Showing papers on "Affinity chromatography published in 1990"

Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.

Abstract: Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neutrophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citrulline requires NADPH and Ca2+. We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affinity column eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer.

Phage antibodies: filamentous phage displaying antibody variable domains

Abstract: NEW ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulm variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities (see ref. 1 for review). Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H).

Abstract: Anti-phospholipid (aPL) antibodies that exhibit binding in cardiolipin (CL) ELISA can be purified to greater than 95% purity by sequential phospholipid affinity and ion-exchange chromatography. However, these highly purified aPL antibodies do not bind to the CL antigen when assayed by a modified CL ELISA in which the blocking agent does not contain bovine serum, nor do they bind to phospholipid affinity columns. Binding to the phospholipid antigen will only occur if normal human plasma, human serum, or bovine serum is present, suggesting that the binding of aPL antibodies to CL requires the presence of a plasma/serum cofactor. Using sequential phospholipid affinity, gel-filtration, and ion-exchange chromatography, we have purified this cofactor to homogeneity and shown that the binding of aPL antibodies to CL requires the presence of this cofactor in a dose-dependent manner. N-terminal region sequence analysis of the molecule has identified the cofactor as beta 2-glycoprotein I (beta 2GPI) (apolipoprotein H), a plasma protein known to bind to anionic phospholipids. These findings indicate that the presence of beta 2GPI is an absolute requirement for antibody-phospholipid interaction, suggesting that bound beta 2GPI forms the antigen to which aPL antibodies are directed. Recent evidence indicates that beta 2GPI exerts multiple inhibitory effects on the coagulation pathway and platelet aggregation. Interference with the function of beta 2GPI by aPL antibodies could explain the thrombotic diathesis seen in association with these antibodies.

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

Abstract: We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme.

Abstract: The target antigen of anti-neutrophil cytoplasm antibodies (ACPA; also known as ANCA) was isolated by affinity chromatography from supernatants of human neutrophils, stimulated with phorbol ester to induce degranulation. Sequence analysis of the antigen revealed 17 NH2-terminal amino acids (IVGGHEAQPHIRPIYMA), which have considerable sequence homology with known serine proteinases. Investigation of the enzymatic activity showed that the antigen is a neutral serine proteinase that is able to cleave elastin. Since the molecular weight of the antigen, its substrate specificity, and its inhibitor profile reported in this study are identical with those reported recently for proteinase 3, we conclude that ACPA are most probably directed against proteinase 3.

Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q.

Abstract: The serum lectin, mannan binding protein (MBP), was isolated in a yield of 40 micrograms/liter from pooled normal human serum by affinity chromatography on mannan-Sepharose, followed by gel-filtration and ion-exchange chromatography and finally by passage down an anti-IgM Sepharose column. A rabbit antiserum was prepared against the purified MBP and an enzyme-linked immunoassay developed that used both the specificity of the polyclonal antibody and the Ca+(+)-dependent carbohydrate binding property of MBP. Assay of the sera from 103 blood-donors showed a wide range of MBP levels, ranging from 0 to 870 micrograms/liter. MBP, after interaction with zymosan, caused efficient activation of a C1r2 125I-C1s2 complex that was prepared by incubation of 125I-C1s2 with serum, from a patient with a complete genetic deficiency of C1q, followed by gel-filtration on Sepharose 6B. The purified MBP is composed of a mixture of trimers, tetramers, pentamers, and hexamers of an approximate 90-kDa structural unit as judged by chromatography, SDS-PAGE and electron microscopy studies. Only the molecules in the pentamer/hexamer fraction, which have a similar overall structure to that of C1q, appeared to cause efficient, zymosan-dependent, activation of C1s within the C1r2C1s2 complex. The pentamer/hexamer form of MBP may therefore play an important role in antibody-independent activation of the C system during the early stages of certain infections.

Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography.

Abstract: We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

[30] DT-diaphorase : purification, properties, and function

Abstract: Publisher Summary DT-diaphorase is a highly active diaphorase in the soluble fraction of rat liver homogenates, which catalyzes the oxidation of nicotine adenine dinucleotide, reduced nicotine adenine dinucleotide hydrogenase (NADH), and nicotinamide adenine dinucleotide phosphate (NADPH) at equal rates This chapter discusses the purification, properties, and function of DT-diaphorase Most of the present knowledge about DT-diaphorase has been obtained in studies of the cytosolic enzyme, which was first purified from both rat and beef liver 44 by employing conventional methods The introduction of affinity chromatography for the purification of DTdiaphorase reduced the number of purification steps considerably In the method presented in the chapter for the purification of DT-diaphorase, the key isolation step is the biospecific adsorption of the enzyme to immobilized dicoumarol, which is a potent competitive inhibitor of the enzyme with respect to NAD(P)H A gel with both a high affinity and a high binding capacity for DT-diaphorase is obtained by coupling dicoumarol through an azo linkage to divinyl sulfonate (DVS)-activated Sepharose 6B In the assay, DT-diaphorase activity is measured routinely with NADH or NADPH as the electron donor and 2,6-dichlorophenol-indophenol (DCPIP) or 2-methyl-l,4-naphthoquinone (menadione) as the electron acceptor The standard assay system contains 50 m M Tris-HC1, pH 75, 008% Triton X-100, 05 m M NADH or NADPH, and 40 μM DCPIP or 10 μM menadione

