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PubMed1995

The expression, affinity purification and characterization of recombinant Pseudomonas exotoxin 40 (PE40) secreted from Escherichia coli.

Procedures have been devised for producing high yields of purified recombinant PE40, a protein important for the development of the anti-AIDS therapeutic, sCD4-PE40. PE40 is a truncated form of the bacterial toxin, Pseudomonas exotoxin A; it lacks the cell-binding domain, but retains domains II and III that are involved in membrane translocation and inhibition of protein synthesis in eukaryotic cells. Expression vectors in Escherichia coli encoding PE40 synthesized the product as a soluble protein under control of the T7 promoter. The expression capabilities of transformants of E. coli BL21(DE3) were highly unstable. Expression levels (secreted and total) were evaluated in shake flasks and at the 10-1 scale at 27 degrees C and 37 degrees C, and following induction by IPTG or lactose. The cell-free media from the batch process was applied directly to a Cibacron blue F3GA-chromatographic medium and PE40 was eluted by nicotinamide in high yield and purity. This purification strategy was based on the structural similarity of the blue dye to NAD, a natural substrate for domain III of PE40. Green and red dye-ligand chromatography steps removed nicotinamide as well as minor residual E. coli proteins from PE40. Reversed-phase liquid chromatography peptide maps of purified PE40 were characterized by electrospray ionization mass spectrometry to determine the sequence and verify disulfide bonding.

ADP Ribose Transferases↗
PubMed1986

Affinity purification of human tissue factor: interaction of factor VII and tissue factor in detergent micelles.

Tissue factor, a known initiator of blood coagulation, was found to be active in Triton X-100. A system consisting of tissue factor, factor VIIa, calcium ions, and coagulation factor X generated activated factor X at an appreciable rate. Based on this observation, we coupled human and bovine factor VII to a solid support. Each column bound tissue factor, solubilized in Triton X-100, in a species-specific manner. These interactions required calcium ions; when the columns were washed with detergent containing calcium ions, no tissue factor was eluted. When calcium ions were omitted from the eluant, tissue factor emerged as a sharp peak. Human tissue factor was extracted from an acetone brain powder into 2% Triton X-100. This extract, made 10 mM in CaCl2, was passed over a factor VII column. Human factor VII (1.2 mg) was coupled to 30 ml of Affi-Gel 15. This column bound approximately equal to 15 micrograms of human tissue factor. The eluted material was approximately equal to 25% pure. Final purification was achieved by gel filtration after chymotryptic digestion of contaminants. The tissue factor activity was stable to this treatment. The molecular weight determined by sodium dodecyl sulfate/PAGE (approximately equal to 46,000) was also unchanged by chymotrypsin. The final material was a single band on PAGE, demonstrated similar resistance to tryptic and chymotryptic digestion as bovine tissue factor, and had approximately the same specific coagulant activity as the previously purified bovine material. Tissue factor was also purified from human placenta, yielding a similar protein. A partial 28-residue sequence of the latter has been obtained.

Biological Assay↗
PubMed1994

Affinity purification of monospecific antibodies from polyclonal sera as a means for the identification of differentially expressed genes and proteins.

The identification and characterization of genes and proteins that are either cell-type specific or exhibit an otherwise spatially or temporally restricted expression pattern is a crucial step toward understanding the complex interactions between the different cell types in an organism. We demonstrate here that monospecific polyclonal antibodies purified from rabbit antisera by a simple Western blotting and reelution technique can be successfully used for the detection of such proteins, as well as the corresponding genes by screening cDNA expression libraries. This strategy is discussed as a supplement to other approaches, such as the generation of tissue-specific monoclonal antibodies and the "subtractive hybridization technique."

Animals↗
PubMed2002

Affinity purification of GST fusion proteins for immunohistochemical studies of gene expression.

Fusion proteins expressed in bacteria are often insoluble or inefficiently purified by standard procedures previously reported to work well for the non-fused proteins. We report here a simple but general procedure that can be used to quickly customize and optimize the purification of milligram quantities of most GST fusion proteins. For each new protein, this procedure determines the optimal conditions for solubilization with detergents in a bacterial lysate, binding to glutathione-agarose beads, and elution with different buffers. This approach was applied to three GST fusion proteins containing large fragments of the Hox transcription factors Lox2, Lox4, and Lox6 that had low solubility and poor elution when purified following published procedures. After optimization, purified proteins were obtained at high yield and successfully used to raise and purify antibodies for the study of the expression patterns of these genes in embryonic tissues.

Animals↗
PubMed2002

Affinity purification of diverse plant and human 14-3-3-binding partners.

