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Fluidized-bed receptor-affinity chromatography.

A multipurpose fluidized-bed receptor-affinity purification system based upon the biological recognition between an immobilized receptor and its soluble protein ligands is described. The fluidized affinity sorbent consists of a soluble form of interleukin-2 receptor chemically bonded to an aldehyde derivative of controlled pore glass beads, which have a pore diameter of 1000 A and a particle density of 1.2-1.3 g/mL. The fluidized-bed separation device used in this study consists of a specially designed column fitted at the inlet end with a perforated distributor plate covered with a screen and the top outlet with an adjustable piston. The fluidized-bed consisting of a loose gel matrix permits the unimpeded passage of cell debris and particulate matter, while the target protein is captured by the affinity beads. Purification of the humanized-anti-Tac monoclonal antibody is used as a model system to determine the operational parameters. Also, fluidized-bed receptor-affinity chromatography has been successfully employed in the purification of recombinant interleukin-2 and single chain anti-Tac(Fv)-Pseudomonas exotoxin immunotoxin from unclarified inclusion body extracts. Overall, fluidized-bed receptor-affinity chromatography is found to be a productive affinity method suitable for the purification of recombinant human interleukin-2 and related molecules.

ADP Ribose Transferases↗
PubMed1987

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography.

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90-95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.

Animals↗
PubMed2003

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags.

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.

Bacterial Proteins↗
PubMed2002

Isolation and detection of human IgA using a streptococcal IgA-binding peptide.

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.

Amino Acid Sequence↗
PubMed1994

Biochemical properties, activities, and presence in biologic fluids of eosinophil granule major basic protein.

The existence of a proform of MBP is predicted from the sequence of MBP cDNA clones. ProMBP has been purified from the supernatants of CHO cells transfected with cDNA encoding prepro MBP. Purification involved heparin-Sepharose affinity purification followed by two sequential size fractionation steps over Sephadex G-100 and yielded proMBP with a molecular mass of 33 kd. Recombinant proMBP from the heparin-Sepharose column was subjected to isoelectric focusing followed by SDS-PAGE and Western blot analysis. The results indicated that most of the 33 kd form of proMBP focused predominantly between pI 4.2 and 5.1, with a major peak at a pI of approximately 4.9. Analyses of the carbohydrates associated with the purified 33 kd form of recombinant proMBP indicated the addition of 4856 to 5150 Da by carbohydrates characteristic of the complex type. Consistent with the hypothesis that the function of the propiece is to neutralize MBP toxicity during granule processing, proMBP lacked MBP cytostimulatory properties and actually blocked the effect of MBP in two different systems, basophil histamine release and neutrophil activation. In addition, as a measure of toxicity, proMBP did not inhibit protein synthesis, whereas MBP markedly reduced protein synthesis. The mechanisms by which MBP exerts its actions both as a cytostimulant and as a toxin are not known; however, it is known that cationic MBP readily reacts with acidic lipids. Using artificial liposomes as targets, MBP caused a disordering of the lipid bilayer membrane, resulting in fusion and lysis. Therefore, MBP may act both as a cytostimulant and as a toxin because of its marked cationicity and its ability to disorder lipid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution.

Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.

Amino Acid Sequence↗
PubMed2006

The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry.

Dengue virus (DV) is a flavivirus and its urban transmission is maintained largely by its mosquito vectors and vertebrate host, often human. In this study, investigation was carried out on the involvement of domain III of the envelope (E) glycosylated protein of dengue virus serotypes 1 and 2 (DV-1 and DV-2 DIII) in binding to host cell surfaces, thus mediating virus entry. Domain III protein of flavivirus can also serve as an attractive target in inhibiting virus entry. The respective DV DIII proteins were expressed as soluble recombinant fusion proteins before purification through enzymatic cleavage and affinity purification. The purified recombinant DV-1 and DV-2 DIII proteins both demonstrated the ability to inhibit the entry of DV-1 and DV-2 into HepG2 cells and C6/36 mosquito cells. As such, the DV DIII protein is indeed important for the interaction with cellular receptors in both human and mosquito cells. In addition, this protein induced antibodies that completely neutralized homologous dengue serotypes although not with the same efficiency among the heterologous serotypes. This observation may be of importance when formulating a generic vaccine that is effective against all dengue virus serotypes.

