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PubMedDate not supplied

The human multicatalytic proteinase: affinity purification using a monoclonal antibody.

A monoclonal antibody, coupled to Sepharose CL-4B, was used for the rapid purification of the human multicatalytic proteinase in a single chromatographic step under mild conditions. The enzyme was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. Electrophoresis under dissociating and reducing conditions revealed at least ten components with molecular masses in the range 22-34 kDa. Affinity-purified enzyme was identical to conventionally purified enzyme with respect to enzymatic properties, molecular mass and subunit composition.

Amino Acid Sequence↗
PubMed1999

Immuno affinity purification of foot and mouth disease virus type specific antibodies using recombinant protein adsorbed to polystyrene wells.

The specificity of foot and mouth disease virus (FMDV) serological tests depends largely on the quality and purity of the antibodies used. Such type specific antibodies can be generated by hybridoma technology. Alternatively, the specific antibodies can be selected from polyclonal serum by immunoaffinity chromatography using recombinant protein/peptide bound affinity matrices. Based on this approach, we purified selectively antibodies against the major epitopes of VP 1 of FMDV serotype Asia 1 using recombinant protein adsorbed to polystyrene wells. Optimum buffer conditions were standardised for efficient elution. Buffer consisting of 4 M MgCl2 with 75 mM HEPES pH 6.5 was found to be optimum with respect to elution efficiency of bound antibodies and integrity of antigen. The specific reactivity of eluted antibodies was confirmed by dot-enzyme linked immunosorbent assay (dot-ELISA) and antigen capture reverse transcription polymerase chain reaction (Ag/RT-PCR). The effect of temperature and repeated elution on the stability of coated protein were studied.

Animals↗
PubMed1991

Affinity purification of the photoreceptor cGMP-gated cation channel.

The cGMP-gated cation channel is a member of a new family of channel proteins that appear to be directly regulated by cyclic nucleotides. A protein with a subunit molecular mass of 78 kDa that exhibits cGMP-gated calcium flux when reconstituted into phospholipid-containing vesicles has been purified using 8-bromo-cGMP-agarose affinity chromatography. This channel activity is sensitive to the inhibitor l-cis-diltiazem. Treatment of the reconstituted channel with trypsin abolishes the l-cis-diltiazem sensitivity. Apparent endogenous proteolysis can also result in smaller molecular weight polypeptides that exhibit cGMP-gated channel activity but are insensitive to l-cis-diltiazem. These results show that the channel can bind cGMP and that it contains a l-cis-diltiazem inhibitory domain that is distinct from the cGMP-binding domain.

Animals↗
PubMed1981

Affinity purification of an acylated and radiolabelled synthetic derivative of residues 75-83 of bovine myelin basic protein, [125I]S79. A model for the purification of picomole quantities of specific peptide fragments of myelin basic protein and antibodies against them.

Among the antibodies contained in a rabbit antiserum to synthetic peptide sequence TTHYGSLPQKAQGHRPQDEG (S82) of bovine myelin basic protein (residues 65-83 plus glycine), was a population reactive with a C-terminal determinant of S82 and cross-reactive with S79 (AQGHRPQDEG) but not S6 (AQGHRPQDENG). This antibody population was purified 153-fold by affinity chromatography from a minicolumn containing S79 coupled to CH-Sepharose 4B(TM) and eluted with 3 M MgCl2. The purified antibodies were then coupled to CNBr-activated Sepharose 4B(TM) and used to purify 125I-labelled, acylated S79 ([125I]S79), 3 M MgCl2 once again having been used to elute the labelled ligand. Sips distribution studies revealed appreciable heterogeneity of binding affinities of unpurified antibodies in their reaction with affinity-purified [125I]S79 or of purified antibodies in their reaction with unpurified [125I]S79 (heterogeneity constant a = 0.34 and 0.36, respectively). In contrast Sips distribution data indicated considerable restriction of binding of the purified antibodies in their reaction with purified labelled ligand (a = 0.92) with an average affinity constant of K0 = 1.56 X 10(8) M-1. The results indicate that the heterogeneous spectrum of binding affinities originally displayed by the unpurified S79-reactive antibodies in their reaction with unpurified labelled S79 was due both to the presence of some antibodies characterized by high affinity binding (K0 greater than 10(9) M-1) and of some labelled ligand with low binding affinity. The affinity chromatographic method as here described should prove advantageous in purifying and eventually characterizing picomolar amounts of serum factors, previously postulated to be fragments of myelin basic protein, that are reactive with reagent antibodies up to an affinity level of 10(8) M-1.

