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At least 451 records · Page 25Linked to original sources
PubMed1989

A newly synthesized selective casein kinase I inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and affinity purification of casein kinase I from bovine testis.

When screening various isoquinolinesulfonamide compounds which we synthesized, CKI-7, N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, was found to have a potent inhibitory action against casein kinase I and a much weaker effect on casein kinase II and other protein kinases. Kinetic analysis indicated that CKI-7 inhibited casein kinase I competitively with respect to ATP and that the Ki values were 8.5 microM for casein kinase I and 70 microM for casein kinase II. An affinity chromatography absorbent was synthesized by coupling CKI-8 (1-(5-chloroisoquinoline-8-sulfonyl], a derivative of CKI-7, to cyanogen bromide-activated Sepharose 4B. Partially purified casein kinase I from bovine testis was subjected to affinity chromatography. Analysis of the purified casein kinase I by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single band with molecular weight 37,000. These newly synthesized compounds, CKI-7 and CKI-8, should serve as useful tools for elucidating the biological significance of casein kinase I-mediated reactions.

Animals↗
PubMed1992

Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.

Animals↗
PubMed1995

Affinity purification of human plasma vitamin D-binding protein.

During the course of our studies to probe the vitamin D ligand-binding domains of vitamin D-binding protein and vitamin D receptor, we developed a synthetic procedure to modify the 3 beta-hydroxyl group of vitamin D3 and its 25-hydroxy- and 1,25-dihydroxy metabolites with a 3'-aminopropylether group. In the present study we have coupled 25-hydroxyvitamin D3-3 beta-3'-aminopropylether to an activated Sepharose matrix. Using this stable and reusable affinity matrix we have purified human vitamin D-binding protein from human plasma to homogeneity.

Binding Sites↗
PubMed1984

Affinity purification of a glucose-containing oligosaccharide using a monoclonal antibody.

Binding of a human urinary tetrasaccharide (Glc alpha 1-6Glc alpha 1-4Glc alpha 1-4Glc) by a mouse monoclonal antibody, 61.1, shows an unusually large dependence upon temperature. Association constants determined by equilibrium dialysis double for each 8 degrees C downward shift in temperature from 37 degrees C (8 X 10(3) M-1) to 4 degrees C (1.7 X 10(5) M-1). Purified 61.1 antibody behaves as a specific temperature-sensitive affinity adsorbent when covalently bound to cyanogen bromide-activated Sepharose or non-covalently bound via its Fc portion to staphylococcal protein A-Sepharose. A column containing 72 mg of antibody 61.1 bound to 5 ml of staphylococcal protein A-Sepharose can bind up to 0.52 mumol of reduced tetrasaccharide. The relatively large combining sites of most antibodies may permit chromatographic selection based upon structural features more complex than those recognized by most plant lectins.

Animals↗
PubMed1994

Affinity purification of GTPase proteins from oat root plasma membranes using biotinylated GTP.

Biotinylated GTP was synthesized and it was demonstrated that this ligand was bi-functional: it competed with [3H]Gpp(NH)p for binding to membrane proteins and it bound to immobilized avidin. Peripheral plasma membrane proteins were solubilized in a low-salt wash, incubated with GTP-biotin and biotinylated proteins were coupled to an avidin column. Elution with excess biotin yielded 10 polypeptides as seen with a silver stained SDS-PAGE gel. Antisera raised against Ras, a small GTPase, strongly interacted with three proteins with MW of 38, 27 and 25 kDa and also with 6 other proteins. G alpha-common antibodies interacted with proteins of MW = 66 and 38 kDa. This method enables the rapid purification of GTP-binding proteins and opens the possibility to assign a role to specific GTPases in signal transduction pathways.

Binding, Competitive↗
PubMed2007

Proteins interacting with Saccharomyces cerevisiae type 1 protein phosphatase catalytic subunit identified by single-step affinity purification and mass spectrometry.

The catalytic subunit of type 1 protein phosphatase (PP1C) interacts with a large number of polypeptides in eukaryotic cells from yeast to man and these regulatory subunits can both modulate the activity of PP1C and target it to different subcellular locations. Thus, PP1 is really a family of protein phosphatases that share a common catalytic subunit, and identifying and characterizing the PP1-associated proteins is therefore critical to understanding the cellular roles of PP1 and its ability to dephosphorylate specific substrates. Here we describe methods for affinity isolation of PP1C-containing protein complexes in the yeast Saccharomyces cerevisiae and the identification of the associated polypeptides by mass spectrometry. The basic method we describe could be easily adapted to study PP1C-associated proteins in other lower or higher eukaryotes and for characterizing the protein-protein interactions of other protein phosphatases in yeast.

Amino Acid Sequence↗
PubMed1996

Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides.

We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5'-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5'-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5'-phosphorylated oligonucleotides into solution. 5'-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50-and 60mer oligonucleotides.