Many proteins that bind to a 14-3-3 column in competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory roles for 14-3-3s in partitioning carbon among the pathways of sugar, amino acid, nucleotide and protein biosynthesis in plants. Our results also suggest that the current perception that 14-3-3s bind predominantly to signalling proteins in mammalian cells is incorrect, and has probably arisen because of the intensity of research on mammalian signalling and for technical reasons.

14-3-3 Proteins↗
PubMed1980

Immunoadsorbent affinity purification of the fifth component (C5) of human complement and development of a highly sensitive hemolytic assay.

The fifth component of complement (C5) has been isolated from human serum in fully hemolytically active form by immunoadsorbent and anion exchange column chromatography. The immunoadsorbent column was prepared by the covalent coupling of the purified IgG fraction obtained from monospecific goat anti-human C5 antiserum to CNBr activated Sepharose 4B. Establishment of appropriate conditions for the dissociation and elution of functionally active C5 from the immunoadsorbent column was of central importance in the development of this purification procedure. The C5 preparations exhibited final yields of 20--50% with 570--710-fold purification factors based on recovery of specific hemolytic activity. These preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide slab gel electrophoretic and immunochemical criteria. A C5-depleted reagent (C5D) was generated from the non-adsorbed protein containing fractions obtained subsequent to the passage of freshly drawn NHS plus 10 mM EDTA through the monospecific anti-C5 Sepharose 4B column. Upon reconstitution of C5D with Ca2+, Mg2+, and C1q, this reagent was utilized for the detection and quantitation of C5 hemolytic activity. The purified C5 preparations contained 1.5--2.5 x 10(12) effective molecules/mg protein and NHS expressed 0.5--2.0 x 10(11) effective molecules/ml.

Animals↗
PubMed1974

The reaction of an alpha-aza-amino acid derivative with chymotrypsin and its use as a ligand for covalent affinity purification.

1. Chymotrypsin is inactivated by N-acetyl-alpha-azaphenylalanine phenyl ester (phenyl N(2)-acetyl-N(1)-benzylcarbazate) in a stoicheiometric reaction. 2. The inactivation is reversible spontaneously (first-order rate constant is 1.2x10(-4)s(-1)) and accelerated by the presence of hydroxylamine. 3. Polymers based on polyacrylamide and carrying ligands containing the alpha-azaphenylalanine phenyl ester group were prepared. 4. Chymotrypsin reacts with these polymers and is removed by them from solution. Trypsin reacts less rapidly. 5. Chymotrypsin is slowly released from the polymer spontaneously and more rapidly on treatment with hydroxylamine. 6. The reaction of trypsin can be inhibited by competitive inhibitors. 7. Chymotrypsin was separated from trypsin by the selective bonding of chymotrypsin on to and its subsequent liberation from one of the polymers described.

Acrylamides↗
PubMed1987

[Production of monoclonal antibodies to an insulin degrading enzyme and affinity purification of the enzyme].

Monoclonal antibodies to a cytosolic insulin-degrading enzyme (IDE) were produced by fusing spleen cells from mouse immunized highly purified human erythrocyte IDE with mouse myeloma cells. Four monoclonal antibodies were identified by their ability to bind to 125I-insulin covalently linked to a cytosolic IDE from human erythrocytes. All four antibodies were found to remove more than 90% of the insulin-degrading activity from erythrocytes extracts, demonstrating that these antibodies were directed against an enzyme which accounts for most of this activity. By immunoprecipitation from metabolically labelled cells and immunoblot procedure, the enzyme from a variety of tissue was shown to be composed of a single polypeptide chain of apparent Mr = 110 kDa. One of these antibodies; 31H7 was coupled to Affi-Gel 10 and used for the purification of this enzyme. Immobilized antigen was eluted at more than 85% efficiency with buffers consisting of either pH2.3, 2.5M MgCl2 or with 6M urea. However, the antigen eluted under 6M urea retained the highest antigenecity (44%) and biological activity (8%) and the yield of the enzyme obtained from this procedure increased up to 17 fold as compared with the conventional method. NaDodSO4/polyacrylamide gel electrophoresis showed a single band of this protein with apparent Mr 110 kDa. These monoclonal antibodies and the purified enzyme will be useful tools for a better understanding of this enzyme, so may lead to the design of specific inhibitors of this enzyme that may be used to treat patients with excessive insulin degradation.

Animals↗
PubMed1989

Monoclonal antibodies against birch pollen allergens: characterization by immunoblotting and use for single-step affinity purification of the major allergen Bet v I.

