Showing papers on "Affinity chromatography published in 1995"

A combinatorial library of an alpha-helical bacterial receptor domain.

Abstract: The construction and characterization of a combinatorial library of a solvent-exposed surface of an alpha-helical domain derived from a bacterial receptor is described. Using a novel solid-phase approach, the library was assembled in a directed and successive manner utilizing single-stranded oligonucleotides containing multiple random substitutions for the variegated segments of the gene fragment. The simultaneous substitution of 13 residues to all 20 possible amino acids was carried out in a region spanning 81 nucleotides. The randomization was made in codons for amino acids that were modelled to be solvent accessible at a surface made up from two of the three alpha-helices of a monovalent Fc-binding domain of staphylococcal protein A. After cloning of the PCR-amplified library into a phagemid vector adapted for phage display of the mutants, DNA sequencing analysis suggested a random distribution of codons in the mutagenized positions. Four members of the library with multiple substitutions were produced in Escherichia coli as fusions to an albumin-binding affinity tag derived from streptococcal protein G. The fusion proteins were purified by human serum albumin affinity chromatography and subsequently characterized by SDS-electrophoresis, CD spectroscopy and biosensor analysis. The analyses showed that the mutant protein A derivatives could all be secreted as soluble full-length proteins. Furthermore, the CD analysis showed that all mutants, except one with a proline introduced into helix 2, have secondary structures in close agreement with the wild-type domain. These results proved that members of this alpha-helical receptor library with multiple substitutions in the solvent-exposed surface remain stable and soluble in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the ligand‐binding domain of the surface‐located fibrinogen receptor (clumping factor) of Staphylococcus aureus

Abstract: The ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign-body and wound infections Recently, we reported the cloning and sequencing of the gene clfA encoding the fibrinogen receptor (clumping factor, ClfA) of S aureus strain Newman and showed that the gene product was responsible for the clumping of bacteria in soluble fibrinogen and for the adherence of bacteria to solid-phase fibrinogen This was confirmed here by showing that antibodies raised against purified Region A inhibited both of these properties Also, immunofluorescent microscopic analysis of wild-type Newman and a clfA::Tn917 mutant of Newman with anti-ClfA Region A sera confirmed that Region A is exposed on the bacterial cell surface Furthermore, polystyrene beads coated with the Region A protein formed clumps in soluble fibrinogen showing that the ClfA protein alone is sufficient for the clumping phenotype Western immunoblotting with anti-ClfA Region A antibodies identified the native ClfA receptor as a 185 kDa protein that was released from the cell wall of S aureus by lysostaphin treatment A single extensive ligand-binding site was located within Region A of the ClfA protein Truncated ClfA proteins were expressed in Escherichia coli Lysates of E coli and proteins that had been purified by affinity chromatography were tested for (i) their ability to bind fibrinogen in Western ligand blotting experiments, (ii) for their ability to inhibit clumping of bacteria in fibrinogen solution and adherence of bacteria to solid-phase fibrinogen, and (iii) for their ability to neutralize the blocking activity of anti-ClfA Region A antibody These tests allowed the ligand-binding domain to be localized to a 218-residue segment (residues 332-550) within Region A

The native structure of intercellular adhesion molecule-1 (ICAM-1) is a dimer. Correlation with binding to LFA-1.

Abstract: In solution, intercellular adhesion molecule-1 (ICAM-1) exhibits extremely low affinity for its receptor, LFA-1, as direct binding to LFA-1 has not been reported. Furthermore, there are conflicting reports on the ability of ICAM-1 in solution to inhibit cell adhesion events. These differences could be due to the valency or an oligomeric native biochemical form of membrane-bound and soluble ICAM-1, which may correlate with its ability to bind to integrins. To test this, stimulated adenocarcinoma (A549) cells or HUVEC were labeled with 35S-methionine/cysteine and treated with a chemical cross-linker. A high m.w. form (200 kDa) of ICAM-1 but not ICAM-2 was specifically immunoprecipitated from cross-linked cell lysates and supernatants. Affinity purification of crosslinked supernatants revealed that the majority of ICAM-1 was dimeric as opposed to recombinant soluble ICAM-1, which contains a minor fraction of dimer. Gel filtration chromatography was used to isolate monomeric and dimer-rich fractions of recombinant soluble ICAM-1, and tested for direct binding to affinity-purified LFA-1. Dimer-rich fractions demonstrated an enhanced ability and estimated affinity, compared with monomeric protein, to bind to purified LFA-1. These data suggest that ICAM-1 exists in its native membrane-bound and shed form as a non-covalent dimer, and that dimerization directly correlates with enhanced binding to LFA-1.

Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme.

Abstract: Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

Generation of Rabbit Monoclonal Antibody Fragments from a Combinatorial Phage Display Library and Their Production in the Yeast Pichia pastoris

Abstract: We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.

Catalytic activity of anti-thyroglobulin antibodies.