Animals↗
PubMed2005

Using deubiquitylating enzymes as research tools.

Ubiquitin is synthesized in eukaryotes as a linear fusion with a normal peptide bond either to itself or to one of two ribosomal proteins and, in the latter case, enhances the yield of these ribosomal proteins and/or their incorporation into the ribosome. Such fusions are cleaved rapidly by a variety of deubiquitylating enzymes. Expression of heterologous proteins as linear ubiquitin fusions has been found to significantly increase the yield of unstable or poorly expressed proteins in either bacterial or eukaryotic hosts. If expressed in bacterial cells, the fusion is not cleaved due to the absence of deubiquitylating activity and can be purified intact. We have developed an efficient expression system, utilizing the ubiquitin fusion technique and a robust deubiquitylating enzyme, which allows convenient high yield and easy purification of authentic proteins. An affinity purification tag on both the ubiquitin fusion and the deubiquitylating enzyme allows their easy purification and the easy removal of unwanted components after cleavage, leaving the desired protein as the only soluble product. Ubiquitin is also conjugated to epsilon amino groups in lysine side chains of target proteins to form a so-called isopeptide linkage. Either a single ubiquitin can be conjugated or other lysines within ubiquitin can be acceptors for further conjugation, leading to formation of a branched, isopeptide-linked ubiquitin chain. Removal of these ubiquitin moieties or chains in vitro would be a valuable tool in the ubiquitinologists tool kit to simplify downstream studies on ubiquitylated targets. The robust deubiquitylating enzyme described earlier is also very useful for this task.

Amino Acid Sequence↗
PubMed1994

The production of a monoclonal T3-antiidiotypic antibody (T3-MAAB) that mimics the effects of T3 on 2-deoxy-D-glucose uptake in chick embryo heart cells.

The Ig fraction of rabbit anti-T3 antibody was injected into the spleens of BALB/c mice. Four days later, the lymphocytes were recovered from their spleens and were fused with cells of the 653 myeloma cell line. Screening of the hybrid colonies was carried out in a T3 RIA system. Positive colonies were those whose supernatant displaced 125I-labeled T3 from its antibody. The positive cultures were recloned and one was injected ip into mice. The crude IgG fraction of the ascites fluid was affinity purified on an affigel-10 column containing a covalently bound rabbit anti-T3-IgG. In order to eliminate possible endogenous T3 contamination during the affinity purification, the column was stripped with a 40% solution of acetonitrile in 0.2 M acetic acid, neutralized, and then the purification proceeded as described. The affinity purified antibody was an IgG2a isotype. This monoclonal antibody (T3-MAAB) displaced labeled T3 from its antibody in an RIA system. It also mimicked T3 in the stimulation of [3H]2-deoxy-D-glucose (2-DOG) uptake in cultured chick embryo heart cells. After 6 h exposure, the dose-response curve of 2-DOG uptake to T3-MAAB was shifted to the left by at least one order of magnitude when compared to the dose-response curve obtained with T3. After 24 h exposure, T3 had the expected additional stimulatory effect that was dependent on neosynthesis of proteins, while T3-MAAB did not. Also at 24 h exposure, T3-MAAB did not stimulate the incorporation of labeled leucine and uridine into the heart cells while T3 at an equivalent concentration did. The MAAB activity could be abolished by boiling, while boiling did not affect the activity of an equivalent concentration of T3, thus excluding a T3 contamination-mediated effect. We conclude, therefore, that (a) a monoclonal hybridoma producing an antibody that mimics T3 was established; (b) this antibody competed with labeled T3 for anti-T3 antibody and, like T3, stimulated sugar uptake into cultured chick embryo heart cells; and (c) this antibody, unlike T3, did not stimulate the neosynthesis of proteins.

