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At least 631 records · Page 35Linked to original sources
PubMed1999

A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate.

The inhibition of Helix pomatia arylsulfatase by the synergistic combination of N-acetyl-l-tyrosine ethyl ester and vanadate has been extended to affinity chromatography for purification. In the presence of vanadate, l-tyrosine ethyl ester (TEE), immobilized on CH-Sepharose 4B retained arylsulfatase from the digestive juice or lyophilized powder of H. pomatia. No enzyme was retained without vanadate or with arsenate or phosphate. Arylsulfatase was eluted from the column matrix by removing the vanadate to less than 50 microM with buffer containing EDTA to chelate the vanadate. Escherichia coli alkaline phosphatase and potato acid phosphatase, two enzymes which are inhibited by vanadate but not by the vanadate-TEE complex, were not retained by the immobilized TEE under any conditions used. The sulfatase activity was completely separated from contaminating glucuronidase activity present in the crude enzyme extracts. The Ki for the immobilized vanadate-TEE system was found to be 5.0 x 10(-7) M with a capacity of 25 mg/ml swollen gel. A purification of greater than 40-fold from the lyophilized powder of H. pomatia (Sigma Type H-5) was achieved using this technique. The Ki/Keq of other phenols with vanadate were determined in a 96-well plate format as an example of a rapid screening technique that could be extended to other phosphoryl and sulfuryl-transfer enzyme classes.

Animals↗
PubMed1977

Complementable fraction and complemented enzyme of mutant M15 from Escherichia coli: partial purification by affinity chromatography.

The technique of affinity chromatography has been used in the partial purification of complementable fractions and complemented enzyme of beta-galactosidase from Escherichia coli mutant M15. The crude extract of mutant M15 was incubated with fragment CM-B. The complemented enzyme and complementable fractions were passed through a small column of p-aminophenyl-beta-D-thiogalactoside to which inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. Phosphate buffer with NaCl was used to elute the complementable fractions from the column. Sodium borate buffer was used to elute the bound complemented enzyme from the affinity support. The results of this study show that 100% of the complemented enzyme was bound to the column. The partially purified enzyme had the same position in disc gel electrophoresis as beta-galactosidase from E. coli.

Chromatography, Affinity↗
PubMed1998

Affinity chromatography purification of mitochondrial inner membrane proteins with calcium transport activity.

Immobilized calcium affinity chromatography was used to obtain a preparation enriched in calcium transporters from Triton X-100 extracts of rat liver mitochondria inner membranes (PPCT). The PPCT were reconstituted into preformed asolectin liposomes which contained 120 mM KCl as internal high K+ medium. 45Ca2+ uptake into proteoliposomes was studied under conditions favoring electrophoretic uptake, and H+i/45Ca2+o or Na+i/45Ca2+o exchange, to test for the presence of the three calcium transport modes present in mitochondria. 45Ca2+ uptake in liposomes was studied in parallel. Na+i/45Ca2+o exchange activity was not detectable. H+i/45Ca2+o exchange activity measured in the presence of a pH gradient (acid inside) obtained after suspension in low K medium in the presence of nigericin, was 100-200 nmoles 45Ca2+ per mg protein in 30 s. 45Ca2+ uptake in voltage-dependent assays (a K+ diffusion membrane potential induced by valinomycin in the presence of methylamine) was not electrophoretic since it was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and probably due to secondary Ca2+/H+ countertransport. H+i/45Ca2+o uptake showed a saturable component at around 80 microM Ca and was coupled to an increase in internal pH in pyranine-loaded PPCT proteoliposomes. 45Ca2+ uptake in PPCT proteoliposomes could also be driven by a pH gradient obtained by raising external pH in high K+ medium. The results are consistent with the presence of a functional nH+/Ca2+ antiporter. Polyclonal antibodies raised against the PPCT were able to immunoprecipitate the H+/45Ca2+ uptake activity and recognized two major bands in the PPCT with molecular masses of about 66 kDa and 55 kDa. This is the first report of a partial purified protein(s) which may represent the H+/Ca2+ exchanger of the inner mitochondrial membrane, and represents an important step towards its identification.

Animals↗
PubMed2001

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins.

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.

Base Sequence↗
PubMed1980

Mouse uterine alkaline phosphatase: improved purification by affinity chromatography and further characterization of the enzyme.

