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PubMed1984

Affinity purification of two populations of antibodies against format determinants of synthetic myelin basic protein peptide S82 from S82-AH- and S82-CH-Sepharose 4B columns.

Two different kinds of immunosorbents were prepared that contained the synthetic myelin basic protein didecapeptide S82 (TTHYGSLPQKAQGHRDQDEG)--one coupled with AH-Sepharose 4B through hexanoate spacers to the C-terminal glycyl residue; the other, with CH-Sepharose 4B through hexanoate spacers to the N-terminal threonine residue. An antiserum rich in antibodies to a format determinant of S82 was passed through each column, and, by means of affinity purification, two homogeneous populations of anti-format antibodies were obtained, each with a binding affinity of 1 X 10(8)M-1 for S82. The population recovered from S82-AH-Sepharose 4B cross-reacted to a considerable extent with synthetic peptide S8 (GSLPQKAQGHRPQDENG) but only to a limited extent with S79 (AQGHRPQDEG). The population recovered from S82-CH-Sepharose 4B cross-reacted poorly, if at all, with S8. An equimoler mixture of S8 + S79, however, reacted well with either population of anti-format antibodies, thus showing that the mixture could mimic the format of S82. It was concluded that secondary structural conformation of S82 could be preserved during the coupling procedure and that the resulting immunosorbents could be used for the affinity purification of anti-S82 antibodies to the format determinants.

Animals↗
PubMed1999

New methods of protein purification. Affinity ultrafiltration.

This review describes a recently developed method for protein purification-affinity ultrafiltration. In affinity ultrafiltration, the protein to be purified is complexed with a macroligand composed of a soluble polymer or an insoluble microparticle with covalently bound, target protein-specific affinity ligands. The complex is trapped by an ultrafiltration membrane, whereas unwanted proteins pass through the membrane. The unwanted proteins are removed from the system by the carrier liquid. The system is then supplemented with an agent eluting the target protein by dissociating it from the microligand complex. The purified protein then passes the membrane, while the macroligand is trapped by it. The macroligand can be re-used after regeneration. Affinity ultrafiltration has a number of advantages over other protein purification techniques: 1) commercial availability of ultrafiltration systems with various high-productivity designs; 2) availability of presynthesized macroligands, which can be supplemented with additional, easily manufactured, commercial latex-based macroligands; 3) rapid separation of large solution volumes; 4) repeated use of equipment, enabling consecutive purification of different proteins; 5) simple scale-up and automation procedures.

Ligands↗
PubMed2004

A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel.

The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.

Animals↗
PubMed1991

Affinity purification of monoclonal antibodies, using a bifunctional oligosaccharide hapten.

A bifunctional hapten was synthesized consisting of a blood group A active tetrasaccharide (A-tetra) and a blood group Le(a) active pentasaccharide lacto-N-fucopentaose II (LNF II), linked to each other with a phenylaminothiourea spacer connecting the reducing ends (A-tetra-LNF II). The hapten was demonstrated to retain both blood group A and Le(a) activity and could be easily bound to both monoclonal anti-A and anti-Le(a) affinity columns. Due to the strong temperature dependence of the two antibodies their binding to oligosaccharides, the bifunctional hapten could be utilized to achieve easy desorption in the final step of affinity purification of either monoclonal anti-Le(a) or anti-A. The system is postulated to have general applicability in affinity purification of any ligate that binds with an avidity too high to achieve non-denaturing desorption.

Antibodies, Monoclonal↗
PubMed1989

Affinity purification of plasma proteins: characterization of six affinity matrices and their application for the isolation of human factor VIII.

For the purification of coagulation factor VIII, (1,1'-carbonyl-diimidazole [CDI]-activated) Sepharose CL-4B was functionalized with two aminoalkyl and four aminoalkyl-carbamylalkyl ligand-spacer combinations. The affinity matrices were contacted with human plasma. All affinity matrices showed complete adsorption of factor VIII (greater than 90%) and three aminoalkyl-carbamylalkyl Sepharoses gave factor-VIII recoveries of 50-65% and a factor-VIII preparation with a specific activity of 1-2 U factor VIII/mg of protein. Furthermore, no fibrinogen, immunoglobulin G and albumin could be detected in the isolated factor VIII. Optimal results were obtained using the di-methyl-aminopropyl-carbamyl-pentyl-Sepharose affinity matrix.

Adsorption↗
PubMed1997

Secretory production of erythropoietin and the extracellular domain of the erythropoietin receptor by Bacillus brevis: affinity purification and characterization.

Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and and EPO-fixed gel for sEPOR. The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities. Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.

Amino Acid Sequence↗
PubMed1994

Identification of epitopes and affinity purification of thyroid stimulating auto-antibodies using synthetic human TSH receptor peptides.

