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Sample records for affinity chromatography purification

  1. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  2. Affinity chromatography for purification of two urokinases from human urine.

    PubMed

    Takahashi, R; Akiba, K; Koike, M; Noguchi, T; Ezure, Y

    2000-05-26

    A new affinity chromatography (hydrophobic-mediated affinity chromatography), which was characterized by the matrix having both affinity site to urokinase and hydrophobic site, was established for the purification of urokinase from human urine. The hydrophobic affinity matrix (tentatively named PAS in the text) was prepared by immobilizing 6-aminocaproic acid on Sepharose CL-6B, followed by a coupling p-aminobenzamidine to a part of the hydrophobic site on the matrix. The PAS matrix was applied to the purification of urokinase from human urine, and high- and low-molecular weight pure urokinases were efficiently obtained in high yield by the present method. PMID:10892585

  3. Purification of glycolytic enzymes by using affinity-elution chromatography.

    PubMed Central

    Scopes, R K

    1977-01-01

    1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures. PMID:192194

  4. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  5. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  6. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Graça, Vânia C; Sousa, Fani; Santos, Paulo F; Almeida, Paulo S

    2015-01-01

    Affinity chromatography (AC) is one of the most important techniques for the separation and purification of biomolecules, being probably the most selective technique for protein purification. It is based on unique specific reversible interactions between the target molecule and a ligand. In this affinity interaction, the choice of the ligand is extremely important for the success of the purification protocol. The growing interest in AC has motivated an intense research effort toward the development of materials able to overcome the disadvantages of conventional natural ligands, namely their high cost and chemical and biological lability. In this context, synthetic dyes have emerged, in recent decades, as a promising alternative to biological ligands. Herein, detailed protocols for the assembling of a new chromatographic dye-ligand affinity support bearing an immobilized aminosquarylium cyanine dye on an agarose-based matrix (Sepharose CL-6B) and for the separation of a mixture o f three standard proteins: lysozyme, α-chymotrypsin, and trypsin are provided. PMID:25749942

  7. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  8. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  9. Production and Purification of Streptokinase by Protected Affinity Chromatography

    PubMed Central

    Babashamsi, Mohammad; Razavian, Mohammad Hossein; Nejadmoghaddam, Mohammad Reza

    2009-01-01

    Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific streptokinase assay. The results showed that in the fed–batch culture, the rate of streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the streptokinase purification to a single step increased the yield over 95% at the chromatography stage. PMID:23407807

  10. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC).

    PubMed

    de Costa, Fernanda; Barber, Carla J S; Pujara, Pareshkumar T; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography. PMID:26843168

  11. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  12. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  13. Purification of phage display-modified bacteriophage T4 by affinity chromatography

    PubMed Central

    2011-01-01

    Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be

  14. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples. PMID:27498022

  15. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

  16. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    PubMed

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  17. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column. PMID:19469504

  18. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  19. Development of a novel affinity chromatography resin for platform purification of lambda fabs.

    PubMed

    Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

    2014-01-01

    Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. PMID:25082738

  20. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  1. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  2. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

  3. Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes

    SciTech Connect

    Marie, J.; Seyer, R.; Lombard, C.; Desarnaud, F.; Aumelas, A.; Jard, A.; Bonnafous, J.C. )

    1990-09-25

    The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH{sub 2}){sub 2}SS(CH{sub 2}){sub 2}CO-(Ala{sup 1}, Phe(4N{sub 3}){sup 8})AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (K{sub d}{approximately}1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

  4. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  5. Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

    PubMed

    Ahirwar, Rajesh; Nahar, Pradip

    2015-08-01

    Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A. PMID:26102634

  6. Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

    PubMed

    Cass, Brian; Pham, Phuong Lan; Kamen, Amine; Durocher, Yves

    2005-03-01

    Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. PMID:15721774

  7. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    PubMed

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

  8. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  9. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies.

    PubMed

    Boulet-Audet, Maxime; Kazarian, Sergei G; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  10. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  11. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  12. Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

    PubMed

    Kuhar, Kalika; Mittal, Anuradha; Kansal, Rekha; Gupta, Vijay Kumar

    2014-02-01

    Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to -14-fold with -84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100 degrees C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50 degrees C and 37 degrees C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.

  13. Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.

    PubMed

    Chand, Karam; Biswas, Sanchay K; Mondal, Bimalendu

    2016-03-01

    An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. PMID:26925450

  14. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    PubMed

    Rimmelzwaan, G F; Groen, J; Juntti, N; Teppema, J S; UytdeHaag, F G; Osterhaus, A D

    1987-03-01

    Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen. The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine serum albumin content in an ELISA and by SDS-PAGE. Over 99% of the contaminating proteins proved to be removed, and 20% or 70-90% of infectious CPV or CPV antigen, respectively, was recovered.

  15. p53-Encoding pDNA Purification by Affinity Chromatography for Cancer Therapy.