Biotin↗
PubMed2004

Soluble expression and affinity purification of functional domain of human acetylcholine receptor alpha-subunit by the modulation of maltose binding protein.

An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR alpha205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR alpha205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR alpha205 and AChR alpha205 were similar to that of AChR alpha-subunit from Torpedo.

Binding Sites↗
PubMed1995

Identification of thyroid blocking antibodies and receptor epitopes in autoimmune hypothyroidism by affinity purification using synthetic TSH receptor peptides.

UNLABELLED: To examine the interaction of immunoglobulins from patients with newly diagnosed hypothyroidism with the TSH receptor (TSHr), we tested protein-A purified IgG in an ELISA assay with a series of peptides representing the entire extracellular domain (ECD) of human TSHr. Antibodies bound, on average, 4.1 peptides (range 0-16) per patient, and antibodies from 26 of 30 patients (86.6%) demonstrated binding to at least one peptide. Six of the 20-mer peptides (61, 151, 181, 301, 361, 376) were most frequently recognized. These were used to construct affinity columns and separate IgGs from 10 patients into bound and unbound fractions. All fractions were tested for their ability to stimulate and inhibit cAMP generation in FRTL-5 cells. Inhibitory IgGs were purified from 9 patients (90%), suggesting that the incidence of blocking antibodies (TBAb) in autoimmune hypothyroidism is higher than previously reported. 7 of 10 patients had antibodies that recognized peptide 361 further supporting the importance of this epitope in TBAb binding. Anti-microsomal and anti-thyroglobulin antibodies did not co-purify with inhibitory antibodies, and were always in the unbound fractions. We found no correlation between the pattern of antibody binding or bioactivity with clinical manifestations of hypothyroidism. CONCLUSIONS: (1) The majority of patients with autoimmune hypothyroidism have antibodies against the TSHr-ECD that recognized linear epitopes. Most have antibodies directed at more than one site and the pattern is quite heterogeneous. (2) Six sites (noted above) are most frequently recognized. (3) Inhibitory antibodies are distinct from anti-microsomal and anti-thyroglobulin antibodies.

Adolescent↗
PubMed2002

Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion.

A major attraction in using Bacillus subtilis as an expression host for heterologous protein production is its ability to secrete extracellular proteins into the culture medium. To take full advantage of this system, an efficient method for recovering the target protein is crucial. For secretory proteins which cannot be purified by a simple scheme, in vitro biotinylation using biotin ligase (BirA) offers an effective alternative for their purification. The availability of large amounts of quality BirA can be critical for in vitro biotinylation. We report here the engineering and production of an Escherichia coli BirA and its application in the purification of staphylokinase, a fibrin-specific plasminogen activator, from the culture supernatant of Bacillus subtilis via in vitro biotinylation. BirA was tagged with both a chitin-binding domain and a hexahistidine tail to facilitate both its purification and its removal from the biotinylated sample. We show in this paper how, in a unique way, we solved the problem of protein aggregation in the E. coli BirA production system to achieve a yield of soluble functional BirA hitherto unreported in the literature. Application of this novel BirA to protein purification via in vitro biotinylation in general will also be discussed. Biotinylated staphylokinase produced in the study not only can act as an intermediate for easy purification, it can also serve as an important element in the creation of a blood clot targeting and dissolving agent.

Avidin↗
PubMedDate not supplied

Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications.

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose Cl 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.

Chromatography, Affinity↗
PubMed1994

Affinity purification of sucrose binding proteins from the plant plasma membrane.

Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonic acid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.

Biological Transport↗
PubMed1990

Affinity purification of nicotinic acetylcholine receptor from rat brain.

Using chromatography on DE-52 and acetylcholine-Affi-Gel columns, nicotinic acetylcholine receptor was purified to approximately 10,000 fold from Lubrol extract of rat brain with a recovery of 15%. The purified preparation contained no cholinesterase activity. alpha-Bungarotoxin did not inhibit [3H]acetylcholine binding to the purified preparation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 4 major protein bands with apparent molecular weights of 53,000, 67,000, 80,000 and 108,000. When nicotinic acetylcholine receptor was eluted with either carbachol or nicotine from the affinity column, these major bands were found on SDS-PAGE gels. Immunoblot analysis showed that the Mr 80,000 protein was an acetylcholine-binding subunit and that the Mr 48,000 protein, a minor band on SDS-PAGE gel, was a structural subunit.

Acetylcholine↗
PubMed1991

Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity.