Two monoclonal antibodies against birch pollen proteins were produced by immunizing BALB/c mice with birch pollen extract. In immunoblotting experiments, antibody BIP 1 reacted with a 17-kilodalton (kD) protein considered to represent the major birch pollen allergen Bet v I. A second monoclonal antibody, BIP 3, reacted with 3 different birch pollen proteins of molecular weights 32, 36 and 68 kD of which the 36- and 68-kD proteins corresponded to minor allergens of birch pollen. Two-dimensional electrophoresis/immunoblotting experiments revealed that BIP 1 reacted with all Bet v I isoallergens, also identified by human IgE antibodies. Using BIP 1 coupled to Sepharose 4B as reverse immunosorbent, Bet v I was obtained in a single-step procedure and characterized as single band by SDS-PAGE.

Allergens↗
PubMed1992

Active-site characterization of S1 nuclease. I. Affinity purification and influence of amino-group modification.

A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3'-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.

Animals↗
PubMed1993

Dye-ligand affinity purification of human complement factor B and beta 2 glycoprotein I.

The rapid purification of human factor B using dye-ligand chromatography is described. The 50% ammonium sulphate supernatant of fresh human serum is equilibrated in pH 7.4, 25 mM Tris buffer containing 0.5 mM CaCl2 and 0.5 mM MgCl2, with 25 mM sodium caprylate and chromatographed on Cibacron Blue F3GA-agarose. Caprylic acid binds to the fatty acid binding site of albumin, preventing it from binding to the resin which thus retains a high capacity for binding factor B. Factor B together with the homologous protein beta 2I are eluted from the column by a linear gradient of KCl. Subsequent NaCl gradient FPLC on Hiload S-Sepharose, equilibrated in 10 mM potassium phosphate, 5 mM EDTA, pH 7.0, provides both factor B and beta 2I in homogeneous form.

Chromatography, Affinity↗
PubMed1985

Monoclonal antibodies to murine interferon-gamma: affinity purification and molecular characterization of murine interferon-gamma.

Monoclonal antibodies (MAb), AN-18.17.24, obtained by fusing the murine myeloma cell line Ag8.653 with spleen cells from rats immunized with murine interferon gamma (IFN-gamma) produced by the phorbol myristic acetate(PMA)-stimulated T-cell lymphoma L12-R4, were shown to be specific for MuIFN-gamma. An immunoadsorbent column was prepared and used for large-scale purification of MuIFN-gamma produced either by PMA-stimulated L12-R4 cells or by normal T-lymphocytes stimulated with Con A and interleukin 2. The purified product from L12-R4 cells, which retained biologic activity, was made up of two proteins (Mr = 16,500 and 17,500) as determined by sodium dodecyl sulfate gel electrophoresis. By contrast MuIFN-gamma produced by normal T-lymphocytes resulted in two proteins with Mr of 20,600 and 21,800 as determined by sodium dodecyl sulfate gel electrophoresis. These results indicate that MuIFN-gamma produced by L12-R4 tumor cells or by normal T-lymphocytes differ in their molecular weight, probably because of different degrees of glycosylation of the same molecule.

Animals↗
PubMed1991

Obviation of drug resistance and affinity purification of P-glycoprotein by isoquinolinesulfonamides.

A newly synthesized isoquinolinesulfonamide named H-85; N-[2-[N-formyl-N-[[3-(4-chlorophenyl)-2-propenyl] amino] ethyl]-5-isoquinolinesulfonamide was found to reverse drug resistance in multidrug resistant P388 murine leukemic cells (P388/ADR). The energy-dependent extrusion of [3H]vinblastine from P388/ADR-cells was significantly suppressed by 10 microM H-85 but not so the efflux from the sensitive P388 cells. A 140-kDa protein overexpressed in P388/ADR cells was photoaffinity labeled with a vinblastine analog; N-(P-azid-[3-125I]salicyl-N'-(beta-aminoethyl) vindesine and H-85 selectively inhibited photolabeling of the 140-kDa protein. This 140-kDa protein was purified to apparent homogeneity by succeeding steps of phosphocellulose, DEAE-cellulose, and W-66 (a derivative of H-85)-coupled sepharose chromatography. The purified 140-kDa protein proved to be immunopositive with the P-glycoprotein-specific monoclonal antibody, C219.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells.

Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3'-dithiopropionyldiaminopentyl methotrexate and/or folate), in which carboxyl groups of the functional components are joined by a disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by high pressure liquid chromatography and mass spectrometry. These bifunctional, dissociable probes were utilized for the single-step purification to homogeneity of two folate transport proteins (43 and 39 kDa) from L1210 cells. Treatment of the 39-kDa protein with peptide N-glycosidase F produced a smaller component (32 kDa); the 43-kDa protein, conversely, was unchanged by this procedure. When the 39-kDa transporter in intact cells was labeled with a fluorescein derivative of folate and then treated with phosphoinositol-specific phospholipase C, complete loss of fluorescence was observed. Alternatively, there was no change in fluorescence when the 43-kDa transporter was labeled with a fluorescein derivative of methotrexate and treated with the enzyme. These results indicate that the 43-kDa transporter is a nonglycosylated, integral membrane protein, whereas the 39-kDa counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.