System for High-Level Production in Escherichia coli and Rapid Purification of Recombinant Proteins: Application to Epitope Mapping, Preparation of Antibodies, and Structure—Function Analysis

Abstract: Publisher Summary This chapter presents an application of the system for high-level production in Escherichia coli and rapid purification of recombinant proteins The advent of gene cloning, the engineering of vectors for efficient expression, and the application of fast and high-flux methods for protein purification made available many recombinant proteins of biological interest This represented a breakthrough for the structure–function analysis of bioactive proteins and cell receptors and facilitated X-ray crystallographic studies for definition of the three-dimensional structures of these proteins The E coli expression system allows the high-level production of recombinant proteins in authentic form, as fusion proteins with the [His] 6 affinity tail and with mouse DHFR and the [His] 6 tail Because of the presence of the affinity tail, proteins that are produced in a soluble form or can be solubilized with GuHCl or urea can be purified almost to homogeneity in one step by nickel chelate affinity chromatography The purified recombinant proteins simplify the production of monoclonal and polyclonal antibodies directed against defined regions of the native proteins

The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin.

Abstract: Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.

Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum.

Abstract: Immunoglobulin light and heavy chains are synthesized in mammalian cells as precursors containing a signal peptide. Processing and assembling result in formation of active antibodies. Chimeric genes have been made containing the coding sequence of the barley alpha-amylase signal peptide which has been fused to cDNAs coding for either the mature light or the mature heavy chain of a monoclonal antibody. A plasmid was constructed linking both chimeric genes under the control of plant active promoters in an expression cassette. This DNA fragment was stably integrated into the genome of Nicotiana tabacum by Agrobacterium tumefaciens mediated gene transfer. Synthesis of light and heavy chains and assembly to antibodies was detected in transgenic tobacco tissue using specific secondary antibodies. By electron microscopic immunogold labeling, the presence of assembled antibody could be detected within the endoplasmic reticulum. Affinity chromatography indicated biological activity of the assembled immunoglobulin produced in plant cells. Unexpectedly, a significant amount of assembled antibodies was found within chloroplasts.

Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue

Abstract: Surmmary A serine proteinase (Alp) from the culture supernate of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-e-amino-caproyl-D-trypto-phan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and α-1-proteinase inhibitor, and it was largely inhibited by α-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of A. fumigatus, which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of A. oryzae. Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 μg/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of A. fumigatus are described.

Purification and characterization of an inhibitor (soluble tumor necrosis factor receptor) for tumor necrosis factor and lymphotoxin obtained from the serum ultrafiltrates of human cancer patients.

Abstract: Serum ultrafiltrates (SUF) from human patients with different types of cancer contain a blocking factor (BF) that inhibits the cytolytic activity of human tumor necrosis factor alpha (TNF-alpha) in vitro. BF is a protein with a molecular mass of 28 kDa on reducing sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The active material was purified to homogeneity by a combination of affinity chromatography, PAGE, and high-pressure liquid chromatography. Amino acid sequence analysis revealed that BF is derived from the membrane TNF receptor. Purified BF blocks the lytic activity of recombinant human and mouse TNF-alpha and recombinant human lymphotoxin on murine L929 cells in vitro. However, BF inhibits the lytic activity of TNF-alpha more effectively than it does that of lymphotoxin. The BF also inhibits the necrotizing activity of recombinant human TNF-alpha when coinjected into established cutaneous Meth A tumors in BALB/c mice. The BF may have an important role in (i) the regulation and control of TNF-alpha and lymphotoxin activity in cancer patients, (ii) interaction between the tumor and the host antitumor mechanisms, and (iii) use of systemically administered TNF-alpha in clinical trials with human cancer patients.

Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate.

Abstract: We have developed polyclonal antibodies to two synthetic peptides corresponding to the amino-(N-)terminal or carboxyl-(C-)terminal segments of the human androgen receptor (hAR) protein, as deduced from the nucleic acid sequence of the androgen receptor cDNA. Immunoreactive antisera were identified by solid phase enzyme-linked immunosorbent assay and purified by peptide affinity chromatography. Specific immunoreactivity with the hAR was confirmed by immunoblotting, using both a fusion protein produced in E. coli that contains the Cterminal 880-amino acid sequence of hAR and the full-length receptor protein produced in COS cells after transfection with a plasmid containing the entire hAR-coding region. Immunohistological evaluation of rat and human prostatic tissue using anti- C-terminal or anti-N-terminal antibodies demonstrated similar patterns of specific staining of the nuclei of epithelial and stromal cells. Castration resulted in a decrease in the amount of nuclear AR detected in the rat prostate afte...

