the targeted aspartate/isoleucine residues may contribute to regulator binding in IDH1 and to cofactor binding in IDH2, i.e. that these homologous residues are located in regions that have evolved for binding the adenine nucleotide components of different ligands. In other mutant enzymes, an alanine replacement of Asp-191 in IDH1 eliminates measurable catalytic activity, and a similar substitution of the homologous Asp-197 in IDH2 produces pleiotropic catalytic effects. A model is presented for the primary function of IDH2 in catalysis and of IDH1 in regulation, with crucial roles for these single aspartate residues in the communication and functional interdependence of the two subunits.

Amino Acid Sequence↗
PubMed2003

A new epitope tag from hepatitis B virus preS1 for immunodetection, localization and affinity purification of recombinant proteins.

Previously, a murine monoclonal antibody (mAb) KR127 (IgG2a/kappa) that binds specifically to the preS1 of hepatitis B virus (HBV) was generated and the fine epitope was mapped to amino acids (aa) 37-45 (NSNNPDWDF). In this current study, the epitope in combination with KR127 was tested for protein tagging. Initially, to evaluate the importance of each residue of the KR127 epitope in antibody binding, alanine substitution mutants of the epitope were constructed and characterized for KR127 binding by immunoblot analysis and competition ELISA. The results showed that substitution of Ser(38) by alanine (S38A) increased the affinity to KR127. The mutated epitope (NANNPDWDF), designated S1 tag, was fused to the amino (N)- or carboxyl (C)-terminus of three human recombinant proteins, soluble B lymphocyte stimulator (sBLyS), the N-terminal domain of thrombopoietin (nTPO), and a mitochondrial ribosomal protein (CGI-113) for expression in mammalian cells, while it was fused to the N- or C-terminus of two proteins, a single-chain antibody fragment (ScFv) and the carboxyl-terminal domain (PAc) of the protective antigen of Bacillus anthracis for expression in Escherichia coli. The immunodetection, immunoprecipitation, and affinity purification of the expressed S1-tagged proteins by KR127 were successfully demonstrated. In addition, a KR127 mutant (AP2) with higher affinity, K(d) (0.9 nM), for the S1 tag compared to that (20 nM) of KR127 was obtained by mutational analysis of the heavy chain CDR3 (HCDR3) of KR127. The AP2 antibody was 4-fold more sensitive in detecting the S1-tagged protein than KR127. The S1 tag-KR127 or AP2 combination could be universally used for monitoring protein expression, localizing proteins, and protein purification, as well as studying protein interactions.

Animals↗
PubMed2005

Two-stage affinity purification for inducibly phosphorylated membrane proteins.

Characterisation of tyrosine phosphorylations induced in immune cells in response to inflammatory stimuli may help elucidate the molecular bases of the diversity of immune responses. We have used anti-phosphotyrosine antibodies in combination with cell surface biotinylation in a two-step affinity purification procedure to recover pervanadate-induced tyrosine phosphorylated proteins from sub-cellular compartments, including the cell surface, of murine T cells and macrophages prior to separation by solution-phase isoelectric focussing and one-dimensional gel electrophoresis and identification by tandem mass spectrometry.

Animals↗
PubMed1998

Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein.

The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells.

Chromatography, Affinity↗
PubMed1988

Antibody-selectable filamentous fd phage vectors: affinity purification of target genes.

Foreign DNA fragments can be inserted into a minor coat protein gene of filamentous phage, creating a fusion protein that is incorporated into the virion; we call these particles "fusion phage". The foreign amino acids are displayed on the surface, allowing fusion phage bearing antigenic determinants from a target gene to be purified in infectious form by affinity to antibody directed against the gene product. Here we introduce fusion-phage vectors that accept foreign DNA inserts with little effect on phage function; and describe affinity purification of virions bearing a target determinant from a 10(8)-fold excess of phage not bearing the determinant, using minute amounts of antibody. These "antibody-selectable" vectors are a promising alternative to conventional expression systems for using antibodies to clone genes, though the ability to isolate rare clones from actual libraries remains to be demonstrated.

Antibodies↗
PubMed2005

An improved expression plasmid for affinity purification of Staphylococcus aureus gyrase A subunit.