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0.8% (v/v) Triton X-100 and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 000 +/- 10 000 was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme. Some metalloenzyme properties of the phosphatase were also studied and it was demonstrated that both Mg2+ and Zn2+ ions are necessary for hydrolytic activity. Treatment with neuraminidases retarded the anodal migration of the enzyme during electrophoresis on cellulose acetate membranes but did not influence its activity or catalytic properties. These results suggest that the uterine alkaline phosphatase is a sialoglycoprotein. The sialic acid residues, however, do not appear to constitute part of the active centre of the enzyme. In addition, the optimum pH for activity depended on substrate concentration and decreased with decreasing substrate concentration. Apparent Km values also depended on variations in pH and decreased with decreasing pH. Plots of pKm versus pH revealed a functional group with a pK value of 9.45. The enzyme also hydrolysed a variety of compounds having either phosphomonoester or pyrophosphate linkages and was inactivated after heating at 60 degrees C for 15 min. The activation energy, determined from a linear Arrhenius plot, was 50.1 kJ mol-1.

Alkaline Phosphatase↗
PubMed1985

Partial purification of dopamine D2 receptors using lectin affinity columns.

Dopamine D2 receptors, detected by [3H]spiperone binding, were solubilized from bovine caudate nucleus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard. The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmacological properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D2 dopamine receptor is therefore a glycoprotein.

Acetylglucosamine↗
PubMed2001

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA.

Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for the removal of proteins without histidine tag from IMAC columns. Four histidine-tagged recombinant proteins/protein complexes were purified to homogeneity from cell culture medium of insect cells by using an EDTA washing buffer. The presence of a low concentration of EDTA in washing buffers during IMAC may have a general application in the purification of histidine-tagged proteins.

Chelating Agents↗
PubMed1993

Purification of vacuolar ATPase with bafilomycin C1 affinity chromatography.

We have developed a fast and simple affinity purification method for vacuolar ATPases (V-ATPases) using bafilomycin C1 -coupled cellulose column. The purified protein from solubilized chicken medullary bone microsomal vesicles contains 8 subunits at sizes of 72, 65, 55, 41, 34, 20, 17 and 15 kDa. This subunit structure is typical to V-ATPases. Although the enzyme obtained is inactive, this method is useful in search of various isoforms of known V-ATPase subunits. We also present further evidence that the binding site of bafilomycin is located in the 17 kDa proteolipid subunit of V-ATPase.

Animals↗
PubMed2005

An artificial protein L for the purification of immunoglobulins and fab fragments by affinity chromatography.

The development and characterization of an artificial protein L (PpL) for the affinity purification of antibodies is described. Ligand 8/7, which emerged as the lead from a de novo designed combinatorial library of ligands, inhibits the interaction of PpL with IgG and Fab by competitive ELISA and shows negligible binding to Fc. The ligand 8/7 adsorbent (Ka approximately 10(4) M(-1)) compared well with PpL in binding to immunoglobulins from different classes and sources and, in addition, bound to IgG1 with K and lambda isotypes (92% and 100% of loaded protein) and polyclonal IgG from sheep, cow, goat and chicken. These properties were also reflected in the efficient isolation of immunoglobulins from crude samples.

Bacterial Proteins↗
PubMed1982

Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli.

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.

Animals↗
PubMed1981

Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield.

Cathepsin D was purified by two-step affinity chromatography on concanavalin A-- and pepstatin--Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin--Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

Animals↗
PubMed1998

The anti-MUC1 monoclonal antibody BCP8 can be used to isolate and identify putative major histocompatibility complex class I associated amino acid sequences.

MHC class I molecules were isolated from the MUC1-positive human breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibodies (MAb) W6/32. Acid-eluted peptides from the class I molecules were separated twice by high-performance liquid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA. A peak with strong and specific BCP8 reactivity was found in fractions eluting at 16.5-17.5 min. The protocol used for the MUC1+ pancreatic adenocarcinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity purifications of class I molecules using MAb W6/32, followed by affinity purification of HLA.A2 molecules by the HLA.A2.1-specific MAb, MA2.1, and high-performance liquid chromatography fractionation of the acid-eluted material. A single peak with MAb BCP8 reactivity was noted at 18-19 min. The protocol for the MUC1+ breast adenocarcinoma cell line SKBr-3 (HLA.A11,B40), which used A11- and B40-specific MAbs, also resulted in the detection of BCP8-specific peaks at approximately 18-19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MUC1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to cause strong class I up-regulation in T2 cells as well as to serve as an epitope for CTL generated in a primary in vitro immune response. These studies suggest that MUC1-derived peptides are processed and presented in the context of MHC class I molecules on the surface of tumor cells and support the use of MAb BCP8 to further define MHC class I associated MUC1 motifs.

Antibodies, Monoclonal↗
PubMed1987

Estrogen receptor purification by affinity chromatography using an orange triazine dye.

