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PubMed2005

Bio-orthogonal affinity purification of direct kinase substrates.

Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second nonenzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the alkylated phosphorothioate epitope recognize these labeled substrates, but not alkylation products of other cellular nucleophiles. This strategy is demonstrated with Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates.

Adenosine Triphosphate↗
PubMed1996

Characterization of monoclonal antibodies recognizing human merosin and their use in affinity purification of native merosin.

Two monoclonal antibodies (MAbs) to human placenta laminin (pl-LAM), 1D8 (IgG1) and 6G5 (IgG2b) were generated and shown by ELISA and immunoblot analysis to recognize only native pl-LAM, but not denatured, reduced pl-LAM or mouse EHS laminin. Intact pl-LAM was easily isolated and purified in large scale from human placenta by 1D8-conjugated affinity chromatography. Electrophoretic analysis of the purified pl-LAM revealed the presence of a major 750-kDa component composed of 320-, 220-, and 200-kDa polypeptides and a minor 800-kDa component composed of 320-, 240-, and 220-kDa polypeptides. Neither molecule had a 400-kDa component corresponding to the A chain. It has already been shown that the 320-kDa polypeptide is identical to the M chain of human merosin (Hori et al. J. Biochem. 1994;116:1212-1219). Electron microscopy revealed that isolated merosin was composed of three short arms and one long arm. By immunohistochemistry, MAbs showed positive staining in human adult kidney and liver. These results indicate that these MAbs recognize only native merosin and can be used to study merosin structure and function by rapid purification of native merosin and by immunohistochemical analysis.

Animals↗
PubMed1985

Purification, affinity to anti-human arginase immunoglobulin-Sepharose 4B and subunit molecular weights of mammalian arginases.

The affinities of anti-human liver arginase antibodies raised in rabbits to liver arginases from man, bovine, pig, dog, guinea pig, rat and mouse were investigated by Scatchard analysis of the binding of the arginases from crude liver extracts to Sepharose-bound immunoglobulins. All arginases bound with good affinity, but the binding capacities of the immunosorbent for the enzymes from various species decreased with decreasing phylogenetic relationship of the species. Arginase from murine peritoneal macrophages did not bind to the immunosorbent at all. A simple two-step purification method for the liver arginases of all species mentioned above is given. All arginases were purified to electrophoretical homogeneity. The molecular weights of their subunits were estimated.

Animals↗
PubMed1989

Affinity purification of IgG subclasses and the distribution of thyroid auto-antibody reactivity in Hashimoto's thyroiditis.

To delineate accurately the IgG subclass distribution of thyroid auto-antibodies, sera from nine patients with Hashimoto's thyroiditis were fractionated into IgG subclasses by complete depletion of the other IgG subclasses on affinity columns. All IgG subclass fractions contained thyroglobulin and microsomal (or thyroid peroxidase) antibody activity, although when compared to the total serum concentrations of IgG subclasses, IgG4 antibodies were overrepresented. However, in contrast to recent studies, this particular subclass never predominated--IgG4 antibody levels being exceeded by those of the IgG1 and IgG2 subclasses; it seems likely that these differences relate to varying sensitivity for different subclasses in previously used assay methods. This pattern of subclass activity differed from that of tetanus toxoid antibodies, which were found in six subjects. There was no light chain restriction within any subclass, showing that the overproduction of IgG4 thyroid antibodies is not of monoclonal origin. The functional affinity of subclasses for both thyroid antigens varied between patients, but IgG2 subclass fractions showed the highest functional affinity in the majority of samples. We also found that IgG2 subclass thyroid antibodies were ineffective in eliciting antibody-dependent cell-mediated cytotoxicity, as distinct from the other three subclasses. Our results show that thyroid antibodies are less restricted in their IgG subclass distribution and patients are less heterogeneous than previously described. Moreover, IgG2 thyroid antibodies are quantitatively important and differ in relative functional affinity and effector function from IgG1 and IgG4 thyroid antibodies.

Adult↗
PubMed1987

Affinity purification of renal dipeptidase solubilized with detergent.

A new method is described for purification of the dehydropeptidase I enzyme, renal dipeptidase (EC 3.4.13.11). The six steps used are homogenization of the tissue, extraction with Triton X-100, sedimentation of insoluble material, and ion-exchange, size-exclusion, and affinity chromatographies. The use of Triton X-100 to solubilize the enzyme is a major advantage over the previously described procedure using n-butanol and results in both improvements in yield and interexperimental consistency. Also, the affinity column method we have employed results in higher recovery of active enzyme at the final step and a product which is apparently homogeneous. The method has general utility for this class of enzymes.

Animals↗
PubMed2000

Cloning of Plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor 1,4-bis[3-[N-(cyclohexyl methyl)amino]propyl]piperazine..

A series of 10 1,4-bis(3-aminopropyl)piperazine compounds was found to display antiplasmodial activity with 50% growth inhibition between 30 and 250 nM, on three Plasmodium falciparum strains differently sensitive to chloroquine. By affinity chromatography using one of these compounds, a 52-kDa protein was isolated from P. falciparum, microsequenced and cloned. It corresponded to a single copy gene encoding a 453 amino acid protein displaying the typical features of protein disulfide isomerases, a thiol metabolizing enzyme belonging to the thiol: disulfide oxidoreductase superfamily, which was not previously described in malarial species.

Amino Acid Sequence↗
PubMed1996

Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae [corrected].

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiella pneumoniae. Chromatography using a novel 5'-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46-50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 microM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 microM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

Amino Acid Sequence↗
PubMed1977

Affinity purification methods. V1. A novel and rapid isolation procedure for lysozymes.

Lysozymes from animal and plant sources have been purified on agarose derivatives containing a phenylacetyl ligand. While this adsorbent can also bind chymotrypsin, the use of imidazole permits separation of lysozymes from the protease. The phenylacetyl-agarose is a general affinity sorbent bearing a ligand of non-biochemical origin in contrast to those, for example, which use nucleotide cofactors. By careful selection of desorption conditions, different enzymes can be purified with the same sorbent. Results also suggest that binding of lysozymes to an affinity ligand does not require a leash structure between ligand and support matrix.

Chromatography, Affinity↗
PubMed2001

Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.

Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.

Alkaline Phosphatase↗
PubMed1989

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase.

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.

Adenosine Triphosphate↗
PubMed2002

Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry.

The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing.

Chromatography, Affinity↗
PubMed1992

Affinity purification of antibodies using immobilized FB domain of protein A.

A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A.

Amino Acids↗
PubMed1992

Affinity purification of human tau proteins and the construction of a sensitive sandwich enzyme-linked immunosorbent assay for human tau detection.

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.

Animals↗
PubMed2004

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry.

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.

Amino Acid Sequence↗