Abstract: Thyroglobulin (Tg)-specific autoantibodies from a patient with Hashimoto's thyroiditis hydrolyzed radiolabeled Tg, shown by production of several smaller sized products on SDS electrophoresis gels The apparent Km value for Tg was in the nanomolar range, a property typical of an Ab combining site The Tg antibodies also hydrolyzed tripeptide-methylcoumarinamide (MCA) substrates with lower affinity, displaying a preference for Arg-MCA and Lys-MCA containing conjugates The hydrolysis of one of these conjugates, Pro-Phe-Arg-MCA, was inhibited competitively by Tg, suggesting a catalytic site located in the Ab combining site In control experiments, 1) the hydrolytic activities were removed by immunoadsorption with immobilized anti-human IgG; 2) IgG depleted of the Tg-specific Abs by affinity chromatography did not display Tg and Pro-Phe-Arg-MCA hydrolyzing activities; and 3) the peptide-MCA hydrolyzing activity tracked exactly with the 150-kDa IgG peak on a gel filtration column run in denaturing solvent (6 M guanidine chloride)

Carcinoembryonic Antigen and Other Glycoconjugates Act as Ligands for Galectin-3 in Human Colon Carcinoma Cells

Abstract: Galectin-1 and galectin-3, galactoside-binding lectins with molecular weights of M r 14,500 and 31,000, respectively, are expressed in normal and malignant cells and have been implicated in regulation of cell growth, adhesion, and metastasis. We analyzed the expression of galectins in 21 cultured human colon carcinoma cell lines by immunoblotting. Galectin-1 was detected in only 7, whereas galectin-3 was found in 20 of the cell lines. KM12 cells, which express only galectin-3, were used to isolate this lectin by affinity chromatography, and the purified lectin was used to identify complementary glycoconjugates by blotting. Galectin-3 was shown to bind to human laminin, carcinoembryonic antigen, and lysosome-associated membrane glycoproteins, which are involved in cell adhesion. Galectin-3 was localized on the KM12 cell surface and colocalized with carcinoembryonic antigen. Several endogenous glycoproteins and cell surface proteins of molecular weights in the range M r 58,000 to >200,000, including carcinoembryonic antigen and lysosome-associated membrane glycoproteins, were identified as galectin-3 ligands by coimmunoprecipitation with and affinity chromatography on immobilized galectin-3. These data demonstrate that galectin-3 interacts with several adhesion molecules and suggest that this lectin may have a role in human colon carcinoma cell adhesion.

Purification of a Glucose/Mannose Specific Lectin, Isoform 1, from Seeds of Cratylia mollis Mart. (Camaratu Bean)

Abstract: A quantitatively main molecular form ofCratylia mollis lectin, isoform 1 (iso 1) was purified by affinity chromatography on Sephadex G-75, followed by ion exchange chromatography on CM-cellulose. Another lectin form was identified in the latter step. Iso 1 is specific for glucose/mannose, with a main subunit of 31 kDa mol wt; the native protein is basic (pI 8.5-8.6) and the constituent polypeptides had a pI range of 5.15–7.75. An antibody to the protein was raised in a rabbit, and the conjugate was active in an immunosorbent assay.

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

Abstract: A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 59-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.

Antigen-binding sites of antibody molecules specific for cancer antigens

Abstract: Novel compositions are provided that are derived from antigen-binding sites of immunoglobulins having affinity for cancer antigens. The compositions exhibit immunological binding properties of antibody molecules capable of binding specifically to a human tumor cell expressing an antigen selected from the group consisting of high molecular weight mucins bound by 2G3 and 369F10, c-erbB-2 tumor antigen, an approximately 42 kD glycoprotein, an approximately 55 kD glycoprotein, and the approximately 40, 60, 100 and 200 kD antigens bound by 113F1. A number of synthetic molecules are provided that include CDR and FR regions derived from same or different immunoglobulin moieties. Also provided are single chain polypeptides wherein V H and V L domains are attached by a single polypeptide linker. The sFv molecules can include ancillary polypeptide moieties which can be bioactive, or which provide a site of attachment for other useful moieties. The compositions are useful in specific binding assays, affinity purification schemes, drug or toxin targeting, imaging, and genetic or immunological therapeutics for various cancers. The invention thus provides novel polypeptides, the DNAs encoding those polypeptides, expression cassettes comprising those DNAs, and methods of inducing the production of the polypeptides.

