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PubMed1999

Identification and characterization of individual cyclin-dependent kinase complexes from Saccharomyces cerevisiae.

In S. cerevisiae, regulation of cell cycle progression is known to be carried out by a single cyclin-dependent kinase homologue, Cdc28p, acting at different stages of the cell cycle in association with various cyclins and other regulatory subunits. However, a still unsolved problem is the identification of the physiologically relevant substrates of the different Cdc28p kinase complexes which participate in this regulation. Purification and characterization of the subunit composition and enzymological properties of these Cdc28p complexes would therefore contribute substantially to our understanding of the molecular mechanisms controlling the cell cycle. We have used a combination of ammonium sulphate fractionation, nickel nitrilotriacetate affinity purification, ATP Sepharose affinity chromatography and Resource Q ion exchange chromatography to purify two different Cdc28p kinase complexes. Using specific clb deletion mutants and plasmid or genomic HA epitope-tagged CLBs, we show that one of these complexes is composed almost exclusively (93% or greater) of Clb2p-Cdc28p, whereas the other is mainly (75% or greater) Clb3p-Cdc28p. These procedures provide the basis for the analysis of regulatory, enzymatic and functional properties of individual Cdc28p kinase complexes.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2003

An improved method for the in vitro evolution of aptamers and applications in protein detection and purification.

One of the key components of proteomics initiatives is the production of high affinity ligands or probes that specifically recognize protein targets in assays that detect and capture proteins of interest. Particularly versatile probes with tremendous potential for use as affinity molecules are aptamers. Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on affinity for a target molecule. Aptamers offer advantages over traditional antibody-based affinity molecules in their ease of production, regeneration and stability, largely due to the chemical properties of nucleic acids versus amino acids. We describe an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. We demonstrate the ability of aptamers that recognize thyroid transcription factor 1 (TTF1) to bind their target protein with high affinity and specificity, and detail their uses in a number of assays. The TTF1 aptamers were characterized using surface plasmon resonance, and shown to be useful for enzyme-linked assays, western blots and affinity purification.

Animals↗
PubMed1984

Affinity chromatography for purification of antibodies to Neisseria gonorrhoeae and Neisseria meningitidis lipopolysaccharides.

Lipopolysaccharides (LPSs) were prepared by phenol-water extraction of the gonococcal strain 8551 and the group B meningococcal strain 44/76, digested with pronase, and purified by ultracentrifugation and Sepharose CL-6B fractionation in the presence of 1.5 per cent sodium deoxycholate. On SDS-PAGE with 10 per cent acrylamide the purified 125I-labelled LPSs migrated as single, low-molecular-weight components. The LPSs were coupled to CNBr-activated Sepharose 4B for affinity purification of antibodies to the common antigenic factor 1 and the sero-type factor 5 of LPS 8551, and antibodies to LPS 44/76. The antibodies eluted showed ELISA activity against wells coated with LPS or whole cells of the bacteria, the antibody activity being inhibited by LPS. SDS-PAGE of whole cells of the strain 8551 and immunoblotting with the anti-factor 1 or -factor 5 antibodies resulted in single, broad bands corresponding to the low-molecular-weight LPS subunits.

Antibodies, Bacterial↗
PubMed1993

Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum.

A novel affinity-purification scheme based on the tight binding of NAD+:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) polymerase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded patches and DNA ends has been developed to facilitate the purification of this enzyme from the lower eukaryote Dictyostelium discoideum. Two homogeneous forms of the enzyme, with M(r) values of 116,000 and 90,000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)-cellulose as affinity material. The Km is 20 microM NAD+ for the 90,000-M(r) protein and 77 microM NAD+ for the 116,000-M(r) protein. The optimum conditions for the enzyme activity in vitro are 6-10 degrees C and pH 8. The time course is linear during the first 10 min of the reaction only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotinamide, theophylline, caffeine and thymidine.

