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Results for “Affinity purification”

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PubMed1998

Affinity purification of recombinant trypsinogen using immobilized ecotin.

Affinity purification of inactive precursors (zymogens) of serine proteases on protease inhibitor columns is not feasible, due to the weak interaction between canonical protease inhibitors and protease zymogens. In this study we demonstrate that immobilized ecotin, a unique protease inhibitor from Escherichia coli, provides a superior affinity matrix for the purification of trypsinogen and possibly other serine protease zymogens as well.

Animals↗
PubMed2002

Design, production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins.

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.

Avidin↗
PubMed1987

Artifactual immunofluorescent labelling in yeast, demonstrated by affinity purification of antibody.

In the course of making antibodies against various yeast (S. cerevisiae) proteins, we have noted that it is common to observe reactivity of rabbit sera with a number of extraneous bands on Western transfers of yeast proteins. The pattern of reactive bands can change within a period of weeks, even when the rabbit has not been injected with antigen. A simple method of affinity purification, using antigen bound to nitrocellulose, is employed to remove the reactivity with these extraneous bands from immune sera. The importance of affinity purification is demonstrated by our attempts to immunolocalize a 55 kd yeast protein (p55). Immune serum stains yeast cells to give a striking pattern of spots and blotches not seen with preimmune serum. However, affinity purification of anti-p55 antibody shows that this pattern is not due to staining by anti-p55 antibody; rather the pattern is due to staining left in the serum depleted of anti-p55 antibody.

Antibodies, Fungal↗
PubMed2005

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications.

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged LacI protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Bacterial Proteins↗
PubMed1995

Affinity purification of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT is described and characterized. Spinach (Spinacia oleracea L. cv Melody) Rubisco, a substrate for pea Rubisco LSMT, was immobilized to polyvinylidene fluoride (PVDF) transfer membranes (Immobilon-P) and used as a ligand for the affinity purification of Rubisco LSMT from pea leaf extracts and chloroplast lysates. Pea Rubisco LSMT specifically bound to PVDF-immobilized spinach Rubisco but not to control PVDF membranes which contained immobilized BSA or pea Rubisco. Rubisco LSMT was not eluted by 1 M KCl but was specifically released by S-adenosyl-L-methionine (AdoMet) or spinach Rubisco. Elution of Rubisco LSMT by AdoMet was a result of catalytic methylation of the PVDF-immobilized spinach Rubisco, and was therefore more efficient than elution by the competitive ligand spinach Rubisco. An increase in the specific activity of Rubisco LSMT of approximately 7000-fold was achieved in one step with this affinity purification technique. Rubisco LSMT is a monomeric protein with a molecular mass of approximately 60 kDa.

Chromatography, Affinity↗
PubMed2003

Affinity purification and diagnostic use of TSH receptor autoantibodies from human serum.

Purification of TSH receptor autoantibodies (TRAb) from the serum of patients with Graves' disease (GD) might help to elucidate the nature of these disease causing autoantibodies. We describe here for the first time the successful affinity purification of human TRAb. Affinity purification was performed in a four step procedure with human recombinant TSH receptor (TSH-R) expressed in K562 cells. Purification from six different serum pools from patients with GD and two individual sera (one with only thyroid stimulating antibodies (TSAb) one with only thyroid blocking antibodies (TBAb)) resulted in a purity of 39.2+/-3.8 IU/mg TRAb or 25.7+/-2.1 microg IgG/IU (about 3.5-13.7 microg TRAb/ml serum). The average enrichment based on the respective original serum was 3420-fold (range 1200-10,000). The kDa of the purified TRAb were in the range of 0.7-2.6 x 10(-10)M. All purified TRAb (except from the TBAb serum which showed blocking activity) showed a more than 1000-fold stronger stimulation in the TSAb bioassay based on the IgG content than the original serum, and similar stimulation based on international units (IU/l) TRAb. When labelled purified TRAb were used in a competitive assay as tracer instead of bovine TSH, their binding to the human recombinant TSH-R on tubes was displaced by 99 of 100 GD sera (selected for TBII activity). Correlation to the standard TSH tracer was r=0.92. Interestingly, the use of TRAb tracer derived from a patient with TSAb and a patient with TBAb gave virtually identical results (r=0.93) with these patients, suggesting similar if not identical binding sites for both TRAb subtypes. In conclusion, this is the first report on the purification of human TRAb from the serum of patients with GD. The purified TRAb are of low concentration with high affinity, strong TBII and TSAb activity. Further characterisation may allow new insights in TRAb epitope localisation, the pathology of GD and the differences between TSAb and TBAb. Also, their use as tracer in a competitive assay is the first report on a completely homogenous assay with high sensitivity for TSH-R autoantibodies.