Herpes simplex virus (HSV) ribonucleotide reductase is formed by the association of two distinct dimeric subunits, R1 and R2. Attempts to purify either the HSV holoenzyme or its R1 subunit in their active form have been unsuccessful until now. The C terminus of the R2 protein being involved in the association with R1, the synthetic nonapeptide corresponding to this terminus, impedes the formation of the holoenzyme by competing with R2 for a critical site on R1. Based upon these observations, we developed an affinity chromatographic procedure to purify the R1 protein from HSV-1-infected baby hamster kidney cells. Specific binding of R1 to an affinity column made by linking the peptide HSV R2-(326-337) to Affi-Gel 10, followed by specific elution with an excess of an analogous peptide exhibiting a higher affinity for R1 yielded, in a single step, highly purified R1 protein. The purified R1 preparations contained approximately 95% of intact R1, the remaining 5% consisting of two R1 copurifying proteolytic breakdown products. The purified R1 protein exhibited a high reductase specific activity when mixed with an excess of the R2 subunit. Moreover, in vitro kinase assays revealed that the purified R1 protein of HSV-1 possesses an autophosphorylating activity also able to phosphorylate alpha-casein and histone II-S. The intrinsic protein kinase activity of HSV R1 is associated with its unique N-terminal domain which is absent from all other reductase subunits 1 and contains consensus motifs found in Ser/Thr protein kinases. A preliminary characterization of the kinase activity of the R1 protein of HSV-1 ribonucleotide reductase is presented.

Animals↗
PubMed1995

Expression in Escherichia coli and affinity purification of a CKS-troponin I fusion protein.

The human cardiac troponin I gene was subcloned and expressed at high levels in Escherichia coli as a fusion protein to CMP-KDO synthetase (CKS). Expression levels of the CKS-troponin I fusion were 8% of total cellular protein 4 h after induction with IPTG. The fusion was expressed primarily as an insoluble protein as shown by SDS-PAGE analysis. Expressed CKS-troponin I fusion from a crude lysate was antigenic against anti-CKS and anti-troponin I monoclonal antibodies in Western blots. The fusion was affinity-purified over a TnC affinity column using a urea-solubilized extract of a crude cell lysate. Serial dilutions of crude soluble extracts of the troponin I fusion were assayed in several microparticle enzyme immunoassays and found to exhibit similar immunogenic responses relative to cardiac troponin I isolated from human heart tissue.

Biomarkers↗
PubMed1992

Artificial immunoglobulin G-binding protein mimetic to staphylococcal protein A. Its production and application to affinity purification of immunoglobulin G.

Staphylococcal protein A consists of a single polypeptide with five immunoglobulin G (IgG)-binding domains, which are linked as E-D-A-B-C in this order from the amino terminal. The DNA coding domains A-B were polymerized one to six times linearly, taking advantage of the non-palindromic nucleotide sequence of the AccI recognition site and the resultant DNAs were inserted in pTRP vector carrying trp promoter. The artificial IgG-binding proteins [pA(AB)1-6], which had been expressed in Escherichia coli JM109, were purified by methods involving IgG-Sepharose affinity chromatography. Among pA(AB)1-6 immobilized on cyanogen bromide-Sepharose, pA(AB)4-Sepharose was the highest in IgG-binding capacity at the same level of mg protein per ml gel, about 30% higher than protein A-Sepharose. At 8 mg protein per ml gel, it bound and eluted about 24 mg of IgG from rabbit serum. Its IgG-binding capacities were the highest with porcine, rabbit, human and guinea pig sera, intermediate with bovine, horse and sheep sera and the lowest with mouse, goat, rat and chicken sera.

Amino Acid Sequence↗
PubMed1994

Preparation and affinity purification of a novel, biologically active, CNTF fusion protein.

A biologically active fusion protein comprising a short hydrophilic leader peptide fused to the N-terminus of rat CNTF was generated using commercially available materials. The coding region for rat CNTF was sub-cloned into the pFLAG-1 vector and transfected into the JM 109 strain of E. coli. The transfected cells expressed high levels of the fusion protein (FLAG-CNTF) following induction by isopropyl beta-D-thiogalactoside (IPTG). FLAG-CNTF is expressed as insoluble material that was resolubilized by extraction with guanidine hydrochloride and purified by immuno-affinity chromatography. Analysis of the purified material by reverse phase HPLC and Western blot analysis indicated that FLAG-CNTF was composed of two closely related species that are greater than 99% pure after affinity chromatography. The purified FLAG-CNTF migrated as a 27 KD doublet on SDS polyacrylamide gel electrophoresis. Both bands of the doublet were shown to contain the FLAG peptide and CNTF by Western blot analysis, and amino acid sequence analysis demonstrated a single amino acid sequence corresponding to FLAG peptide and the N-terminus of CNTF. The purified fusion protein was tested for biological activity using the IMR-32 human neuroblastoma cell line. Treatment of IMR-32 cells with FLAG-CNTF increased the level of choline acetyltransferase (ChAT) in these cells 2-3-fold over that of control cells in a dose dependent manner. A direct comparison of the effects of FLAG-CNTF and recombinant human CNTF on IMR-32 ChAT activity showed that both factors exhibited similar potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents.

Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 X 10(8) minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 X 10(8) MLD/mg protein N).

Animals↗