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PubMedDate not supplied

Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis.

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.

Chromatography, Affinity↗
PubMed2003

Optimisation of expression and immobilized metal ion affinity chromatographic purification of recombinant (His)6-tagged cytochrome P450 hydroperoxide lyase in Escherichia coli.

Fatty acid hydroperoxide lyase (HPL) is a cytochrome P450 acting on fatty acid's hydroperoxides in many plants. The optimisation of the expression of recombinant (His)(6)-tagged HPL in Escherichia coli is described: the highest HPL production yield were obtained with TB medium supplemented with 2.5 mM delta-aminolevulinic acid and 0.5 mM IPTG. For the first time, the time course expression of a plant P450 in a bench-scale fermentor is detailed and the amount of recombinant HPL production is 16.3 mg/l. The UV-Visible spectrum of the recombinant (His)(6)-tagged HPL have been recorded after a Ni(2+)-based affinity chromatography (IMAC).

Aldehyde-Lyases↗
PubMed2005

Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans.

Nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory neurotransmission in neurons and muscles. To identify nAChR accessory proteins, which may regulate their expression or function, we performed tandem affinity purification of the levamisole-sensitive nAChR from Caenorhabditis elegans, mass spectrometry of associated components, and RNAi-based screening for effects on in vivo nicotine sensitivity. Among the proteins identified was the calcineurin A subunit TAX-6, which appeared to function as a negative regulator of nAChR activity. We also identified five proteins not previously linked to nAChR function, whose inactivation conferred nicotine resistance, implicating them as positive regulators of nAChR activity. Of these, the copine NRA-1 colocalized with the levamisole receptor at neuronal and muscle plasma membranes, and, when mutated, caused reduced synaptic nAChR expression. Loss of SOC-1, which acts in receptor tyrosine kinase (RTK) signaling, also reduced synaptic levamisole receptor levels, as did mutations in the fibroblast growth factor receptor EGL-15, and another RTK, CAM-1. Thus, tandem affinity purification is a viable approach to identify novel proteins regulating neurotransmitter receptor activity or expression in model systems like C. elegans.

Affinity Labels↗
PubMed1990

Application of receptor-affinity chromatography to bioaffinity purification.

Receptor-affinity chromatography based upon the receptor-ligand interactions has been utilized for the purification of recombinant human interleukin-2 (rIL-2) from microbial and mammalian sources. The receptor-affinity purification process of rIL-2 is used as a model system to demonstrate the utility of this approach for the purification of recombinant proteins. The receptor-affinity purified biomolecule is shown to be biochemically and biologically more homogeneous than the immunoaffinity purified material.

Amino Acids↗
PubMed2006

Expression of the Blomia tropicalis paramyosin Blo t 11 and its immunodominant peptide in insect cells.

Blo t 11, a dust-mite (Blomia tropicalis) paramyosin, is an allergen with significant IgE reactivity that has potential as a diagnostic/therapeutic reagent for house-dust-mite allergy. The present study describes the successful expression of Blo t 11 and its immunodominant peptide fD in insect cells using a baculovirus expression system. The Blo t 11 and fD genes were cloned into the pMelBacA vector and the resulting vectors were co-transfected into Sf9 insect cells with Bac-N-Blue DNA. Plaque assay was used to select for recombinant virus that was then used to infect High Five insect cells for protein expression. Secreted proteins were harvested by immuno-affinity purification using monoclonal antibodies to Blo t 11. Purified proteins were analysed by immunoblotting, N-terminal sequencing and ELISA. Immunoblot analyses revealed the full-length Blo t 11 cDNA expressed as a minor protein band of approx. 200 kDa and two major protein bands of approx. 60 and 70 kDa. Clones expressing fD cDNA fragment produced a protein of approx. 30 kDa that was confirmed to be fD by N-terminal sequencing. Approx. 4-7.5 mg/l of fD and 1 mg/l of Blo t 11 were obtained by affinity purification. ELISA results showed that human IgE reactivity to these recombinant allergens was lower as compared with that of the native Blo t 11, suggesting that these baculovirus-expressed allergens exhibiting reduced allergenicity could be useful for the development of immunotherapeutic reagents for mite allergy.

Allergens↗
PubMed2006

Analysis of the ribosomal protein S19 interactome.

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Glutathione Transferase↗
PubMed1992

Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms.

Nitric oxide (NO) is generated from L-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Anti-sera A and B reacted with a approximately 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of M(r) between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the approximately 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed2003

One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1.

The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis. However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature. Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria. The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L). The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.

Chromatography, Affinity↗
PubMed2006

RANKL treatment releases the negative regulation of the poly(ADP-ribose) polymerase-1 on Tcirg1 gene expression during osteoclastogenesis.