Animals↗
PubMed2005

Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform.

Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations.

Chromatography, Affinity↗
PubMed2005

An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation.

Tandem affinity purification (TAP) strategies constitute an efficient approach for protein complex purification from many different organisms. However, the application of such strategies for purifying endogenous Arabidopsis multi-protein complexes has not yet been reported. Here, we describe an alternative TAP (TAPa) system that successfully allows protein complex purification from Arabidopsis. In our newly generated TAPa tag we have replaced the tobacco etch virus (TEV) protease cleavage site with the more specific and low-temperature active rhinovirus 3C protease site. In addition, the second purification step can now be performed through two different affinity tags: a six His repeat or nine copies of a myc repeat. To examine our purification procedure we generated a C-terminal fusion between the TAPa tag and CSN3, a component of the multi-protein COP9 signalosome (CSN) complex. Subsequent analysis showed that CSN3-TAPa could rescue a csn3 mutant, and that the components of the CSN complex could be co-purified with CSN3-TAPa. As part of our long running interest in light signaling in Arabidopsis we have generated Arabidopsis transgenic lines harboring, both N-terminal and C-terminal TAPa fusions of many different light signaling pathway regulators. Molecular characterization of these transgenic lines showed fusion expression in 88% of the genes analyzed and that this expression is largely independent of the fusion orientation. Mutant complementation analysis showed that most of the TAPa fusions analyzed retained function of the wild-type proteins. Taken together, the data demonstrate the suitability of the TAPa system to allow efficient multi-protein complex isolation from stably transformed Arabidopsis.

Amino Acid Sequence↗
PubMed2003

New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification.

We report the first use of tandem affinity purification (TAP) in a prokaryote to purify native protein complexes, and demonstrate its reliability and power. We purified the acyl carrier protein (ACP) of Escherichia coli, a protein involved in a myriad of metabolic pathways. Besides the identification of several known partners of ACP, we rediscovered ACP/MukB and ACP/IscS interactions already detected but previously disregarded as due to contamination. Here, we demonstrate the specificity of these interactions and characterize them. This suggests that ACP is involved in additional previously unsuspected pathways. Furthermore, this study shows how the TAP method can be simply used in prokaryotes such as E. coli to identify new partners in protein-protein interactions under physiological conditions and thereby uncover novel protein functions.

Acyl Carrier Protein↗
PubMed1994

The fractionation of human plasma proteins. I. Affinity purification of human complement properdin.

A method was developed for the affinity purification of human complement properdin. The preparation is part of an integrated scheme in which over 20 human plasma proteins can be recovered in a highly purified form. The yield of properdin was 5.9 mg from 3 liters of plasma, amounting to a 28% recovery. The crucial step in the purification was the application of affinity chromatography using C3b-ester-Sepharose.

Blood Proteins↗
PubMed2005

DNA affinity purification of Epstein-Barr virus OriP-binding proteins.

DNA affinity purification has been used to identify cellular and viral proteins associated with the Epstein-Barr virus origin of plasmid DNA replication. This approach allows for a one- or two-step purification scheme of high-affinity DNA binding proteins from crude nuclear extracts. Additionally, this approach may be useful for isolation of proteins that are found in the insoluble fractions of the nuclear matrix or scaffold.

Blotting, Western↗
PubMed1991

Affinity purification of plasminogen by radial-flow affinity chromatography.

A method for the purification of plasminogen using immobilized L-lysine on a membrane, the whole system being constructed in a radial flow cartridge, is described. Human plasma was applied to the cartridge at 20 ml/min. The results showed that under the chromatographic conditions chosen, in a single pass, greater than 85% recovery of plasminogen was attained with a 110-fold increase in specific activity.

Chromatography, Affinity↗
PubMed1999

Affinity purification of Alu-DNA-repeat-binding proteins from human somatic cells.

A 66-kD Alu-DNA-repeat binding protein was identified in human somatic cell nucleoplasm. Gel shift assay, southwestern blotting, and affinity purification on DNA attached to a carrier were used. A 60-kD protein copurified with the 66-kD protein during affinity purification, probably due to protein--protein interactions. The gel shift assay reveals multiple complexes with exponential dependence of their relative mobility. The short binding site of the 66-kD protein was defined with the help of synthetic oligonucleotides. It is localized between the A and B boxes of RNA polymerase III promotor and is the same as that reported for the Alu-binding protein from human spermatozoids. The same short binding site, the similarity of the isolation procedure from germ and somatic cells, and similar binding properties and molecular masses suggest homology of the two proteins. The relationship of the proteins we studied and the Alu-DNA-binding proteins described in the literature is discussed.