Animals↗
PubMed2002

Identification of delta helicase as the bovine homolog of HUPF1: demonstration of an interaction with the third subunit of DNA polymerase delta.

Delta helicase is a 5' to 3' DNA helicase that partially co-purifies with DNA polymerase delta (pol delta) from fetal bovine thymus tissue. We describe the resolution of delta helicase from pol delta on heparin-agarose chromatography and its purification to apparent homogeneity by affinity purification on single-stranded DNA-cellulose chromatography, unique-sequence RNA-agarose chromatography, and ceramic hydroxyapatite chromatography. Delta helicase isolated from fetal bovine thymus had an apparent M(r) of 115 kDa in SDS-PAGE, and photo-crosslinked to [alpha-32P]ATP. Tandem mass spectrometry peptide mass data derived from the bovine polypeptide matched to human UPF1 (HUPF1), a 5' to 3' RNA and DNA helicase, and a requisite component of the mRNA surveillance complex. Antisera against HUPF1 cross-reacted with delta helicase on western analysis, and delta helicase activity was immunoinactivated by pre-incubation with antibodies to HUPF1, suggesting that delta helicase is the bovine homolog of HUPF1. Immunoprecipitation experiments demonstrated that HUPF1 interacts with the 66-kDa third subunit of pol delta in vivo.

Adenosine Triphosphatases↗
PubMed2004

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Animals↗
PubMed1983

Purification of a bacterial organophosphate-hydrolysing phosphatase by Cibacron 3GA-Sepharose affinity chromatography.

A phosphatase catalysing the hydrolysis of organophosphorus pesticides was purified to homogeneity using Cibacron 3GA-Sepharose CL 6B affinity chromatography. The enzyme which is localized in the periplasm of the bacterium Alcaligenes NC5 was extracted by treating with 0.2M MgCl2, pH 8.4. The enzyme was adsorbed to the Cibacron-Sepharose at pH 7.0 and eluted with Tris-HCl buffer at pH 8.0, with 47 per cent recovery. The enzyme thus obtained was electrophoretically homogeneous. This simple affinity purification procedure enhances the potential for its use in large scale detoxification systems.

Alcaligenes↗
PubMed2004

Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope.

Beyond the rewards of plant genome analysis and gene identification, characterisation of protein activities, post-translational modifications and protein complex composition remains a challenge for plant biologists. Ideally, methods should allow rapid isolation of proteins from plant material achieving a high degree of purity. We tested three purification strategies based on the eight-amino acid StrepII, six-amino acid His(6) and 181-amino acid Tandem Affinity Purification (TAP) affinity tags for enrichment of a membrane-anchored protein kinase, Nt CDPK2, and a soluble protein, At SGT1b, from leaf extracts. Transiently expressed StrepII-tagged Nt CDPK2 was purified from Nicotiana benthamiana to almost complete homogeneity in less than 60 min and was directly suitable for enzymatic or mass-spectrometric analyses, allowing the identification of in planta phosphorylation sites. In contrast, purification of Nt CDPK2 via His(6) tag yielded partially oxidised protein of low purity. At SGT1b could be isolated after transient expression from N. benthamiana or from transgenic Arabidopsis thaliana as either TAP-tagged or StrepII-tagged protein. While StrepII-tag purification achieved similar yield and high purity as the TAP-tag strategy, it was considerably easier and faster. Using either tagging strategy, a protein was co-purified with At SGT1b from N. benthaniana and A. thaliana leaf extracts, suggesting that both the StrepII and TAP tags are suitable for purification of protein complexes from plant material. We propose that the StrepII epitope, in particular, may serve as a generally utilizable tag to further our understanding of protein functions, post-translational modifications and interaction dynamics in plants.

Amino Acid Sequence↗
PubMed2005

Purification and characterization of a single-chain chimeric anti-p185 antibody expressed by CHO-GS system.

