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PubMed2005

An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies.

Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor.

Antibodies↗

PubMedDate not supplied

Protein A mimetic peptide ligand for affinity purification of antibodies.

A peptide mimicking protein A for its ability to recognize the Fc immunoglobulin portion has been identified through screening of a synthetic multimeric peptide library. Screening of the multimeric library, composed of randomized synthetic tripeptide tetramers, has been carried out using a very simple assay, measuring the library ability to interfere with the interaction between protein A and biotinylated immunoglobulins, monitored on solid phase using an enzyme-linked immunosorbent assay format. The tetrameric tripeptide identified after three screening cycles was produced in larger amounts and then immobilized in high yield on preactivated solid support for the preparation of affinity columns, which proved useful for a very convenient one-step purification of antibodies directly from crude sera. Antibody purity after affinity purification was close to 95 per cent, as determined by densitometric scanning of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of purified fractions, and up to 2 mg of antibody could be purified from 1 ml of peptide-derivatized affinity support. The ligand was stable to treatment with a vast array of sanitation agents, such as ethanol and 0.1 M sodium hydroxide, and to repeated use, thus making the ligand applicability extremely attractive for the purification of monoclonal antibodies for therapeutic use. Column binding selectivity was similar to that of protein A-affinity columns, since immunoglobulin G from several sources (rabbit, goat, sheep, mouse) was conveniently purified, with no detection of leaked ligand fragments in the purified preparations.

Animals↗

PubMed2005

Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor.

Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.

Chromatography, Affinity↗

PubMed1998

Affinity purification of immunoglobulin M using a novel synthetic ligand.

While monoclonal antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several problems, since specific and cost-effective ligands for IgM are not available. A synthetic peptide (TG19318), deduced from the screening of a combinatorial peptide library, was characterized previously by our group for its binding properties for immunoglobulins of the G class and its applicability as a synthetic ligand for polyclonal and monoclonal IgG purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobilizing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very convenient one-step purification of monoclonal IgMs directly from crude sources, loading the samples on the columns equilibrated with saline buffers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a buffer change to 0.1 M acetic acid or 0.05-0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85-95%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gels of purified fractions, and by gel permeation analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support.

Animals↗

PubMed1991

Affinity purification of a major and a minor allergen from dog extract: serologic activity of affinity-purified Can f I and of Can f I-depleted extract.

Most dog-allergic patients react to a major 25 kd component on sodium dodecyl sulfate blots, Can f I (Ag 13). We initially raised monoclonal antibodies (Cf-3 and Cf-2) reactive with IgE-binding components distinct from Can f I. After a slight modification, we immunized other strains of mice and produced monoclonal antibodies coded Cf-1a and Cf-1b reactive with Can f 1. We affinity purified the allergens, Can f I and "dog allergen 2" with Cf-1a and Cf-2 ascites, respectively, and house dust-rich dog dander. Comparison of purified Can f I with dog saliva in RAST demonstrated that Can f I is a potent allergen for most dog-allergic patients (average response, 70%). After depletion of dog saliva of Can f I, a slightly lower contribution for Can f I was found, but the overall results supported the conclusion that Can f I is a major allergen in dog saliva. Comparison of purified dog allergen 2 with dog dander in RAST demonstrated that dog allergen 2 is less important for dog-allergic patients (average response, 23%). We radiolabeled the purified allergens and developed assays to measure Can f I and dog allergen 2 in allergen extracts and dust samples. Dog saliva was a strong allergen source, dog urine and feces contained very little of the allergens, and both allergens were found to a variable degree in the nine dog breeds tested.

Allergens↗

PubMed1985

Affinity purification of an interferon-induced human guanylate-binding protein and its characterization.

Among the proteins that are synthesized only in interferon-treated human cells, a Mr = 67,000 protein has been previously identified by its binding to guanylate agaroses. After a 24-h treatment of human diploid fibroblasts with 200 units/ml of interferon-gamma, about 3 X 10(5) molecules of guanylate-binding protein (GBP) accumulate in each cell. We have developed a one-step purification procedures for GBP using guanylate affinity chromatography. To further elucidate the specific binding of this protein to guanylates, we have used a photoactive probe, 8-azidoguanosine [alpha 32P] triphosphate for the labeling of the GBP. Photolysis of the 8-azido-[alpha-32P]GTP in the presence of GBP results in the covalent attachment of the 32P-guanylate to the GBP. This photolabeling reaction can be inhibited only by guanylates but cannot be inhibited by other nucleotides, suggesting a specific association of GBP to guanylates. Using the purified GBP as an immunogen, we have successfully made rabbit antiserum for GBP. Both the GBP antigen and its guanylate-binding activity are detected only in the cytoplasm of interferon-treated human fibroblasts. The synthesis of the mRNA of GBP is also found in mice exposed to endogenous interferon and in interferon-treated human lymphocytes.

Animals↗

PubMed1996

Affinity purification of cellulose-binding enzymes of Clostridium stercorarium.