Animals↗
PubMed2002

Purification, characterization, and immunogenicity of the refolded ectodomain of the Plasmodium falciparum apical membrane antigen 1 expressed in Escherichia coli.

The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83(Gly) to 531(Glu)) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.

Animals↗
PubMed1994

Epstein-Barr virus nuclear antigen 2 exerts its transactivating function through interaction with recombination signal binding protein RBP-J kappa, the homologue of Drosophila Suppressor of Hairless.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays a crucial role in B cell immortalization by Epstein-Barr virus (EBV), most probably by its ability to transactivate several cellular and viral genes. Recently, we showed that EBNA-2 interacts with the TP1 promoter of EBV through a cellular protein. In this report we provide evidence that this protein is recombination signal binding protein (RBP)-J kappa, highly conserved in evolution, and originally isolated by its ability to bind to the J kappa-type V(D)J recombination signal sequence. To identify the cellular protein interacting with the TP1 promoter, we performed electrophoretic mobility shift assays using binding sequences of known transcription factors, that carry partial homology to the crucial sequences of the EBNA-2 responsive element (EBNA-2RE), as competitor. Competition assays revealed the RBP-J kappa recognition site as a very efficient competitor of cellular TP1 promoter binding protein. In parallel, we purified the protein to homogeneity from Raji cells by two ion-exchange columns and affinity purification using the EBNA-2RE coupled to magnetic beads. Affinity purified fractions separated on SDS-PAGE revealed a single predominant band after silver staining which was recognized by anti-RBP-J kappa monoclonal antibody. These purified fractions exhibited binding specificity for EBNA-2RE and EBNA-2. In vitro-translated murine RBP-2 cDNA reacted with EBNA-2RE and EBNA-2 in the same fashion as the affinity purified protein. The interaction between RBP-J kappa and EBNA-2 is a prerequisite for EBNA-2-mediated transactivation of the TP1 promoter.

3T3 Cells↗
PubMed1988

Monoclonal antibodies to the major feline allergen Fel d I. I. Serologic and biologic activity of affinity-purified Fel d I and of Fel d I-depleted extract.

Monoclonal antibodies were raised against a major feline allergen, Fel d I. The specificity of the antibodies for Fel d I was demonstrated by a modified crossed immunoelectrophoretic procedure. These antibodies were used in affinity purification and depletion. Fed d I was eluted from the affinity matrix at a low pH. It was tested in serologic and biologic assays. Results of both the RAST and the histamine release test with affinity-purified Fel d I confirmed that it is a very potent allergen for the majority of patients allergic to cats. In the extract depleted with the monoclonal antibody, the ratio Fel d I/cat albumin was reduced by a factor of 20. With this depleted extract, 18 of 25 patients showed a reduction greater than 50% of the bound anti-IgE in the RAST compared with the RAST with crude cat extract. In the histamine release test with cells from two patients, the activity of the depleted extract was reduced by factors of 30 and 40, respectively. Depletion of Fel d I by polyclonal antibodies resulted in a reduction of the ratio Fel d I/cat albumin by a factor of 330. In the RAST, a reduction of the IgE response greater than 50% was found in 23 of 25 patients; in 12 patients the reduction was greater than 90%. In the histamine release test, the polyclonally depleted extract was 200 to 300 times less potent than the crude cat-dander extract. These results indicate that a very large proportion of the allergenic activity of cat-dander extract is caused by a single molecule: Fel d I.

Allergens↗
PubMedDate not supplied

Chemical coupling of a monoclonal antisurfactant protein-B antibody to human urokinase for targeting surfactant-incorporating alveolar fibrin.