Alu Elements↗
PubMed2003

Statistical optimization for immobilized metal affinity purification of secreted human erythropoietin from Drosophila S2 cells.

We used a novel approach to affinity purify human erythropoietin (hEPO) following its secretion from Drosophila melanogaster S2 cells. Immobilized metal affinity purification of hEPO was optimized using a two-step serial statistical optimization strategy. After determining the elution conditions (based on preliminary batch-type purification experiments), the first optimization step considered three purification factors; resin, equilibrium, and washing. The results of this analysis showed that the resin amount was the major factor influencing yield and purity in both model equations and the washing factor lowered the confidence limits of the acquired model equations. The washing conditions were then set based on the results of the first step optimization and the second step then optimized three factors; resin, equilibrium, and elution. The yield and purity of hEPO were then compared following purification using three different approaches; batch-type purification based upon the conditions determined by serial statistical optimization, batch-type purification performed in preliminary experiments, and FPLC column chromatography-type purification. We found that the serial statistical optimization approach provided the best combination of yield and purity. These findings indicate that serial statistical optimization strategies can be successfully employed for immobilized metal affinity protein purification using either batch-type or column approaches.

Animals↗
PubMed2005

A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells.

The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.

Affinity Labels↗
PubMed1994

Affinity purification of angiotensin type 2 receptors from N1E-115 cells: evidence for agonist-induced formation of multimeric complexes.

The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (AngII) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT2-receptor subtype, whereas the density of the AT1 receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) selectively solubilized AT2 receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (AngII) during affinity purification of AT2 receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar1,Ile8-AngII was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar1,Ile8-AngII, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1-receptor antagonist, was completely ineffective in eluting any AngII receptors from the affinity column, further confirming their AT2 identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1998

Immunoglobulin specificity of TG19318: a novel synthetic ligand for antibody affinity purification.

A synthetic ligand [TG19318], able to mimic protein A in the recognition of the immunoglobulin Fc portion, has been previously identified in our laboratory through the synthesis and screening of multimeric combinatorial peptide libraries. In this study we have fully characterized its applicability in affinity chromatography for the downstream processing of antibodies, examining the specificity and selectivity for polyclonal and monoclonal immunoglobulins derived from different sources. Ligand specificity was broader than protein A, since IgG deriving from human, cow, horse, pig, mouse, rat, rabbit, goat and sheep sera, IgY obtained from egg yolk, and IgM, IgA and IgE were efficiently purified on TG19318 affinity columns. Adsorbed antibodies were conveniently eluted by a buffer change to 0.1 M acetic acid or 0.1 M sodium bicarbonate pH 9, with full retention of immunological properties. Monoclonal antibodies deriving from cell culture supernatants or ascitic fluids were also conveniently purified on TG19318 affinity columns, even from very diluted samples. The affinity constant for the TG19318-IgG interaction was 0.3 microM, as determined by optical biosensor measurements. Under optimized conditions, antibody purity after affinity purification was close to 95%, as determined by densitometric scanning of SDS-PAGE gels of purified fractions, and maximal column capacity reached 25 mg Ig/ml support. In vivo toxicity studies in mice indicated a ligand oral toxicity greater than 2000 mg kg-1 while intravenous toxicity was close to 150 mg kg-1. Validation of antibody affinity purification processes for therapeutic use, a very complex, laborious and costly procedure, is going to be simplified by the use of TG19318, which could reduce considerably the presence of biological contaminants in the purified preparation, a very recurrent problem when using recombinant or extractive biomolecules as affinity ligands.

Animals↗
PubMed2005

A combined approach for the localization and tandem affinity purification of protein complexes from metazoans.

An understanding of a protein's function is greatly aided by the knowledge of its localization in vivo and identification of any interacting partners. Here, we describe a method, based on expression of a genetically encoded fusion protein, that allows for protein localization and affinity purification (LAP) in metazoans. This method makes it possible to rapidly identify transformants and to conduct live imaging of protein localization and dynamics. In addition, the same tag can be used in a modified tandem affinity purification (TAP) procedure to isolate native protein complexes of high purity. The efficacy of this purification procedure allows for the characterization of rare protein complexes that are largely inaccessible to classical biochemical purifications. The LAP strategy should be widely applicable to the characterization of protein function in cellular and developmental contexts in various metazoans.

Amino Acid Sequence↗