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PubMedDate not supplied

[Synthesis of sorbent from cross-linked starch for affinity purification of glucose(mannose)-specific lectins].

Cross-linked starch gel for the affinity chromatography of D-glucose (D-mannose)-specific lectins is suggested. In order to optimize hydrodynamic properties of gel 30% starch has been hydrolysed by HCI at 70 degrees C during 60 min and then cross-linked by epichlorohydrin under alkaline conditions. Every 100 g of starch require 18 ml of epichlorohydrin and 36 ml of 8 N KOH. The gel obtained has been successfully used for the purification of lectins from Pisum sativum L., Lens culinaris L., Vicia sativa L., and Vicia faba L. seeds. These lectins, purified on starch gel do not differ from sephadex-purified samples.

Chromatography, Affinity↗
PubMed2005

Rapid affinity purification processes for cyclodextrin glycosyltransferase from Bacillus circulans.

Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM alpha-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.

Bacillus↗
PubMed1988

Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies.

Shiga-like toxin (SLT-II) was purified to apparent homogeneity from Escherichia coli K-12 strain NM522 containing the cloned toxin genes on recombinant plasmid pEB1. Purification was accomplished by a series of column chromatography techniques: anion-exchange, chromatofocusing, cation-exchange, and monoclonal antibody affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the pure toxin showed that SLT-II consisted of A and B subunits with apparent molecular weights of 32,000 and 10,200 +/- 800, respectively. A band of molecular weight 25,000 was also observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as the A1 subunit by Western immunoblot analysis with toxin-specific monoclonal antibodies (MAbs). The pI of the purified toxin was 5.2. Approximately 1 pg of pure SLT-II, but was not neutralized by polyclonal antibodies or MAbs to SLT-I. Five hybridomas against SLT-II were produced (BC5 BB12, DC1 EH5, EA5 BA3, ED5 DF3, and GB6 BA4). Culture supernatant fluids containing MAbs from these hybridomas did not neutralize the cytotoxicity of SLT-I or Shiga toxin. Western blot analysis showed that two MAbs (MAb DC1 EH5 and MAb GB6 BA4) recognized the A and A1 subunits of SLT-II and three MAbs (MAb BC5 BB12, MAb EA5 BA3, and MAb ED5 DF3) recognized the B subunit of SLT-II. MAb BC5 BB12 was used to prepare an affinity column for toxin purification.

Animals↗
PubMed2004

Affinity purification of plasmid DNA by temperature-triggered precipitation.

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.

Chemical Precipitation↗
PubMed1983

Detection and affinity purification of beta-endorphin precursors using a monoclonal antibody.

A monoclonal antibody to porcine beta-lipotropin has been produced which binds to the N-terminal (gamma-lipotropin) portion of the molecule. The antibody can be used to detect beta-lipotropin as well as other beta-endorphin precursors (predominantly a Mr 38 000 polypeptide) using radiobinding assay or the immunoblotting technique. Purification of the peptides can be readily achieved by affinity chromatography using the monoclonal antibody covalently bound to Sepharose 4B. As the antibody recognises the N-terminal part of beta-lipotropin, it can be used to detect and purify beta-lipotropin and other beta-endorphin precursors in the presence of beta-endorphin.

Animals↗
PubMed1985

Single-step monoclonal antibody affinity purification of human urogastrone from urine.

The use of a monoclonal antibody (MAb) affinity column to purify epidermal growth factor/urogastrone from human urine in a single-step process is described. The MAb was raised against purified recombinant human urogastrone derived from the expression of a cloned synthetic urogastrone gene. The MAb was characterised and shown to cross-react fully with recombinant and native human urogastrones. The material eluted from the column was shown to have retained biological activity. When the eluted material was run on SDS/PAGE under reducing conditions an apparently homogeneous species of 5400 Da apparent molecular weight was seen. When examined on acid PAGE, 2 molecular species corresponding to beta and gamma urogastrone were observed.

Antibodies, Monoclonal↗
PubMed1990

Affinity purification of retinoic acid-binding proteins using immobilized 4-(2-hydroxyethoxy)retinoic acid.

An affinity chromatographic matrix that purifies cellular retinoic acid-binding protein to near homogeneity from rat testes cytosol has been developed. The three-step procedure includes an acid precipitation, a batch treatment with CM Bio-Gel, and affinity chromatography on 4-(2-hydroxyethoxy)retinoic acid coupled to epoxy-activated Sepharose 6B. The binding protein was purified approximately 8500-fold based on total soluble testicular protein and with a recovery in excess of 80%. In addition, further enhancement of the purity of the protein can be attained by size-exclusion HPLC to increase purification to 21,000-fold. The recovered protein has an apparent M(r) 14,300 as determined by size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is isolated in the apo-form and retains its ability to bind retinoic acid as evidenced by the binding of [3H]retinoic acid. An apparent retinoic acid-binding protein of M(r) 18,000 has also been isolated from rat testes nuclei by the affinity chromatography step. The affinity phase has been used for 6 months without any detectable loss in its ability to purify cellular retinoic acid-binding protein.

Animals↗
PubMed2006

Affinity purification of eukaryotic 48S initiation complexes.