We prepared a series of overlapping peptides (29 in total, 20 amino acids each) containing the sequence of the entire extracellular domain of the human TSH receptor. Three peptides (181-200, 376-394, and EC3 (629-639)) bound IgG from patients with Graves' disease in an enzyme linked immunoassay. Peptide 181-200 bound IgG from 9 of 10, EC3 from 8 of 10, and 376-394 from 6 of 10 patients respectively, compared to 0 of 9 controls. We affinity purified TSHr auto-antibodies from four Graves' patients using the three above noted peptides bound to epoxy-activated sepharose. Thyroid stimulating activity was enriched in the bound fraction from at least two of the three peptide affinity columns in each of the four patients, although the pattern of affinity enrichment differed between patients. One patient was found to possess a combination of stimulatory and inhibitory TSHr antibodies and, after affinity purification, the anti-376-394 and anti-EC3 fractions were enriched in stimulatory activity, suggesting that those regions of the receptor were epitopes for stimulatory antibodies. However, affinity purification against peptide 181-200 produced an IgG preparation that was not stimulatory, but was a potent thyroid inhibitor. Thus, we have not only partially purified TSHr auto-antibodies, but also successfully separated stimulatory and inhibitory antibodies from a single patient using combination TSHr peptide affinity.

Blotting, Western↗
PubMed1984

Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots.

An affinity-purification method has been developed for the rapid, efficient, and precise elution of antibodies specifically bound to antigens immobilized on nitrocellulose after blot transfer from SDS polyacrylamide gels. The applicability of this technology has been demonstrated using antisera raised against the nuclear matrix-pore complex-lamina fraction prepared from Drosophila melanogaster embryos. In so doing, we have established the existence in whole embryo lysates, of two nearly identical forms of the predominant 74-kilodalton polypeptide previously identified in lower resolution studies of the nuclear matrix-pore complex-lamina fraction. These species, distinguishable on the basis of a slight difference in SDS PAGE mobilities on low concentration polyacrylamide gels, are immunochemically cross-reactive and have been localized exclusively to the nuclear periphery (nuclear envelope) by indirect immunofluorescence analyses of cryosections. The steady-state levels of these two polypeptides have been examined in total embryo lysates both as a function of embryogenesis and in response to heat shock. The larger species is not detectable in early embryos but approaches levels approximately equal to that of the smaller form by about the temporal midpoint of embryonic development. In response to heat shock, this larger form appears to be converted nearly quantitatively into the lower molecular weight polypeptide. These results, as well as the general reliability of the nitrocellulose blot immunoaffinity-purification methodology, have been substantiated through the use of monoclonal antibodies.

Animals↗
PubMed1993

Affinity purification of a somatostatin receptor-G-protein complex demonstrates specificity in receptor-G-protein coupling.

The inhibitory neuropeptide somatostatin (SRIF) initiates many of its physiological effects by binding to membrane receptors which are coupled to pertussis toxin-sensitive G-protein(s). We have solubilized such a SRIF receptor-G-protein complex and purified it using a biotinylated SRIF analog and guanine nucleotide-dependent affinity chromatography. Following [125I-Tyr11]SRIF binding to membranes from AR4-2J pancreatic acinar cells, only two detergents, dodecyl-beta-D-maltoside (D beta M) and sucrose monolaureate, extracted greater than 70% of the prebound peptide in association with receptor. The D beta M-solubilized ligand-receptor complex was extremely stable: the half-time (t1/2) for dissociation was 11 days at 4 degrees C. However, guanosine 5'-3-O-(thio)triphosphate (10 microM) elicited rapid dissociation of the [125I-Tyr11]SRIF-receptor complex (t1/2 < 30 s), and this effect was concentration-dependent (ED50 = 4.0 + 0.3 nM). [125I-Tyr11]SRIF dissociation was also stimulated by GDP (ED50 = 4.1 +/- 0.3 microM), and the potency of GDP was increased 4-fold by 30 microM AlF4-. Thus, the solubilized receptor was functionally associated with G-proteins. Cross-linking of the soluble [125I-Tyr11]SRIF-receptor complex resulted in the covalent labeling of a 70-90-kDa band, the same band that was specifically labeled in membranes. Affinity purification of the SRIF receptor-G-protein complex was accomplished by prebinding a biotinylated SRIF analog, [N-biotinyl-Leu8,D-Trp22,Tyr25]SRIF28, to membranes followed by solubilization of the ligand-receptor-G-protein complex, adsorption to streptavidin-agarose, and specific elution with 100 microM GDP, 30 microM AlF4-. G-proteins were identified in the eluate by immunoblotting with specific antipeptide antisera. Using this protocol, the G-protein subunits alpha i, alpha i-3, and beta 36 were shown to be specifically associated with the AR4-2J cell SRIF receptor. Thus, we have developed a new, generally applicable, procedure for the efficient solubilization and affinity purification of a stable SRIF receptor-G-protein complex and have characterized the specific G-protein subunits associated with pancreatic SRIF receptors.

Animals↗
PubMed2005

Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination.

Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBP-calmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.

Animals↗
PubMed1993

A simple method for affinity purification of radiolabeled monoclonal antibodies.

A simple method is described for affinity purification of radiolabeled antibodies using glutaraldehyde-fixed tumor target cells. The cell-bound antibody fraction is removed from the cells by an acid wash and then immediately subjected to buffer-exchange chromatography. The method was applied to the D3 murine monoclonal antibody which binds to a 290 kDa antigen on the surface of Line 10 guinea pig carcinoma cells. No alteration in the molecular size profile was detected after acid washing. Purification resulted in a significant increase in immunoreactivity by an average of 14 +/- 47% (SD; range 4-30%).

Animals↗
PubMed2004

Isolation of a peptide ligand for affinity purification of factor VIII using phage display.

Polypeptides for use in affinity chromatography of factor VIII were identified using phage display technology. Phage libraries were designed to express polypeptide fusions containing five to seven residues flanked by two cysteines that form a disulfide bond. Individual bacteriophage were selected for the ability of these polypeptides to bind factor VIII, and then release the protein under mild elution conditions. Strong consensus sequences were observed that appear to be necessary for this reversible interaction. Chemically synthesized ligands identified by this screening were immobilized onto a chromatographic support and used for affinity purification of factor VIII from a complex feedstream. A chromatographic step was developed that provided a 10000-fold reduction in host cell proteins and DNA, while providing exceptional product recovery.

Bacteriophages↗
PubMed1989

The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein.

A method of affinity purification of a regulatory protein that binds specific RNA sequences is described. RNAs containing the regulatory sequences are transcribed in vitro from oligonucleotide templates, biotinylated, and incubated with unfractionated cytosol. Specific RNA-protein complexes are bound in solution to avidin, and the resulting complex is bound to biotin-agarose beads. The cytosolic binding protein is released from the RNA in high salt, and a second round of purification yields an essentially homogeneous protein. Using this method, we have identified the protein in human liver that binds iron-responsive RNA regulatory sequences. Iron-responsive elements (IREs) are RNA stem-loops present in the mRNAs encoding ferritin and the transferrin receptor. IREs form the basis for the translational regulation of ferritin gene expression and the regulation of transferrin receptor mRNA degradation rates. The IRE binding protein purified by this technique migrates as a 90-kDa polypeptide on SDS/PAGE. The interaction of the purified protein with IRE-containing RNAs can be detected by gel-mobility shift assays or by covalent crosslinking induced by UV irradiation.

Base Sequence↗
PubMed1986

Affinity purification of sequence-specific DNA binding proteins.

We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.

Base Sequence↗
PubMed1993

A multifunctional prokaryotic protein expression system: overproduction, affinity purification, and selective detection.

A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized. Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal. Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins. Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols. Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography. Purification was independent of recombinant protein solubility in sonicated or freeze-thawed E. coli lysates. Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitation, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.

Amino Acid Sequence↗
PubMed2001

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries.

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.

Amino Acid Sequence↗
PubMed2002

Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective.

We employed a combination of tandem affinity purification and mass spectrometry for deciphering protein complexes and the protein interaction network in budding yeast. 53 genes were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates. 38 baits pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. We identified four proteins shared between complexes of different functionality thus charting segments of a protein interaction network. Concordance with the results of genome-wide two-hybrid screening was poor (14% of identified interactors overlapped) suggesting that the two approaches may provide complementary views on physical interactions within the proteome.

Chromatography, Affinity↗
PubMedDate not supplied

Reversible biotinylation phosphoramidite for 5'-end-labeling, phosphorylation, and affinity purification of synthetic oligonucleotides.

A fluoride/amine-cleavable phosphoramidite designed for biotinylation, phosphorylation, and affinity purification of synthetic oligonucleotides was synthesized and coupled efficiently to the 5'-end of DNA on a solid-phase automatic synthesizer. The two hydroxyl groups of diethyl bis(hydroxymethyl)malonate were used to link biotin and the 5'-end of DNA together through a diisopropylsilyl acetal functionality and a phosphate ester group, respectively. The DNA was cleaved from solid support and fully deprotected by treating with a mixture of MeNH(2) ( approximately 40%) and NH(4)OH ( approximately 29%) (1:1, v/v, 65 degrees C, 30 min), and the linkage between biotin and DNA was found completely stable under these conditions. The biotinylated full-length DNA was efficiently attached to NeutrAvidin coated microspheres and failure sequences and other impurities were simply removed by washing with buffer and water. The microspheres were then treated with HF/pyridine/THF (rt, 1 h) and MeNH(2) ( approximately 40%, rt, 15 min) sequentially to yield high quality full-length 5'-end phosphorylated unmodified DNA as revealed by HPLC analysis. It is anticipated that this method will find applications in areas that require efficient isolation of 5'-end phosphorylated DNA from a complex mixture.

Affinity Labels↗