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An immunospecific affinity chromatographic method for purification of human pregnancy zone protein (PZP) directly from serum is described. Highly purified goat-anti-human PZP-immunoglobulin was applied as a ligand. Recovery of PZP varied from 56--75%. The impurities constituted maximally 5--10% of the total protein in the eluate. The purification factor was approximately 100.
An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.
The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated 'reactive' lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.
Seven nucleotides linked through the gamma-phosphate to diamine hydrocarbons were synthesized and coupled to Sepharose for use in protein purification affinity chromatography. The synthesis involved converting the nucleotides to nucleoside-5'- trimetaphosphates using dicyclohexyl carbodiimide, followed by nucleophilic ring opening of the trimetaphosphate with an alpha, omega-diamino hydrocarbon to generate a gamma-phosphoamide linkage in each nucleotide.
BACKGROUND: Due to the absence of alphaGAL epitopes, humans and galactosyltransferase knock-out (GALT/ KO) mice express high levels of anti-Gal antibodies. We describe the properties of mouse anti-GAL antibodies. METHODS: Anti-GAL IgG antibodies were quantified by affinity purification. Antibody affinities and avidities were determined in direct binding and competition assays. Antibody-mediated rejection was investigated using hyperimmunized GALT/KO mice as recipients of GAL+ heart allografts. RESULTS: In young GALT/KO mice the levels of anti-GAL antibodies were low. Immunization of GALT/KO mice resulted in increased anti-GAL antibody expression. In mouse serum 0.6% of IgG was specific for alphaGAL compared to 0.5% in human serum. The avidity of purified mouse and human anti-GAL IgG was 30 and 6 nM, the affinity 15 and 50 microM, respectively. The isotype distribution in mouse and human anti-GAL IgG appeared to be similar to the isotype distribution in normal sera. The affinity of mouse and human anti-GAL IgM was 150 and 750 microM, respectively. Immunized GALT/KO recipients of GAL+ heart transplants rejected their grafts within 2 hr although nonimmunized GALT/KO mice retained their grafts for up to 6 days. Immunohistological examination of the rejected GAL+ hearts revealed massive deposition of IgM and IgG on endothelial cells of the graft with a concomitant deposition of complement. CONCLUSIONS: Our studies demonstrate that anti-GAL antibodies from immunized GALT/KO mice bind alphaGAL with an avidity/affinity similar to human anti-GAL antibodies and are able to induce hyperacute rejection of GAL+ heart allografts.
We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA--Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9-2.4% of the amount of protein applied to the column. They contained 20 +/- 1.3% hexose and 1.7 +/- 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate--polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50-55, 75, 95, 130, and 155 kdaltons along with minor bands of 20-40, 42, 45, 60-65, 175, and 200-250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 10(6) cells/mL in the presence of PHA. A 50% inhibition was observed when 20 micrograms/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 microgram/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line.(ABSTRACT TRUNCATED AT 250 WORDS)
Monoclonal antibodies against streptokinase as ligands were coupled to different matrices. Both CNBr-activated Sepharose and macroporous bead cellulose activated by carbonochloridate revealed as suitable for purification of streptokinase by affinity chromatography. Immunoadsorbents with a higher concentration of the coupled ligand were more effective than those with a lower. For streptokinase optimal conditions of binding and elution without negative influence on structure and activity were ascertained. A buffer with slight alkaline pH was successful for desorption. Using this method it was possible to obtain pure streptokinase from several streptokinase containing media.
Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.
The reinvestigation of the affinity chromatographic method of purifying papain has been carried out. It has been reported that papain could be purified by taking advantage of the affinity of the enzyme for the insolubilized peptide inhibitor, agarose-Gly-Gly-Tyr(Bz)-Arg. Using pure tetrapeptide obtained commercially and standard coupling procedures, a significant purification of papain could not be achieved. Both active and nonactivatible enzyme bound to a column prepared in this manner were eluted together by the use of deionized water. An affinity medium with properties similar to those reported by Blumberg et al. was obtained by removal of the benzyl group on tyrosine prior to coupling with agarose. The deprotected tetrapeptide was also synthesized by an independent route and inhibition constants for the binding of the protected and deprotected tetrapeptide to papain were determined in kinetic experiments.
A simple and efficient activation-affinity purification system was developed to obtain thrombin from recombinant CHO cells expressing human prothrombin. In this method, a controllable process for the activation of recombinant prothrombin is directly coupled with a purification strategy for the recombinant thrombin generated. At a constant flow rate and with a contact time limited to few seconds, recombinant prothrombin was filtered through immobilized trypsin. In a closed flow system, the recombinant thrombin generated was filtered through newly designed thrombin-specific affinity gels. Hirudin, the most specific thrombin inhibitor, and hirudin-based peptides were covalently immobilized to Sepharose, thus creating thrombin-specific affinity gels that immediately absorb the thrombin generated from the activation mixture. Prothrombin and incompletely activated molecules did not bind to the affinity gel and were recirculated for a further activation cycle. Due to the specificity of the affinity gels for thrombin and the elimination of thrombin from the activation mixture, proteolytic degradation and autocatalytic inactivation of the recombinant thrombin was prevented. Recombinant thrombin was isolated from the hirudin-based affinity gels by chaotrope salt elution, resulting in high yields of highly pure, active thrombin. Affinity purification of thrombin was not deleteriously affected by contamination of the starting material with other proteins. Activation and affinity purification were equally effective for recombinant and human plasma-derived prothrombin as well as for human and recombinant thrombin.