Monoclonal antibody A21 reacts specifically with the extracellular domain of p185c-erbB-2 oncoprotein, a member of the epidermal growth factor receptor family. In a previous study, we constructed a single-chain chimeric antibody, assembled using an A21 single-chain Fv antibody and a human IgG1 Fc fragment. In this study, we expressed this chimeric antibody using a CHO-GS system, and developed a simple and efficient method for its purification. After only one step using affinity purification, the recovery rate and purity of the antibody attained was 60 and 91%, respectively. After a second step, using reverse phase HPLC purification, the purity was above 99%. The high purity of the recombinant antibody allowed us to identify a number of its intrinsic molecular properties, including antigen binding activity, measurement of affinity constant, N-terminal sequencing, and mass spectrometer analysis. These results further augment the potential of this recombinant antibody to be a drug candidate for cancer therapy.

Amino Acid Sequence↗
PubMed2003

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins.

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.

Blotting, Western↗
PubMed2004

Applications of affinity chromatography in proteomics.

Affinity chromatography is a powerful protein separation method that is based on the specific interaction between immobilized ligands and target proteins. Peptides can also be separated effectively by affinity chromatography through the use of peptide-specific ligands. Both two-dimensional electrophoresis (2-DE)- and non-2-DE-based proteomic approaches benefit from the application of affinity chromatography. Before protein separation by 2-DE, affinity separation is used primarily for preconcentration and pretreatment of samples. Those applications entail the removal of one protein or a class of proteins that might interfere with 2-DE resolution, the concentration of low-abundance proteins to enable them to be visualized in the gel, and the classification of total protein into two or more groups for further separation by gel electrophoresis. Non-2-DE-based approaches have extensively employed affinity chromatography to reduce the complexity of protein and peptide mixtures. Prior to mass spectrometry (MS), preconcentration and capture of specific proteins or peptides to enhance sensitivity can be accomplished by using affinity adsorption. Affinity purification of protein complexes followed by identification of proteins by MS serves as a powerful tool for generating a map of protein-protein interactions and cellular locations of complexes. Affinity chromatography of peptide mixtures, coupled with mass spectrometry, provides a tool for the study of protein posttranslational modification (PTM) sites and quantitative proteomics. Quantitation of proteomes is possible via the use of isotope-coded affinity tags and isolation of proteolytic peptides by affinity chromatography. An emerging area of proteomics technology development is miniaturization. Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. More applications of affinity-based purification can be expected, including increasing the resolution in 2-DE, improving the sensitivity of MS quantification, and incorporating purification as part of multidimensional liquid chromatography experiments.

Chromatography, Affinity↗
PubMed1994

Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates.

We have characterized, by ligand blotting, solubilized tumor necrosis factor receptors (TNFR) from K562 cells. Preparations that had been partially purified by gel filtration chromatography yielded two prominent bands of M(r) 60,000 and 75,000 corresponding to the two known TNFR (types I and II, respectively). In addition to these, types I and II TNFR-related species of M(r) > 100,000 were detected after purification by tumor necrosis factor (TNF)-affinity chromatography, suggesting that TNF had driven receptor aggregation during this step. To test this hypothesis ligand blots were performed on receptor preparations that had been partially purified by gel filtration chromatography and incubated with TNF before electrophoretic separation. Indeed, type II TNFR aggregates, but not type I TNFR aggregates, were generated at optimal TNF concentrations. Formation of type II TNFR aggregates in this last experimental setting and of both type I and type II TNFR aggregates during affinity purification could be prevented if an alkylating agent (N-ethylmaleimide) was added during the TNFR-TNF incubation step. Similar results were obtained when intact K562 cells were incubated with TNF and then analyzed for receptor aggregation; type II TNF receptor aggregates were generated at TNF concentrations ranging from 10(-9) to 10(-10) M and their formation was prevented in the presence of N-ethylmaleimide.

Blotting, Western↗
PubMed2004

Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors.

Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC(50) values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC alpha or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome.

Animals↗