Of the bacterial topoisomerases, the gyrase A subunit (GyrA) of Staphylococcus aureus is particularly difficult to purify because of its tendency to form inclusion bodies. Previous attempts at purification yielded low concentrations of protein with reduced specific activity. To overcome this problem, we modified the commercially available plasmid expression vector, pBAD/Thio-TOPO, via the addition of DNA sequences encoding a hexahistidine tag upstream and a cleavage site for tobacco etch virus protease downstream of the gene encoding thioredoxin. The resulting expression system consisting of the modified plasmid, pSAGA7, and the recommended host strain, Escherichia coli TOP 10, facilitated high level expression of soluble GyrA and its affinity purification to over 95% homogeneity. Purified GyrA had high biological activity as evidenced by a specific activity of 4.3x10(5)U/mg. The pSAGA7/TOP10 expression system also facilitated the expression and purification of a subunit of S. aureus topoisomerase IV, ParE, and a recently discovered protein unrelated to topoisomerases, QnrB, two "hard to purify" proteins. We conclude that pSAGA7 might be useful for high-level soluble expression and purification of diverse microbial proteins.

Chromatography, Affinity↗
PubMed2005

Affinity purification of Ypt6 effectors and identification of TMF/ARA160 as a Rab6 interactor.

Rab/Ypt GTPases are key regulators of intracellular traffic in eukaryotic cells. One important function of Rab/Ypts is the nucleotide-dependent recruitment of downstream effector molecules onto the membrane of organelles. In budding yeast Ypt6 is required for recycling of membrane proteins from endosomes back to the Golgi. A biochemical approach based on the affinity purification of Ypt6:GTP-interacting proteins from yeast cytosol led to the identification of two conserved Ypt6 effectors, the tetrameric VFT complex and Sgm1. The mammalian homolog of Sgm1, TMF/ARA160, contains a short conserved coiled-coil motif that is sufficient for the binding to the three mammalian orthologs of Ypt6, Rab6A, Rab6A', and Rab6B.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Affinity purification of antigen-specific serum immunoglobulin from the European eel (Anguilla anguilla).

Immunization of specimens of the European eel with a hapten-carrier conjugate resulted in a significant rise in anti-hapten titres. Antigen-specific immunoglobulin was purified on a matrix onto which the hapten-carrier conjugate had been immobilized. Rabbit antisera raised against the product of the affinity purification recognized two molecular moieties. The first was eel immunoglobulin as inferred by the polypeptide composition, i.e. disulphide-linked heavy and light chains of 72 kDa and 25 kDa respectively. The second was a 110-kDa protein. A 800-kDa and a 400-kDa molecular form of eel immunoglobulin were disclosed by a combination of gel filtration and immunoelectrophoretic detection. The latter was the more abundant serum form. The 110-kDa protein was found non-covalently associated with the 400-kDa immunoglobulin. Both molecular forms of immunoglobulin from immune sera exhibited antigenic specificity and reactivity.

Anguilla↗
PubMed2004

Tandem affinity purification and identification of protein complex components.

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.

Multiprotein Complexes↗
PubMed2001

Cyclic AMP affinity purification and ESI-QTOF MS-MS identification of cytosolic glyceraldehyde 3-phosphate dehydrogenase and two nucleoside diphosphate kinase isoforms from tobacco BY-2 cells.

The soluble protein fraction of tobacco bright yellow 2 cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-binding activity, detected with both a conventional binding assay and a surface plasmon resonance biosensor. A cAMP-agarose-based affinity purification procedure yielded three proteins which were identified by mass spectrometry as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and two nucleoside diphosphate kinases (NDPKs). This is the first report describing an interaction between cAMP and these proteins in higher plants. Our findings are discussed in view of the reported role of the interaction of cAMP with GAPDH and NDPK in animals and yeast. In addition, we provide a rapid method to isolate both proteins from higher plants.

Animals↗
PubMed1986

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5.

Adenylyl Cyclases↗
PubMed1991

Use of a monoclonal antibody (APE-1) directed against the conserved GTPEYLAPE motif present in protein kinases as an affinity purification reagent.

cDNA sequence studies have predicted the existence of protein kinases that have not been characterized at the protein level. This laboratory has previously shown that polyclonal rabbit antibodies directed against a highly conserved sequence motif, GTPEYLAPE, present in the catalytic domain of many protein kinases will react during immunoblots with several protein kinases containing the same or homologous motifs. In this report I describe the development of a mouse monoclonal antibody, designated APE-1, that reacts with the GTPEYLAPE motif. In addition, the use of this monoclonal antibody in the affinity purification of kinase activities from rabbit reticulocyte lysate is described. This approach may provide a general method for the purification of previously uncharacterized protein kinases that share regions which are homologous to the GTPEYLAPE motif. Refinements in this method may permit the rapid purification of nonabundant and/or rapidly inactivated protein kinases that have not been isolated using other approaches.

Amino Acid Sequence↗
PubMedDate not supplied

[Obtaining of ScFv-CBD fusion protein and its application for affinity purification of recombinant human interferon alpha2b].

The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.

Antibodies, Monoclonal↗