UNLABELLED: The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. INTRODUCTION: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for approximately 60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. MATERIALS AND METHODS: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. RESULTS: The -3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to -1589 retained this response to RANKL. A deletion up to -1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the -1589/-1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. CONCLUSIONS: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function.

Amino Acid Sequence↗
PubMed2005

Phosphorylation of glutathione-S-transferase by protein kinase C-alpha implications for affinity-tag purification.

Expression and purification of proteins as fusions with glutathione S-transferase (GST) is a standard and widely employed system. In more than 2,500 published studies, GST has been used to facilitate the purification of recombinant proteins, assess protein-protein interactions, and establish protein function. In this report, we provide evidence that GST can be phosphorylated in vitro by protein kinase C-alpha (PKC-alpha) at Ser-93. Therefore, since GST itself may be a target for a number of catalytic enzymes, failure to remove the GST tag from the recombinant protein may lead to inaccurate conclusions.

Animals↗
PubMed1988

Purification of Forssman and human blood group A glycolipids by affinity chromatography on immobilized Helix pomatia lectin.

A method for the affinity purification of intact glycolipids having nonreducing terminal alpha 1-3 linked N-acetylgalatosamine residues has been developed. This technique relies on the retention of the carbohydrate-binding specificity of immobilized Helix pomatia lectin in aqueous solutions of tetrahydrofuran. Both Forssman glycolipid and a mouse blood group A-active hexaosylceramide were bound by columns of the lectin equilibrated in a solvent containing 95% tetrahydrofuran and 5% water. After application of a step gradient of increasing water content up to 50%, the specifically bound glycolipids were eluted in solvent containing N-acetylgalactosamine. The Forssman and A-active glycolipids were similarly purified in a single chromatographic step from total lipid extracts of sheep and human type A erythrocyte stroma, respectively. Nonspecifically bound lipids and glycolipids were eluted from this column by simply increasing the water content of the eluting buffer. The extension of this method to other carbohydrate-binding proteins including lectins and monoclonal antibodies may provide a rapid purification of glycolipids based on their carbohydrate structures.

Animals↗
PubMed1993

Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.

Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells.

Adenovirus E1B Proteins↗
PubMed2004

Overexpression and affinity chromatography purification of the Type III restriction endonuclease EcoP15I for use in transcriptome analysis.

The Type III restriction endonuclease EcoP15I is a multifunctional hetero-oligomeric enzyme that recognizes the non-symmetric DNA sequence 5'-CAGCAG. For efficient cleavage, EcoP15I needs the interaction with two copies of the recognition sequence that have to be inversely oriented in the DNA double strand. The enzyme cuts the upper DNA strand 25-26 bp and the lower DNA strand 27-28 bp, respectively, downstream of the recognition sequence-a distinct feature that makes the enzyme particularly valuable for gene expression profiling methods relying on the SAGE procedure (Matsumura et al., PNAS 100, 15718, 2003). Because the broader use of this transcriptome analysis method requires the availability of larger amounts of restriction endonuclease EcoP15I and the enzyme is not commercially available, we have cloned the genes coding for the EcoP15I restriction endonuclease into pQE-16 plasmid vector that provides the enzyme with a C-terminal 6xHis-tag. After Ni-NTA affinity chromatography and ion exchange chromatography on heparin sepharose, we obtained 5mg homogeneous EcoP15I per gram cell pellet within 1-2 day(s). Moreover, the C-terminally 6xHis-tagged EcoP15I restriction endonuclease shows comparable enzymatic activity as the untagged enzyme.

Amino Acid Sequence↗
PubMed1997

Purification of amylases and other enzymes by a forced-affinity chromatography method.

An affinity matrix of soluble starch gel was prepared by cross-linking catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA) indicated that the amount of enzyme bound to the starch gel column increased with increases in the ammonium sulfate (AmS) concentration in the equilibrating buffer. TAA had an affinity for the gels with a starch structure, and desorbed from the column with the buffer containing no AmS. Bound TAA was also eluted with starch and cyclodextrin solution. The AmS stimulative effect was partially replaced by polyethylene glycol and surfactants. Besides TAA, various, other amylases bound satisfactorily to the starch gel. Moreover, affinity purifications of dextranase, cellulase, and pectinase were done by gels with dextran, cellulose, and pectin structures, respectively. By the aid of forced effects of AmS, various carbohydrases could be purified by the affinity gels of polysaccharide linked by epichlorohydrin.

Adsorption↗
PubMed1990

Two groups of bullous pemphigoid antigens are identified by affinity-purified antibodies.