Animals↗
PubMed2004

Two-step affinity purification of the hepatitis C virus ribonucleoprotein complex.

Positive-strand RNA viruses replicate their RNA genome within a ribonucleoprotein (RNP) complex that is associated with cellular membranes. We used a two-step method of purification to isolate hepatitis C virus (HCV) RNP complexes from human hepatoma cell line Huh7, which stably expresses HCV subgenomic replicons. The procedure involved hybridization of replicon-expressing cellular lysates with oligonucleotides tagged with biotin and digoxigenin at their respective termini complementary to subgenomic replicon RNA followed by avidin-agarose enrichment of the mixture and subsequent immunoprecipitation of biotin-eluted material with anti-digoxigenin antibody. The immunoprecipitates were immunoblotted with antisera against HCV nonstructural (NS) proteins. The analysis revealed the association of all the HCV NS proteins (NS3, NS4a, NS4b, NS5a, and NS5b) that are encoded by the subgenomic replicon RNA. The HCV RNP complex migrated in a native polyacrylamide gel with an approximate molecular mass of 450 kD. The association of these viral proteins in the RNP complex reinforces the widely acknowledged notion that RNA viruses accomplish replication within a membranous RNP complex.

Chromatography, Affinity↗
PubMed1991

Immuno-affinity purification and characterization of the alpha subunits of G0 type G proteins from various bovine tissues.

Polyclonal antibodies to the alpha subunits of G0 type G proteins (G0 alpha) were coupled to agarose gel and used to isolate G0 alpha from solubilized membranes of various bovine tissues. The cholate extract of membranes was applied to the anti-G0 alpha-agarose gel column. The column was washed extensively, then bound proteins were eluted at a neutral pH using a commercial ActiSep Elution Medium. The proteins in the eluate displayed a single band of 39 kDa on SDS-polyacrylamide gel electrophoresis. They bound to GTP gamma S and were ADP-ribosylated by pertussis toxin. The yield of the immunoreactive G0 alpha from the extract was about 40%. Isoelectric focusing, immunoassay and peptide mapping analysis of the G0 alpha-like proteins purified from the heart and adrenal medulla indicated that these proteins were very similar to the alpha subunit of a minor subtype of G0 in the brain which was previously referred to as G0 * alpha.

Adrenal Medulla↗
PubMed1998

Use of a heterodimeric coiled-coil system for biosensor application and affinity purification.

The two-stranded alpha-helical coiled-coil is now recognized as one of nature's favorite ways of creating a dimerization motif. Based on the knowledge of protein folding studies and de novo design model systems, a novel heterodimeric coiled-coil protein was synthesized. The heterodimeric E/K coiled-coil was constructed with two distinct peptides (E and K) that will spontaneously associate into a full helical coiled-coil structure in solution. Equilibrium CD, NMR and real time biosensor kinetics experiments showed that the E/K coiled-coil is both structurally (deltaG(unfold)=11.3 kcal/mol) and kinetically (Kd approximately 1 nM) stable in solution at neutral pH. The engineered coiled-coil had been applied as a dimerization and capture domain for biosensor based applications and used in an expression/detection/affinity chromatography system. Specific test examples demonstrated the usefulness of the E/K heterodimeric system in these applications. The universality of coiled-coil as a dimerization motif in nature and our ability to design and synthesize these proteins suggest a wide variety of applications.

Amino Acid Sequence↗
PubMed1987

Monoclonal antibodies to human tumor necrosis factors alpha and beta: application for affinity purification, immunoassays, and as structural probes.

Monoclonal antibodies were produced against two structurally related tumor necrosis factors (TNFs), TNF-alpha (previously called tumor necrosis factor) and TNF-beta (previously called lymphotoxin). The potential of these antibodies for the purification of TNFs, the development of specific immunoassays, and for defining the antigenic and functional domains of these cytokines was investigated. None of the monoclonal antibodies cross-reacted with both TNF-alpha and TNF-beta, or reacted with synthetic peptides which represented several of the regions of homology between these cytokines. Neutralizing monoclonal antibodies were utilized as immunoadsorbents to purify recombinant TNF-alpha and TNF-beta from E. coli lysates. TNFs purified by this method were greater than 98 percent pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited specific activities that were the same as TNFs isolated from natural sources using conventional chromatographic techniques. In addition, specific ELISA assays were developed that could detect less than 1 ng/ml of TNF-alpha or TNF-beta, and in contrast to bioassays, could discriminate between these related cytokines.

Animals↗