Cellulose-affinity chromatography proved to be a fast and efficient purification procedure for exoenzymes of the thermophilic anaerobe Clostridium stercorarium, suitable for the preparation of large amounts of enzymes for technical applications. The cellulose-binding enzymes could be identified as the C. stercorarium; cellulolytic enzymes Avicelase I and Avicelase II characterized previously as endo-1,4-beta-glucanase and exo-1,4-beta-glucanase. A third protein was identified as xylanase A, the major endo-1,4-beta-xylanase of this organism.

Amino Acid Sequence↗

PubMed1994

One-step affinity purification of bacterially produced proteins by means of the "Strep tag" and immobilized recombinant core streptavidin.

The "Strep tag" is a nine amino acid peptide with intrinsic streptavidin-binding activity. If this sequence is genetically fused to the C-terminus of a polypeptide the recombinant protein can be directly purified by affinity chromatography from the host cell extract on immobilized streptavidin. However, variations were observed in the suitability of different commercial streptavidin-agarose preparations for this purpose. Therefore, the influence of the source of streptavidin, the coupling chemistry, and the nature of the affinity chromatography resin was investigated. A procedure was developed for the production of recombinant core streptavidin in Escherichia coli, followed by its efficient refolding and purification with an overall yield of up to 140 mg functional protein per 11 bacterial culture. When coupled to activated CH-Sepharose 4B this truncated form of streptavidin showed a performance in the affinity chromatography of Strep tag fusion proteins that was superior to all other combinations tested. In contrast to its conventional preparation from Streptomyces strains the recombinant core streptavidin was produced without a proteolytic processing step. Thus, deleterious effects during the streptavidin affinity purification of proteins due to residual proteolytic activity in the immobilized streptavidin were avoided. The versatility of the optimized purification system was demonstrated with five different Strep tag fusion proteins.

Bacterial Proteins↗

PubMed1998

Screening of combinatorial peptide libraries: identification of ligands for affinity purification of proteins using a radiological approach.

Peptides deduced from peptide libraries may serve as affinity ligands for protein purification. Identification of a ligand that binds the protein of interest depends highly on the screening method used. One approach which offers simple and direct detection involves screening a solid-phase peptide library against a radiolabeled target protein. We have developed a radiological screening method, using 14C as a radioactive label, that offers high resolution and sensitivity. Less than 100 DPM/bead are detectable after a one-day exposure using autoradiography. The validity of the technique was illustrated by screening a solid-phase hexameric-peptide library spiked with YNFEVL-beads against 14C-labeled ribonuclease S-protein. For this particular system, the amount of protein bound to a single bead was estimated to be in the femtomolar range with a peptide:protein ratio of 500:1. Finally, a portion of the library was screened against 14C-labeled fibrinogen. Three peptides deduced from the library, WQEHYN, WQETYQ, and YENYGY, purified fibrinogen from a mixture with albumin.

Autoradiography↗

PubMed1984

Detergent solubilization and affinity purification of a local anesthetic binding protein from mammalian axonal membranes.

Previously, we identified a local anesthetic receptor in the membranes of nerve cells, especially enriched in axonal membranes, by the binding assay of a fluorescence local anesthetic probe, quinacrine (Greenberg, M., and Tsong, T.Y. (1982) J. Biol. Chem. 257, 8964-8971). We report here affinity chromatography purification of the quinacrine binding protein from axonal membranes of bovine brain. One per cent sodium cholate solubilized more than 90% of the membrane proteins, and the quinacrine binding activity was preserved in the extract; KD remained at 1.0 microM. An affinity column of quinacrine mustard covalently linked to Affi-Gel 10 (Bio-Rad) retained about 5% of the total solubilized proteins, and the subsequent elution with 1.0 M lidocaine revealed a protein peak which coeluted with the lidocaine peak. After removal of lidocaine, the protein peak retained the high affinity to quinacrine (KD = 1.5 microM). Furthermore, bound quinacrine could be displaced with dibucaine in the range of its nerve-blocking concentration, around 10 microM. Sodium dodecyl sulfate-gel electrophoresis of the peak protein fractions gave a predominant band of approximately 16 kDa. A major protein band with the same molecular weight was also seen in the sodium dodecyl sulfate gel of the total axonal membrane proteins. The receptor concentration in the axonal membranes was estimated to be 2-5 nmol/mg protein. With a molecular weight of 16 kDa, the receptor should constitute 3-8% of total proteins, consistent with the finding that it is a major protein constituent of the axonal membranes. The purity of the binding protein after the affinity chromatography step was about 90%. The purified protein contained roughly 60 nmol of quinacrine binding sites/mg.

Animals↗

PubMed1996

Affinity purification of von Willebrand factor using ligands derived from peptide libraries.

The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6) (M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.

Adsorption↗

PubMed1998

Affinity purification of mouse monoclonal IgE using a protein A mimetic ligand (TG19318) immobilized on solid supports.