Anti-enterotoxin immunoglobulins immobilized on CH-Sepharose or CNBr-Sepharose were used for affinity chromatography purification of Clostridium perfringens enterotoxin. Cell extracts containing enterotoxin or partially purified toxin preparations were applied to the column and nonspecifically-bound protein was eluted. NaOH was used to elute specifically bound toxin. The purity of enterotoxin purified by Sephadex G-100 chromatography followed by affinity chromatography appears similar to toxin highly purified by conventional means. The procedure can be used successfully for the rapid (less than 2 h) purification of small amounts of enterotoxin.
A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.
An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.
In order to further molecular investigations on the binding capacity of acetylcholine receptors, a method was developed for the affinity chromatography of the nicotinic acetylcholine receptor. Reversibly binding cholinergic ligand groups were used as affinity ligands, instead of the well known snake venom alpha-toxins. These ligands are small in size, chemically well defined and fixed to long spacer chains (at least 40 nm). One ligand, with a pharmacologically stabilizing effect on the receptor, was a derivative of gallamine. Another, with a depolarizing effect, resembled carbamoylcholine and a third was a derivative of decamethonium. The receptor proteins were isolated from Torpedo marmorata electric organs. Preparation included solubilization with a non-ionic detergent, alkaline treatment to extract peripheral membrane proteins and affinity purification. The receptor proteins eluted from the three affinity resins were identical in their assembly of subunits (alpha, beta, gamma and delta) but of different purity. Receptor proteins were obtained on a large scale within a short time and under mild conditions for elution with the affinity ligands of the decamethonium or the gallamine type. This was a considerable advantage compared to the use of alpha-bungarotoxin.
The specific interaction between biotin and avidin was exploited in the affinity purification of solid-phase synthesized peptide libraries. During peptide library synthesis, by means of the single-resin method in which coupling on variable positions is carried out using an equimolar mixture of amino acids, biotin was used to cap the unreacted amino groups remaining after coupling of the equimolar amino acid mixture. The following synthesis and deprotection procedures were performed as usual in tert,-butyloxycarbonyl chemistry. The purification of the peptide mixture containing N-biotinylated sequences was performed by affinity chromatography on an avidin-agarose column. The unwanted terminated sequences were retained in the avidin column while the purified peptide mixture was eluted as indicated by reverse-phase HPLC and MS analysis monitoring. The avidin column was regenerated and the biotinylated sequences were released under reversible denaturing conditions. The usefulness of biotinylation for peptide library purification is demonstrated here for the first time for a peptide mixture containing by-products that cannot be separated from the mixture by classical HPLC purification. This purification technique could be applied to all syntheses, presenting difficult reacting steps.
A one-pot affinity precipitation purification of carbohydrate-binding protein was demonstrated by designing thermally responsive glyco-polypeptide polymers, which were synthesized by selective coupling of pendant carbohydrate groups to a recombinant elastin-like triblock protein copolymer (ELP). The thermally driven inverse transition temperature of the ELP-based triblock polymer is maintained upon incorporation of carbohydrate ligands, which was confirmed by differential scanning calorimetry and (1)H NMR spectroscopy experiments. As a test system, lactose derivatized ELP was used to selectively purify a galactose-specific binding lectin through simple temperature-triggered precipitation in a high level of efficiency. Potential opportunities might be provided for enhanced proteomic, cell isolation as well as pathogen detection applications.
This study describes the identification of over 150 target proteins of the five 14-3-3 isoforms in 7-d-old barley (Hordeum vulgare) cv Himalaya seedlings using yeast two-hybrid screens complemented with 14-3-3 protein affinity purification and tandem mass spectrometry. Independent experiments for a subset of genes confirmed the yeast two-hybrid interactions, demonstrating a low false positive identification rate. These combined approaches resulted in the identification of more than 150 putative targets; 15% were previously reported to be 14-3-3 interactors, including, for example, Serpin, RF2A, WPK4 kinase, P-type proton-translocating adenosine triphosphatase, EF1A, glutamine synthetase, and invertases. The affinity purification resulted in 30 interactors, of which 44% function in metabolism, while the yeast two-hybrid screens identified 132 different proteins, with 35% of the proteins involved in signal transduction. A number of proteins have a well-described function in hormonal signaling, such as the auxin transport protein PIN1 and NPH3 and components of the brassinosteroid pathway, such as the receptor kinase BAK1 (OsPERK1) and BRI1-kinase domain-interacting protein 129. However, 14-3-3 interactions with these signal mediators have not been confirmed in the affinity purification. Confirmations of the 14-3-3 interaction with the three ABF-like transcription factors are shown using far western analysis. Also, a REPRESSION OF SHOOT GROWTH ortholog named RF2A was identified; these transcription factors play important roles in the abscisic acid and gibberellin pathways, respectively. We speculate that 14-3-3 proteins have a role in cross talk between these hormonal pathways. The specificity and complementary nature of both the affinity purification and the yeast two-hybrid approaches is discussed.
An affinity column was devised for the purification of a large number of amylases inhibited by the albumin from wheat kernel. The procedure involved linking the protein inhibitors from wheat to Sepharose and then specifically eluting the amylase adsorbed to the gel with a high concentration of maltose. By this procedure, the amylases from Tenebrio molitor L. (yellow mealworm) larvae and chicken pancreas were purified to homogeneity with good yields for the first time, as shown by both alkaline and acidic electrophoresis. Human saliva alpha-amylase, purified by the same procedure, showed specific activity and electrophoretic patterns similar to those obtained by other workers with different techniques.