The bullous pemphigoid antigen was originally described as a 240-kD protein extracted from human epidermis, but a subsequent report has described patients' sera which react with epidermal proteins of molecular masses 240, 200, 180, 97, and 77 kD. We have evaluated the heterogeneity of the pemphigoid antigens identified by the sera of 10 patients with clinically typical bullous pemphigoid. We used indirect immunofluorescence and Western immunoblots of epidermal extracts prepared from epidermis separated by either 1 M salt or 20 mM EDTA to characterize the reactivity of both crude sera and affinity-purified antibodies. Affinity purification of antibodies was performed with either normal human epidermis or protein bands blotted onto nitrocellulose as immunoabsorbents. The anti-basement membrane antibody titers determined by indirect immunofluorescence on the saline- and EDTA-separated epidermis were identical. Despite this, Western blots of extracts prepared from EDTA-separated epidermis demonstrated greater amounts of the 240-kD antigen than saline split skin. Multiple antigens were recognized in epidermal extracts on Western blots by most crude BP sera, including bands at 240, 200, 160, and 100 kD. Different sera reacted with these antigens with a markedly different intensity, falling into two major groups, those bearing antibodies to the 240-200-kD antigens and those with antibodies to the 160-100-kD components. When epidermis was used as a substrate for affinity purification of bullous pemphigoid antibodies, the eluted antibodies reacted with multiple bands on Western blots, demonstrating the reactivity of anti-basement membrane zone antibodies with multiple proteins. Antibodies eluted from several individual bands of immunoblots were found to react with the basement membrane on indirect immunofluorescence. When these nitrocellulose-purified antibodies were reapplied to Western blots, they cross-reacted within two groups, the 240-200 kD antigens and the 160-100 kD antigens. We conclude that pemphigoid antigens are best demonstrated when EDTA-split skin is used for extraction and that different pemphigoid sera may contain antibodies to two separate groups of basement membrane zone antigens.

Antibodies↗
PubMed1980

Affinity-chromatographic purification of human alpha 2-antiplasmin.

A new simple and efficient purification method for alpha 2-antiplasmin is described that is based on the interaction between alpha 2-antiplasmin and a fragment from elastase-digested plasminogen constituting the three N-terminal triple-loop structures in the plasmin A-chain (LBSI). After a single-step adsorption of the alpha 2-antiplasmin from plasminogen-depleted plasma to LBSI-Sepharose and elution with 6-aminohexanoic acid, an 80-90% pure preparation with a yield of 50-60% is obtained. The major impurity is fibrinogen, which can easily be removed by gel filtration, and, as a result, a homogeneous fully active alpha 2-antiplasmin preparation is obtained that has the same properties as previously described for alpha 2-antiplasmin. Evidence is put forward that a form of alpha 2-antiplasmin with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma.

Chromatography, Affinity↗
PubMed1997

Characterization of a beta-actin mRNA zipcode-binding protein.

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.

Actins↗
PubMed1994

Characterization of a testis specific protein localized to endoplasmic reticulum of spermatogenic cells.

BACKGROUND: In order to understand the mechanism of spermiogenesis, it is important to characterize germ cell specific genes and proteins expressed during spermatogenesis. We previously reported that a mouse monoclonal antibody, 1C9, raised against golden hamster testis homogenate, recognized a 103 kDa protein in hamster spermatogenic cells (Ohsako et al.; J. Vet. Med. Sci., 53:969-974, 1991). In the present study, we have determined the precise stage and intracellular localization of this protein. MATERIALS AND METHODS: Hamster, mouse, and rat tissues were used for immunocytochemistry, SDS-PAGE, and immunoblotting. Immunoelectron microscopy was performed using Lowicryl K4M embedded hamster testis and colloidal gold conjugated second antibody. Furthermore, immuno-affinity purification was carried out using a 1C9-Sepharose column. RESULTS: In immunoblot analysis, 1C9 also recognized a 103 kDa protein and a 101 kDa protein in the rat and the mouse testes, respectively. Ten different hamster tissues other than testis did not show reactivity against 1C9. In immunostained paraffin sections of hamster testis, the initial staining appeared in middle pachytene spermatocytes and persisted until maturation phase spermatids (step 15). However, it was no longer detectable in the subsequent steps of spermatids. In addition, strong staining was observed in the post-nuclear region of elongated spermatids. Immunoelectron microscopic analysis showed that the protein was localized to the endoplasmic reticulum (ER) and nuclear envelope of spermatogenic cells, but not in the other organelles, such as Golgi apparatus and acrosome of the spermatids. This protein appears to be associated with ER membrane. Furthermore, this protein is found exclusively in the testicular microsomal fraction, not in the cytosol. By affinity purification, approximately 320 micrograms of the 103 kDa protein was obtained from 10 hamster testes. The purified 103 kDa protein was unaffected by N-glycanase, indicating it does not have asparagine-linked glycoconjugates. CONCLUSIONS: These results indicate that the protein recognized by 1C9 appears to be a unique protein that is localized in the ER and nuclear envelope of spermatogenic cells.

Animals↗