A synthetic ligand (TG19318), deduced from the screening of a combinatorial peptide library, has been previously characterized by our group for its applicability in affinity chromatography for polyclonal and monoclonal IgG purification from crude sources. In this study we have extended the characterization of its recognition properties for other immunoglobulin classes, evaluating its ability to purify mouse monoclonal IgE from ascitic fluid. TG19318 affinity columns proved useful for a very convenient one-step purification of IgE directly from crude ascites, by loading the samples on the columns equilibrated with 50 mM sodium phosphate at pH 7 and eluting and adsorbed IgE by a buffer change to 0.1 M acetic acid. Antibody purity after affinity purification was very high and no albumin traces were detected, as determined by SDS-PAGE analysis. Antibody activity was fully recovered after purification, as determined by immunoassays on antigen-coated plates, and up to 5 mg of IgEs could be purified on a 1 ml column in a single run.

Affinity Labels↗

PubMed1984

Affinity purification and characterization of glutathione S-transferases from bovine liver.

Conditions have been investigated for the use of triazine dye agarose as an affinity matrix for the purification of glutathione S-transferases from bovine liver. Orange A agarose was most suitable for this purpose among various dye agaroses tested. The enzymes were adsorbed on the dye agarose column and then completely eluted with the buffer containing 1 mM reduced glutathione. Thus, a simple and rapid method for purification of bovine liver transferases was developed, which uses column chromatographies on orange A agarose followed by DEAE-Sephacel. One step of the affinity chromatography provided 40-fold purification. Upon chromatography on DEAE-Sephacel, the enzyme activity separated into two major forms (I and II), which were purified to apparent homogeneity as examined by polyacrylamide gel electrophoreses. The pI values of the two forms, I and II, were 6.7 and 6.1, respectively. The overall extents of purification of I and II were about 40-fold and 50-fold, respectively. The activities of the two major enzymes toward various substrates were roughly similar. The optimum pH values of these enzymes were 7.5 as measured with o-dinitrobenzene as a substrate. The activities were significantly inhibited by Cu2+, Hg2+, Cd2+, and Zn2+. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both enzymes showed one band with a molecular weight of about 27,000. Both enzymes, however, were eluted as a single peak from a Sephadex G-150 column at a position corresponding to a molecular weight of about 49,000. These results show that each enzyme consists of two subunits bound to each other non-covalently. The amino acid compositions showed characteristically high contents of leucine and aspartic acid residues. Double immunodiffusion showed complete identity of the two forms reacting with both rabbit anti-enzyme sera. The two enzymes had an identical amino-terminal amino acid sequence as follows: H-Pro-Met-Ile-Leu-Gly-Tyr-Trp-Asp-Ile-Arg-Gly-Leu-Ala-His-Ala-Ile-Ser-Le u-Leu-Leu.

Amino Acids↗

PubMed1979

Properties and affinity purification of the mixed-type putative acetylcholine receptor from wild and a mutant strain of hose flies.

Binding of decamethonium to a soluble preparation from house fly head (either wild or a mutant strain) showed a single kind of binding with values for wild strain of Kd = 0.095 micrometers and Bmax = 0.22 nmol/mg protein. The mutant had a four-fold greater affinity and a seven-fold lesser amount. The binding was blocked by both nicotinic and muscarinic drugs. The decamethonium binding migrated in sucrose gradients as a single peak, with sedimentation coefficient s20,w = 12.5 S and therefore a molecular weight of 342 000. Purification by affinity chromatography was achieved with only partial loss of activity, andthe purified material demonstrated a single band on analytical disc gel electrophoresis. Electrophoresis in sodium dodecyl sulphate gels showed two subunits of molecular wegiths 94 000 and 64 000. Both subunits had an isoelectric point of 4.8.

Animals↗

PubMed1991

Dihydrotestosterone derivatives: relative binding affinity versus affinity purification.

Mono esters of a homologous series of diacids of dihydrotestosterone were synthesized and converted to the corresponding n-butyl amides. The relative binding affinities of these amides to androgen receptor were compared with the degree of purification of rat prostate androgen receptor by affinity columns prepared by linking the steroidal acid to amino Sepharose. There was good correlation between binding of the amide model to androgen receptor and the extent of purification by the affinity resin.

Animals↗

PubMed1991

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.

Amino Acid Sequence↗

PubMed1987

Affinity purification of synenkephalin-containing peptides, including a novel 23.3-kilodalton species.

Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1-70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7-3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6-kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182-203) and [Met5]-enkephalin-Arg6-Gly7-Leu8. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3-kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1-206), containing [Leu5]-enkephalin at the C-terminus.

Animals↗

PubMed1987

Affinity purification of hexosaminidases.

Hexosaminidases A and B were purified by affinity chromatography from normal gastric mucosa, after preliminary separation of isozymes by anion exchange chromatography. Heparin and mannosamine were coupled to Sepharose 4B and used as affinity matrices and the purified enzymes were found to be homogeneous when analysed by polyacrylamide slab gel electrophoresis. This combination of 2 novel affinity chromatographic procedures is superior to existing methods in that a final yield of over 70% could be achieved. Also, the number of steps required to obtain homogeneity are less in contrast to the conventional methods used previously.

Chromatography, Affinity↗