Antibodies↗
PubMed2002

Identification of surface proteins of Helicobacter pylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis.

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.

Bacterial Proteins↗
PubMed1991

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification.

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.

Angiotensin II↗
PubMed1992

Affinity purification of histidine-tagged proteins transiently produced in HeLa cells.

In order to produce eukaryotic proteins in a functional state, it is often necessary to use eukaryotic instead of prokaryotic expression systems. We have designed vectors which can be employed to express either N- or C-terminally histidine-tagged proteins in transiently transfected eukaryotic cells. The histidine tag allows the rapid enrichment of these proteins by metal chelate affinity chromatography in a native and functional state. Yields of up to 5 micrograms protein/5 x 10(7) cells were achieved.

Base Sequence↗
PubMed1989

Monoclonal antibody to microtubule-associated STOP protein: affinity purification of neuronal STOP activity and comparison of antigen with activity in neuronal and nonneuronal cell extracts.

Microtubules, ordinarily cold-labile structures, are made entirely resistant to cold temperature by the presence of substoichiometric amounts of STOP (stable tubule only polypeptide), a microtubule-associated protein. We have produced a monoclonal antibody which specifically recognizes a 145-kDa protein previously implicated in STOP activity in rat brain extracts. An antibody affinity column removes both the 145-kDa protein and STOP activity from solution. A urea eluate from the affinity column contains the 145-kDa protein and exhibits substantial STOP activity. We conclude the 145-kDa protein accounts for all measurable STOP activity in rat neuronal extracts. For this work, we have developed an assay of microtubule cold stability which is generally applicable to the detection of STOP activity in various tissues. Using this assay, we show STOP activity is most abundant in neuronal tissue but is detectable in all tissues tested, with the exception of heart muscle. In all tissues that we have examined, STOP activity elutes as a single peak from heparin affinity columns, and in common with brain STOP, all activity is Ca2+-calmodulin sensitive. The monoclonal antibody recognizes the 145-kDa STOP in rat neuronal extracts but reacts with no protein in active fractions from other tissue. A similar, but not identical, analogue of brain STOP thus appears to be widespread in mammalian tissues.

Animals↗
PubMed1990

Affinity purification of a 70K protein, a membrane protein relevant to sexual cell fusion in Dictyostelium discoideum.

Sexual cell fusion occurs between HM1 and NC4, heterothallic strains in Dictyostelium discoideum. A membrane component of HM1 cells with a molecular mass of 70 kDa (70K protein) has been shown to be implicated in cell fusion (Urushihara et al. (1988) Cell Differ. Dev. 25, 81-88). In the present study, 70K protein was partially purified using affinity Sepharose on which membrane proteins of NC4 cells were immobilized. Through this process, involvement of Ca2+ in the interaction of 70K protein with its receptor was suggested. Lectin staining of partially purified 70K protein indicated it to be a glycoprotein containing D-mannose and/or D-glucose residues.

Calcium↗
PubMed1996

Affinity purification of mannose 6-phosphate receptor proteins. Purification and partial characterisation of goat liver receptors.