In vitro assembly of translation initiation complexes from higher eukaryotes requires purification of ribosomal subunits, eukaryotic initiation factors, and initiator tRNA from natural sources, and therefore yields only limited material for functional and structural studies. Here we describe a robust, affinity chromatography-based purification of eukaryotic 48S initiation complexes from rabbit reticulocyte lysate (RRL), which significantly reduces the number of individual purification steps. Hybrid RNA molecules, consisting of either a canonical 5' UTR or an internal ribosome entry site (IRES) RNA followed by a short open reading frame and a streptomycin aptamer sequence, are incubated in RRL to form 48S complexes. The assembly reaction is then applied to a dihydrostreptomycin-sepharose column; bound complexes are washed and specifically eluted upon addition of streptomycin. The eluted fractions are further purified by centrifugation through a sucrose density gradient to yield pure 48S particles. Using this purification scheme, properly assembled IRES-mediated as well as canonical 48S complexes were purified in milligram quantities.

5' Untranslated Regions↗
PubMed1999

Strep-tag II for one-step affinity purification of active bHLHzip domain of human c-Myc.

The c-Myc protein, the product of the c-myc protooncogene, is a nuclear phosphoprotein with DNA-binding properties when heterodimerized with the Max protein. It contains an amino-terminal transcriptional activation domain and a carboxy-terminal basic helix-loop-helix leucine zipper (bHLHzip) domain that directs heterodimerization and promotes DNA binding. Here, we describe the isolation of the bHLHzip domain of human c-Myc with a technique for efficient single-step purification. Using a C-terminal Strep-tag II affinity peptide and a novel Streptactin-Sepharose matrix, elution is performed under mild conditions by competition with the biotin analog desthiobiotin. No significant influence of the affinity tag on the activity of the bHLHzip domain was observed when the fusion protein was subjected to glutathione S-transferase (GST) pull-down assays for investigating its in vitro-binding properties with GST-Max. The use of the C-terminal Strep-tag II was shown to be more suitable for obtaining pure product fractions than use of the N-terminal GST affinity tag.

Bacterial Outer Membrane Proteins↗
PubMed2002

Affinity purification and characterization of the Escherichia coli molecular chaperones.

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB). Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized beta-casein (30 mg/g beads) column. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP. The cold water and Mg-ATP eluates were analyzed by SDS-PAGE. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB. The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented.

Bacterial Proteins↗
PubMed1989

One-step affinity purification of Bacillus neutral proteases using bacitracin-silica.

A purification procedure for neutral proteases from bacilli is described, in which bacitracin-silica was used as affinity medium. This enabled a one-step purification of the proteases directly from culture supernatant. Since neutral proteases are extremely sensitive towards autodigestion, conditions were chosen such, that autodigestion was largely prevented. Isopropanol appeared to be useful in both eluting the enzymes from the affinity medium, and inhibiting enzymatic activity during this step. The bacitracin-silica medium allowed high flow rates: with columns prepared for use in an FPLC system flow rates up to one column volume per minute were feasible, and still gave satisfactory results. The neutral proteases purified by this method were found to be homogeneous both by SDS-PAGE and analytical gel filtration.

Bacillus subtilis↗
PubMed1990

Affinity purification of tissue plasminogen activator using transition-state analogues.

The search for a simple affinity ligand to purify tissue plasminogen activator (tPA) was facilitated by a solid-phase synthesis approach. A large variety of tripeptide ligands containing argininal were synthesized on agarose gels containing a spacer with carboxy terminal. The immobilized ligands were easy to test with urokinase, and tPA. While a number of sequence combinations showed initial binding by tPA, only a few resulted in tight binding corresponding to a hemiacetal linkage with the active site serine. Hydrophobic residues, especially aromatics, flanking the N-side of argininal gave rise to ligands which were bound strongly by tPA. A gel containing D-Phe-D-Phe-Argal (an aldehyde derivative of arginine) was very effective in purifying tPA derived from cell culture media at small scale (milligrams) and at large (multi-grams).

Amino Acid Sequence↗
PubMed2002

One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.

We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.

Base Sequence↗
PubMed1989

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions.

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.

Amino Acid Sequence↗
PubMed1994

Affinity purification of the major bovine allergen by a novel monoclonal antibody.

A monoclonal antibody was developed to the 20 kd major allergen of cow by immunizing mice with crude dander extract. The monoclonal antibody did not exhibit cross-reactivity to cat, dog, and horse dander extracts when studied by ELISA inhibition. The antibody was used in affinity chromatography for the purification of the 20 kd allergen from cow dander extract. Purity of the allergen was estimated to be 88%, and allergenic reactivity was verified by IgE immunoblotting and skin prick tests. After further purification with size-exclusion chromatography, the allergen was almost 100% pure. The isoelectric point of the double-purified allergen was determined to be 4.1. The amino acid composition was characterized by the predominance of acidic amino acids.

Adult↗
PubMed1992

Dye-affinity purification of transthyretin from an unexploited by-product of human plasma chromatographic fractionation.

Dye-affinity chromatography of human plasma transthyretin on Remazol Yellow GGL-Sepharose from an unexploited by-product of chromatographic fractionation of plasma was optimized for large-scale preparation of a therapeutic product. With this system, transthyretin is only weakly bound to the gel. The residence time on gel and the transthyretin level in the by-product were observed to have no influence on the binding capacity of gel, and the optimum amount of transthyretin to be applied to the gel was found to be 1 g/l of gel. The adsorbent can be used more than ten times. The procedure resulted in the isolation, with a 30% yield with respect to plasma, of an 80% pure protein, which retained its thyroxine-binding capacity. Although the purity is acceptable for substitutive therapy, it can be improved further with a second chromatography on Cibacron Blue-Sepharose.

Adsorption↗