Thermostable β-galactosidase from the archaebacterium Sulfolobus solfataricus Purification and properties

Abstract: A thermophilic and thermostable P-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Suljiulobus sovuturicus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 'C with o-nitrophenyl P-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus P-galactosidase was a tetramer of 240 Ifr S kDa composed of similar or identical subunits. Comparison of the amino acid composition of pgalactosidase from S. solfataricus with that from Eschevichia culi revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not crossreact with P-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for P-galactosidases from other mesopbilic and thermophilic sources.

Evidence that insulin and isoprenaline activate the cGMP-inhibited low-Km cAMP phosphodiesterase in rat fat cells by phosphorylation.

Abstract: Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (less than 10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively.

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method.

Abstract: Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.

Tumor promoter-stimulated Mr 92,000 gelatinase secreted by normal and malignant human cells: isolation and characterization of the enzyme from HT1080 tumor cells.

Abstract: A M r 92,000 metalloprotease, originally observed in neutrophils, has been found to be secreted by various normal and malignant cells of fibroblastic, hematopoietic, and epithelial origin. The reponsiveness of the various cell types to the tumor promoter phorbol ester (phorbol myristate acetate) to secrete this enzyme and a corresponding M r 72,000 gelatinase has been determined using gelatin zymograms. The latent zymogen form of the M r 92,000 enzyme has been purified from phorbol myristate acetate-stimulated HT1080 human fibrosarcoma cells using sequential gelatin-Sepharose affinity chromatography and gel filtration. Selective elution from gelatin-Sepharose allows for a distinct separation of the M r 92,000 gelatinase from the M r 72,000 gelatinase. A fraction of the tumor cell derived latent M r 92,000 enzyme is isolated as an apparent complex with human tissue inhibitor of metalloproteases, which is partially dissociated in sodium dodecyl sulfate and completely dissociated upon reduction of disulfide bonds and upon p -aminophenylmercuric acetate treatment. Organomercurial treatment rapidly allows for autoactivation of the proenzyme to active M r 83,000 and M r 75,000 species. At physiological pH, the enzyme rapidly degrades gelatin into small fragments and slowly cleaves native type V collagen at an apparent single site. Native type IV collagen is degraded to a much lesser extent. The NH2-terminal amino acid sequence of the M r 92,000 proenzyme has been determined and is distinct from the M r 72,000 gelatinase/type IV collagenase which is constitutively produced by fibroblasts. The M r 92,000 enzyme is also immunologically distinct from the M r 72,000 enzyme but immunologically cross-reactive with the neutrophil, high molecular weight gelatinase. The M r 92,000 enzyme constitutes a distinct member of the matrix metalloprotease family. Its substrate specificity implies a broad physiological role, acting on basement membrane type V collagen as well as on denatured (gelatinized) collagens and thus may be involved in the invasive and migratory phenotype of human cells.

Purification of recombinant proteins with metal chelate adsorbent.

Abstract: In 1975 Porath and co-workers introduced immobilized metal ion affinity chromatography (IMAC) for the purification of peptides and proteins (1) The principle of this technique is the coordination between the electron donor groups on a protein (peptide) surface and immobilized transition metal ions The tridentate chelator, iminodiacetic acid, is coupled via a spacer arm to a solid support and used for the immobilization of metal ions such as Ni(II), Cu(II) or Zn(II) Porath postulated that the histidine, cysteine and tryptophan residues in proteins (peptides) are most likely to form stable coordination bonds with metal chelates at neutral pH Present experience (2) indicates that histidine residues on protein surfaces are the predominant electron donor groups

Specific suppression of allograft rejection by soluble class I antigen and complexes with monoclonal antibody

Abstract: In this experiment, we investigated the effect of daily injection or continuous slow infusion of either DA (MHC haplotype, RT1a) serum or soluble DA class 1 MHC antigen or its complexes with monoclonal antibody on rejection of heterotopic heart allograft in the combination of PVG.RT1a (RT1a) donor into PVG (RT1c) recipient. DA serum delayed significantly both the early and late rejection of PVG.RT1a heart grafts in PVG recipients. Removal of soluble class I MHC antigen from DA serum by affinity chromatography on a monoclonal anti-class I antibody column completely abolished the immunosuppressive effect. Continuous infusion of purified soluble class I antigen from DA rat liver, even from day 4 after heart grafting, induced a significant prolongation of graft survival. This effectiveness was donor-specific and amplified by the mixture of monoclonal anti-class 1 (RT1a) antibody with DA serum--this being induced only by using continuous infusion but not by daily injection. The results indicate that soluble class I antigen can act as a specific immunosuppressive agent in allograft rejection and that its effect is amplified by monoclonal anti-class 1 antibody.