The response regulators cheb and chey exhibit competitive binding to the kinase chea

Abstract: The autophosphorylating kinase CheA of the bacterial chemosensory signaling pathway donates a phosphoryl group to either of two regulator proteins, CheY or the receptor methylesterase (CheB). With isothermal titration calorimetry, it was demonstrated that CheA and CheA fragment composed of amino acid residues 1-233 (CheA1-233) bound to CheY with similar dissociation constants of 2.0 and 1.2 microM at 298 K, respectively, indicating that the CheY binding site is wholly within the 1-233 amino acid locus. CheB bound to CheA1-233 with a KD of 3.2 microM, and also bound to intact CheA with the same affinity. CheY was found to complete with CheB for binding to CheA1-233, in spite of the low level of sequence identity between CheY and the regulatory domain of CheB. The competitive nature of CheY and CheB binding was determined in two independent sets of experiments: titration experiments in which either a CheB-CheA1-233 complex was titrated with CheY or CheB was titrated with a CheY-CheA1-233 complex, and competitive affinity chromatography experiments that used a Ni-NTA-chelating resin as an affinity matrix for complexes of the histidine-tagged CheA1-233 fragment and CheY or CheB. The effects of phosphorylation, binding-site mutations, and active-site mutations were also studied to probe the influence of conformational changes in CheY as a regulatory mechanism of CheY-CheA Interactions. Phosphorylated CheY, in the presence of excess EDTA, was found to have a 2-fold lower affinity for CheA1-233, and 6 mM Mg2+ further reduced the affinity of phosphorylated CheY for CheA1-233 (ca. 3-fold), although Mg2+ on its own had no effect on the interactions of either CheB or CheY with CheA1-233. The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA under physiological conditions. The idea that phosphorylation may induce a significant conformational change, reducing the strength of the CheY-CheA interaction, is supported by the relative values of the association constants measured for CheY active-site and binding-site mutants. A binding-site mutation (A103V) in CheY, which is remote from the site of phosphorylation produced a 10-fold reduction in Ka, whereas active-site mutations produced a modest (2-fold) reduction.

Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli

Abstract: The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.

Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase

Abstract: Publisher Summary This chapter discusses the purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Metal chelate chromatography is now finding widespread use in the purification of human immunodeficiency virus (HIV) reverse transcriptase (RT) directly from the high-speed supernatant of bacterial homogenates. Metal chelate affinity chromatography offers a rapid and highly reproducible means of preparing (1) the individual p66 and p51 HIV RT subunits, (2) heterodimer p66/p51, and (3) reconstituted, selectively modified heterodimer directly from bacterial homogenates. The application of a highly selective affinity matrix as the primary purification step has the advantage that bacterial proteases are eliminated at an early stage, avoiding proteolysis of the reconstituted protein. The ease with which HIV RT can be purified by metal chelate affinity chromatography has prompted to develop methodologies for protein mini preparation, where four small columns can be run simultaneously and RT eluted in a batch wise fashion. This procedure has been proven successful in cases where individual domains of the enzyme have been analyzed by insertional mutagenesis.

Single-chain antibody streptavidin fusions: Tetrameric bifunctional scFv-complexes with biotin binding activity and enhanced affinity to antigen

Abstract: To increase the avidity of single-chain antibodies (scFv) for their antigen, we have fused them to core-streptavidin. The chimeric protein, expressed by the vector pSTE (plasmid for streptavidin-tagged expression) from Escherichia coli, can form tetrameric complexes, binds its antigen and contains four biotin binding sites per tetrameric complex. An additional cysteine inserted near the carboxy terminus further stabilised the complex. The scFv fusion protein tetramers could be enriched by affinity chromatography using the biotin analog 2-iminobiotin from periplasmic inclusion bodies after refolding. We have also shown that the scFv fusion protein could be used for direct detection of its antigen in ELISA when stained with biotinylated horseradish peroxidase. The affinity of the scFv-antibody complex was substantially increased by avidity effects due to the tetrameric structure. The biotin binding sites may be used for coupling other antibodies and molecules to form bispecific and bifunctional reagents.

Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli.

Abstract: tRNA pseudouridine 55 (psi 55) synthase, the enzyme that is specific for the conversion of U55 to psi 55 in the m5U psi CG loop in most tRNAs, has been purified from Escherichia coli and cloned. On SDS gels, a single polypeptide chain with a mass of 39.7 kDa was found. The gene is a previously described open reading frame, p35, located at 68.86 min on the E. coli chromosome between the infB and rpsO genes. The proposed name for this gene is truB. There is very little protein sequence homology between the truB gene product and the hisT (truA) product, which forms psi in the anticodon arm of tRNAs. However, there was high homology with a fragment of a Bacillus subtilis gene that may produce the analogous enzyme in that species. The cloned gene was fused to a 5'-leader coding for a (His)6 tract, and the protein was overexpressed > 400-fold in E. coli. The recombinant protein was purified to homogeneity in one step from a crude cell extract by affinity chromatography using a Ni(2+)-containing matrix. The SDS mass of the recombinant protein was 41.5 kDa, whereas that calculated from the gene was 37.3. The recombinant protein was specific for U55 in tRNA transcripts and reacted neither at other sites for psi in such transcripts nor with transcripts of 16S or 23S ribosomal RNA or subfragments. The enzyme did not require either a renatured RNA structure or Mg2+, and prior formation of m5U was not required. Stoichiometric formation of psi occurred with no requirement for an external source of energy, indicating that psi synthesis is thermodynamically favored.