Mannose 6-phosphate receptor (MPR) proteins designated as MPR 300 and MPR 46 have earlier been purified from some mammals on phosphomannan coupled to cyanogen bromide activated Sepharose. In a recent study, the goat liver MPR 300 has been directly purified using Sepharose-divinylsulfone-pentamannosyl phosphate matrix (Sivakumar N. 1996, J. Biochem. Biophys. methods, 31, 181-184(1)). In the present report, we describe the preparation of another affinity matrix Sepharose-divinylsulfone-phosphomannan and its utility in purifying the MPR proteins from goat liver. While the MPR 300 from goat liver showed an electrophoretic mobility similar to other mammalian MPRs, the small receptor showed a molecular weight of 36 kDa. Antibodies raised against goat liver MPR 300 react specifically with the large receptor protein. Additionally affinity purified peptide specific antibody corresponding to amino-acid residues 26-42 (ADGCDFVCRSKPRNVPA) of the cytoplasmic tail of the human liver MPR 46 (Pohlmann et al, 1988. Proc. Natl. Acad. Sci. USA, 84, 5575-5579 (2)) cross-reacts with the purified small receptor.

Animals↗
PubMed1996

Affinity purification of a correctly folded fragment of synthetic HIV-1 mRNA using a HIV-1 Rev peptide-ligand.

Formation of a macromolecular complex between the RNA binding protein HIV-1 Rev and HIV-1 mRNA is an essential prerequisite for nuclear export and subsequent expression of HIV-1 mRNA. The arginine rich peptide TRQARRNRRRRWRARQR, corresponding to residues 34-50 of HIV-1 Rev, contains the mRNA binding motif. We prepared a thioether linked Rev34-50-cellulose conjugate to affinity purify a fragment of synthetic mRNA corresponding to the high affinity binding site for Rev. The correctly folded fraction of mRNA (27.5%) was isolated from a crude synthetic mixture.

Amino Acid Sequence↗
PubMed1986

Lactoferrin: affinity purification from human milk and polymorphonuclear neutrophils using monoclonal antibody (II 2C) to human lactoferrin, development of an immunoradiometric assay using II 2C, and myelopoietic regulation and receptor-binding characteristics.

Several investigators have now confirmed our original report demonstrating the myelopoietic suppressive activity of lactoferrin (LF) in vitro. In order to further clarify this activity, we used the recently produced and purified neutralizing antibody (II 2C) to LF to set up an immunoradiometric assay specific for LF and to affinity purify LF from lysates of peripheral blood polymorphonuclear neutrophils (PMN) obtained from healthy donors. Iron-saturated purified PMN LF was as active as iron-saturated affinity purified milk LF as a suppressor of the release of granulocyte-macrophage colony stimulating factors (GM-CSF) from mononuclear human peripheral blood leukocytes. The activities of both the PMN LF and milk LF were inactivated by preincubation with monoclonal anti-LF antibody (II 2C). In order to evaluate the methods of iron saturation of LF in vitro as measures of their functional activities, milk LF was iron saturated by four different methods, including ferric citrate, ferric ammonium sulphate, ferric chloride with nitriloacetate, and ferric chloride alone. The functional characteristics of all four preparations of LF saturated with iron in vitro were relatively equal and were more active than native LF. Resident mouse peritoneal macrophages separated into subpopulations of GM-CSF-producing cells by velocity sedimentation were evaluated for their LF-receptor binding capacity and for sensitivity to the suppression of GM-CSF release by LF. Iron saturated LF suppressed release of GM-CSF from only those fractions containing LF-receptor bearing cells, although not all fractions containing cells bearing receptors for LF responded to the suppressive activity of LF. These studies provide further evidence for the myelopoietic regulatory activity in vitro of PMN-derived LF, which is mediated through populations of mononuclear phagocytes having receptors for LF.

Animals↗
PubMed1987

Antibody-affinity purification of novel alpha-L-fucosidase from mouse liver.