Intraalveolar fibrin formation is a common histopathological finding in acute inflammatory and chronic interstitial lung diseases. Incorporation of hydrophobic surfactant components into polymerizing fibrin results in a severe loss of surface activity, altered mechanical and structural clot properties, and a reduced susceptibility toward fibrinolytic degradation. Such events have been implicated in atelectasis formation, impairment of gas exchange, and provocation of fibroproliferative changes. In an effort to address the unique features of alveolar fibrin, we designed a hybrid molecule consisting of a monoclonal antibody against surfactant protein SP-B (8B5E) and the catalytic domain of urokinase (B-chain), which was termed MABUC. The urokinase B-chain was prepared by limited reduction of human two-chain-urokinase and subsequent affinity purification and coupled to the antibody using a heterobifunctional cross-linker. Purification of the chimeric protein included gel filtration chromatography and affinity chromatography. An ELISA-like microtiter plate assay, based on the immunological detection of the SP-B moiety and the fibrinolytic activity of the u-PA domain, was developed for the detection of the hybrid molecule. Chromogenic substrate assays, (125)I-based fibrin plate assays, and active site titration were performed to analyze the specific fibrinolytic activity of the conjugate. MABUC was found to fully retain the ability of SP-B binding and the fibrinolytic activity of u-PA. In addition, MABUC was noted to be 1.5-2-fold more effective in the dissolution of surfactant embedding clots and to be approximately 3-fold more resistant against PAI-1, the predominant fibrinolysis inhibitor in the alveolar compartment, as compared to the native u-PA. The superiority of MABUC was particularly prominent (>5-fold efficacy) when investigating clot material incorporating both PAI-1 and surfactant, as a mimicry of alveolar fibrin. We conclude that urokinase and 8B5E can be cross-linked chemically, thus yielding a fibrinolytic enzyme with enhanced substrate specifity for surfactant-containing clots and higher PAI-1 resistance as compared to native u-PA.

Animals↗
PubMed1997

Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor.

Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

Animals↗
PubMed2002

RNA affinity tags for purification of RNAs and ribonucleoprotein complexes.

Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA.

Amino Acid Motifs↗
PubMedDate not supplied

Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads.

A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)(n), on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.

Amino Acid Sequence↗
PubMed1993

Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis.

The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and > 200-fold, respectively. The conservative D51E mutant was still > 100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa.

Amino Acid Sequence↗
PubMed1995

Preparation and characterization of a D-myo-inositol 1,4,5-trisphosphate-specific antibody.

Antibodies against Ins(1,4,5)P3 were raised by immunizing rabbits with two types of InsP3-BSA conjugates which were synthesized by covalently coupling Ins(1,4,5)P3 to the carrier protein via alkyl linkages. The anti-Ins(1,4,5)P3 antibody was detected by a novel ELISA using Ins(1,4,5)P3-immobilized microtitre plates. Both antiserum preparations showed specific binding with Ins(1,4,5)P3, with titres of 1:4000. Most inositol phosphates, including Ins1P, Ins(4,5)P2, Ins(1,3,4)P3, Ins(1,5,6)P3, Ins(1,2,5,6)P1, Ins(3,4,5,6)P4, Ins(1,3,4,5,6)P5, InsP6, and PtdIns(4,5)P2, did not exhibit significant molecular interactions with the antibodies. Ins(1,3,4,5)P4, however, cross-reacted with these antibodies with one-third of the affinity as that of Ins(1,4,5)P3, in part due to the largely shared structural motifs. The differential affinity was significantly improved by affinity purification on Ins(1,4,5)P3-agarose. The affinity-purified antibody displayed IC50 values of 12 nM and 730 nM for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 respectively, according to a competitive ELISA; these values are in line with those reported for the Ins(1,4,5)P3 receptor. The modes of ligand recognition at the binding sites of these two types of biomolecules are, however, different. Moreover, although the ligand binding was interfered with by multivalent anions such as ATP4-, HPO4(3-) and SO4(2-) at high concentrations, no inhibition was noted with heparin, an antagonist of the Ins(1,4,5)P3 receptor.

Animals↗
PubMed1989

Translation in Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system.