A rapid two-step procedure was devised for the purification of the estrogen receptor from the calf uterus. A 900- to 1700-fold purification of the estrogen receptor was obtained using ammonium sulfate precipitation followed by dye affinity chromatography with Reactive Orange 14 immobilized to Sepharose. The Reactive Orange 14-Sepharose was used to purify the estrogen receptor in the presence or absence of estradiol as well as to purify the progesterone receptor. The purified estrogen receptor retained its estradiol- and DNA-binding properties and sedimented into sucrose gradients as the 5 S receptor dimer. The Reactive Orange 14-Sepharose is easily prepared and offers a higher yield and purity of the estrogen receptor than that afforded by estrogen- or heparin-Sepharose chromatography.

Animals↗
PubMedDate not supplied

Immunoglobulin purification by affinity chromatography using protein A mimetic ligands prepared by combinatorial chemical synthesis.

Affinity chromatography using protein A from Staphylococcus aureus as the ligand has been widely used for the isolation of immunoglobulin G (IgG) from various species. Since ligand leakage from the affinity support can occur, time consuming analytical controls are required to detect the presence of contaminants associated with the isolated IgG prior to its use for therapeutic purpose in humans. Besides, protein A is an expensive bacterial product, whose isolation involves complex and labor intensive procedures. Combinatorial chemistry enables the synthesis of a wide variety of ligands within a short period of time. Therefore, chemically defined, stable and inexpensive ligands, which can mimic the action of protein A, have been developed to isolate immunoglobulins. Two different types of ligands, synthesized following the techniques of combinatorial chemistry, have been used to isolate immunoglobulins. One of them is a synthetic peptide (TG 19318) comprising four identical tripeptide chains linked to a central polylysine core. Immobilized TG 19318 has been used to isolate polyclonal and monoclonal antibodies of different classes (IgG, IgM, IgA, IgE) from different sources (serum, ascities and cell supernatants) and species. The ligand has a binding capacity that can reach upto 25mg IgG/mL of the support. It is stable when treated with sanitation agents such as ethanol and 0.1 M sodium hydroxide. Computer-aided molecular design and combinatorial chemistry have been used to develop an IgG binding affinity ligand (22/8), which consists of two organic aromatic amines (3-aminophenol and 4-amino-1-napthol) linked to a scaffold of cyanuric chloride (triazine). Ligand 22/8 displayed wider specificity than protein A, as it isolated IgG from a number of species, the order of adsorption being human> chicken > cow > rabbit > pig > horse > rat > goat > sheep > mouse. It showed an apparent binding capacity of 51.9 mg IgG/g moist gel and can isolate human IgG from plasma in 60% yield with a purity of 92%. The ligand is stable, as it withstood incubation in 1M NaOH for a week without loss of binding capacity for IgG. These findings suggest that synthetic affinity ligands, which are inexpensive, stable and specific can facilitate the purification of immunoglobulins in a cost-effective manner.

Animals↗
PubMed2006

A biosensor-based approach toward purification and crystallization of G protein-coupled receptors.

Biacore technology was used to develop an affinity purification method and screen cocrystallization conditions for the chemokine receptor CCR5. We characterized the binding of nine HIV gp120 variants and identified a truncated construct (YU2DV1V2) that bound CCR5 independent of CD4. This construct was used in an affinity purification step to improve the activity of detergent-solubilized receptor by approximately 300%. The biosensor was also used to screen receptor binding activity automatically under 50 different crystallization conditions. We found that high-molecular-weight polyethylene glycols (PEGs 4,000 and 8,000 Da) most often stabilized the receptor and improved complex formation with potential cocrystallization partners such as conformationally sensitive monoclonal antibodies and gp120. Our results show how biosensors can provide unique insights into receptor purification methods and reveal the effects of crystallization conditions on complex formation. Importantly, these methods can be readily applied to other systems.

Antibodies, Monoclonal↗
PubMed1997

Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification.

Several methods have been developed for the measurement of gelatinase activity from various tissues using detergent extraction. Gelatin-affinity chromatography has been employed for the large-scale purification of gelatinases from conditioned medium obtained from cultured cells. The objective of this paper was to develop a rapid method whereby gelatinase activity could be extracted from regional brain tissues without tedious, intervening purification steps. After Triton X-100 extraction and gelatin-Sepharose 4B purification of rat brain tissue extracts, two major activities were observed on gelatin zymograms. These were identified as gelatinase A and B using co-migration with astrocyte-derived enzymes and inhibition of activity by tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). The non-ionic detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) were equally effective in extracting activities from brain tissue. Little difference in recovery was observed among 0.1, 1 and 10% concentrations of Triton X-100. The method developed here was capable of recovering gelatinase activities from rat brain tissue over a 4-10-fold range using gelatin zymography for the measurement of activity. It is possible that this method may be modified for the measurement of gelatinases in tissues such as biopsy samples of gliomas or astrocytomas or other cancers where gelatinases are thought to play a role in tumor invasion and/or metastasis.

Animals↗