Previous studies have documented the presence of a novel alpha-L-fucosidase in mouse liver that contains unique basic isoelectric forms and that is antigenically similar to, but not identical with, human liver alpha-L-fucosidase [Laury-Kleintop, Damjanov & Alhadeff (1985) Biochem. J. 230, 75-82]. In the present investigation, mouse liver alpha-L-fucosidase was purified approx. 26,500-fold in 10% overall yield by antibody-affinity chromatography with the IgG fraction of goat anti-(human alpha-L-fucosidase) antibody coupled to Sepharose 4B. Native polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis indicated that the mouse fucosidase is highly purified if not homogeneous. Isoelectric focusing demonstrated that all enzymic forms found in crude mouse liver supernatant fluids were purified by the antibody-affinity procedure.

Animals↗
PubMed1998

Affinity purification of yeast cytochrome oxidase with biotinylated subunits 4, 5, or 6.

Null mutants in COX4, COX5a, or COX6, which encode subunits 4, 5, and 6 of yeast cytochrome oxidase are blocked in assembly of the enzyme. The mutants are complemented by gene constructs expressing cytochrome oxidase subunits with a carboxyl terminal extension containing a biotinylation signal sequence. Spectra and enzyme activities of mitochondria from transformants expressing a biotinylated subunit indicate restoration of a functional cytochrome oxidase. Biotinylated cytochrome oxidase can be affinity-purified from mitochondrial extracts by fractionation on a monomeric avidin column. This method can be used to purify the enzyme from small amounts of starting material.

Alleles↗
PubMedDate not supplied

Affinity purification and characterization of anti-Tac(Fv)-C3-PE38KDEL: A highly potent cytotoxic agent specific to cells bearing IL-2 receptors.

A chimeric, single chain antibody fused immunotoxin, denoted anti-Tac(Fv)-C3-PE38KDEL, was engineered and expressed in Escherichia coli. The microbially expressed anti-Tac(Fv)-C3-PE38KDEL was solubilized from inclusion bodies using guanidine hydrochloride, and subsequently refolded in a redox buffer via thiol/disulfide exchange. The recombinant immunotoxin from the crude extract was purified employing receptor-affinity chromatography, which is based upon biological function and involved the immobilized p55 subunit of human IL-2 receptor. The cytotoxic activity of this immunotoxin was measured by the IL-2 dependent phytohemagglutinin (PHA) blast proliferation inhibition and HUT-102 protein synthesis inhibition assays, in which the IC50 values were 41.5 and 0.8 pM, respectively. The biochemical homogeneity and authenticity of the purified material were determined by gel permeation chromatography, amino acid composition and N-terminal sequence analyses, SDS-PAGE, isoelectric focusing, and Western blotting. The receptor-affinity-purified immunotoxin was shown to be highly effective in specifically killing cells bearing IL-2 receptors. Anti-Tac(Fv)-C3-PE38KDEL is a powerful immunosuppressant which may be a potentially useful therapeutic agent in the prevention of allograft rejection and in the treatment of autoimmune diseases. Another anticipated application of this fusion protein is as a chemotoxin in the treatment of some forms of cancer.

ADP Ribose Transferases↗
PubMed2005

Optimised affinity purification of polyclonal antibodies from hyper immunised ovine serum using a synthetic Protein A adsorbent, MAbsorbent A2P.

This report describes the applicability of a synthetic chromatography adsorbent for large-scale purification of polyclonal immunoglobulin G from hyper immunised ovine serum. Under optimised conditions, MAbsorbent A2P was shown to bind approximately 27 mg mL(-1) of ovine immunoglobulin from undiluted serum, with eluted IgG purities of >95%, minor levels of albumin (approximately 1%) and undetectable levels of leached ligand in the purified preparations. The results presented here indicate that the optimised affinity capture of immunoglobulin from ovine serum using MAbsorbent A2P is a feasible alternative to Protein A chromatography or sodium sulphate precipitation for the initial capture of antibodies from undiluted serum.

Animals↗