Yeast Saccharomyces cerevisiae genes TIF1 and TIF2 (translation initiation factor) encode a protein tentatively called translation initiation factor (Tif) due to the similarity of its amino acid sequence and its molecular weight to mammalian eukaryotic initiation factor 4A. To clarify whether Tif is involved in translation, we produced an affinity-purified anti-Tif antibody by using Tif isolated from a Tif-overproducing yeast strain as immunogen and an Escherichia coli strain expressing Tif from an expression vector to provide the extract for affinity purification of the antibody. By using chromatographic procedures and the affinity-purified anti-Tif antibody as probe to identify Tif-containing fractions, we purified Tif from wild-type yeast cells. When yeast cells containing the only TIF1 gene on a plasmid under the control of the galactose-inducible CYC1-GAL10 promoter were grown in medium containing glucose as the carbon source, the production of Tif was shut off and growth was arrested. Lysates made from these cells were inactive in in vitro translation. Addition of Tif to these lysates restored in vitro protein synthesis. These results show that Tif is a translation factor, the yeast homologue of mammalian translation initiation factor 4A.

Blotting, Western↗
PubMed1997

A region of vitamin K-dependent protein S that binds to C4b binding protein (C4BP) identified using bacteriophage peptide display libraries.

Vitamin K-dependent protein S, a blood coagulation inhibitor, interacts with the C4b-binding protein (C4BP) in human plasma with high affinity (KD = 0.1 nM). Identification of a portion of protein S that binds to C4BP has been approached using random libraries of 6- and 15-mer peptides displayed on bacteriophage surfaces. Bacteriophage binding to the beta-chain of C4BP were selected in several rounds of affinity purification with intervening amplification in E. coli. Homology searches of the affinity purified peptide sequences against protein S led to the identification of four regions in protein S that were similar to several of the selected peptides. These regions were synthesized as linear peptides and tested in inhibition experiments. Only one distinct peak (around position 450) was observed when the homology scores versus human protein S sequence were averaged over all affinity purified peptides. A synthetic peptide comprising residues 439-460 in human protein S was found to inhibit protein S binding to C4BP. The same result was found with two overlapping peptides (residues 447-468 and 435-468, respectively) in a second set of synthetic peptides. Direct binding of the peptides to C4BP was inferred from titrations monitored by recording the near UV circular dichroism spectra or the polarization of tryptophan fluorescence. The results suggest that residues 447-460 constitute a portion of protein S that is important for the interaction with C4BP. These findings may have implications for patients suffering from thrombosis, due to the lack of free protein S, by directing the design of drugs that disrupt protein S binding to C4BP.

Amino Acid Sequence↗
PubMed2002

Preparation and characterization of monoclonal antibody against digoxin.

Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.

Animals↗
PubMed1986

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice.

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.

Animals↗
PubMed1984

Immunoaffinity purification of bovine factor VII.

Factor VII has been purified to homogeneity from bovine plasma by a procedure that includes affinity purification on an immunoadsorbent column. Recovery was determined by both coagulant assay and liquid scintillation counting, using 3H-factor VII as an internal standard. The purification factor calculated by both methods was approximately 120,000-fold, with a final yield of approximately 18%. Homogeneity was assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The material migrated as a single polypeptide chain of 53,000 daltons, and following activation by factor Xa, the one-chain zymogen was quantitatively converted to two-chain factor VIIa. Conversion of affinity-purified factor VII to factor VIIa resulted in up to a 119-fold activation of the coagulant activity, which is 2.7-4 times greater than the activatability reported for factor VII prepared by other methods. Zur et al. calculated that pure factor VII, uncontaminated by traces of factor VIIa, would be activated 123-fold upon conversion to factor VIIa. The close agreement between observed activatability of affinity-purified factor VII and the theoretical prediction suggests that we have isolated factor VII essentially free of factor VIIa. The purification data from three lots of bovine plasma yield an estimate for the plasma concentration of factor VII from 10.1 nM to 18.5 nM.

Animals↗