    PubMed

    Sousa, Ângela; Queiroz, João A; Sousa, Fani

    2015-01-01

    The gene therapy approach based on reestablishment of p53 tumor suppressor, which acts as a prevailing guardian against malignant cell transformation, is raising new prospects on the outcome of an effective anticancer treatment. It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Therefore, several downstream methods have been proposed to achieve high quantities of supercoiled plasmid DNA with pharmaceutical grade purity. Affinity chromatography with amino acids as ligands has recently yielded interesting results because these ligands take advantage of their biological function or chemical structure to promote specific interactions with different nucleic acids. Here, we describe detailed procedures for the preparation and purification of supercoiled plasmid DNA, with the purity degree required by regulatory agencies, by using arginine affinity chromatography. With this methodology pure pDNA is obtained, efficient on eukaryotic cell transfection and biologically active, resulting in the reestablishment of the p53 protein levels in cancer cell lines. PMID:26072404

  16. Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bering, C. Larry; Kuhns, Jennifer J.; Rowlett, Roger

    1998-08-01

    We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.

  17. Ligand affinity chromatography, an indispensable method for the purification of soluble cytokine receptors and binding proteins.

    PubMed

    Novick, Daniela; Rubinstein, Menachem

    2012-01-01

    Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity. PMID:22131033

  18. Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

    PubMed Central

    Locatelli-Hoops, Silvia C.; Yeliseev, Alexei A.

    2016-01-01

    Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. PMID:24943318

  19. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    PubMed

    Salehi, Nasrin; Peng, Ching-An

    2016-07-01

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. PMID:27110670

  20. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    PubMed

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

  1. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  2. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  3. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  4. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  5. Purification of Hemoglobin from Red Blood Cells using Tangential Flow Filtration and Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Elmer, Jacob; Harris, David; Palmer, Andre F.

    2011-01-01

    Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are examined and compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter. PMID:21195679

  6. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    PubMed

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. PMID:26657801

  7. Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography.

    PubMed

    Bartnicki, Filip; Kowalska, Ewa; Pels, Katarzyna; Strzalka, Wojciech

    2015-10-30

    Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin. PMID:26427325

  8. Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors

    PubMed Central

    Kuester, Miriam; Becker, Gero L.; Hardes, Kornelia; Lindberg, Iris; Steinmetzer, Torsten; Than, Manuel E.

    2013-01-01

    In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied – studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)2-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members. PMID:21875402

  9. Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel.

    PubMed

    Beyaztaş, Serap; Arslan, Oktay

    2015-06-01

    A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150 kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60 °C and incubated for 60 min. These results indicated that the enzyme was heat stable. PMID:25089709

  10. Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Voráčková, Irena; Suchanová, Šárka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomáš

    2011-01-01

    Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state. PMID:21600288

  11. Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

    PubMed Central

    Wright, C L; Warsy, A S; Holroyde, M J; Trayer, I P

    1978-01-01

    The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography

  12. Purification of phosphinothricin acetyltransferase using Reactive brown 10 affinity in a single chromatography step.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2013-08-01

    The expression of phosphinothricin N-acetyltransferase (PAT) protein in transgenic plants confers tolerance to the herbicide glufosinate. To enable the characterization of PAT protein expressed in plants, it is necessary to obtain high purity PAT protein from the transgenic grain. Because transgenically expressed proteins are typical present at very low levels (i.e. 0.1-50 μg protein/g grain), a highly specific and efficient purification protocol is required to purify them. Based on the physicochemical properties of PAT, we developed a novel purification method that is simple, time-saving, inexpensive and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein was purified to homogeneity from cottonseed with high recovery efficiency. As expected, the Reactive brown 10-produced PAT was enzymatically active. Other applications of the method on protein expression and purification, and development of PAT enzymatic inhibitors were also discussed. PMID:23748142

  13. One-step purification of lactoperoxidase from bovine milk by affinity chromatography.

    PubMed

    Atasever, Ali; Ozdemir, Hasan; Gulcin, Ilhami; Irfan Kufrevioglu, O

    2013-01-15

    Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) of 0.848.10(-5)M. The K(i) for sulphanilamide was determined to be 3.57.10(-5)M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R(z) (A(412)/A(280)) value. The R(z) value for the purified LPO was found to be 0.7. Maximum binding was achieved and K(m) and V(max) values were determined.

  14. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    PubMed

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

  15. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    PubMed

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants. PMID:18307162

  16. Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.

    PubMed Central

    Callaghan, J M; Toh, B H; Simpson, R J; Baldwin, G S; Gleeson, P A

    1992-01-01

    We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized

  17. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 1: Theory

    PubMed Central

    2015-01-01

    We present a novel technique that couples isotachophoresis (ITP) with affinity chromatography (AC) to achieve rapid, selective purification with high column utilization. ITP simultaneously preconcentrates an analyte and purifies it, based on differences in mobility of sample components, excluding species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization, and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into nondiffusing zones, thus achieving high resolution in a short distance and time. We present an analytical model for spatiotemporal dynamics of ITP-AC. We identify and explore the effect of key process parameters, including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our analytical approach shows collapse of these variables to three nondimensional parameters. The analysis yields simple analytical relations for capture length and capture time in relevant ITP-AC regimes, and it demonstrates how ITP greatly reduces assay time and improves column utilization. In the second part of this two-part series, we will present experimental validation of our model and demonstrate ITP-AC separation of the target from 10,000-fold more-abundant contaminants. PMID:24937679

  18. "Clickable" agarose for affinity chromatography.

    PubMed

    Punna, Sreenivas; Kaltgrad, Eiton; Finn, M G

    2005-01-01

    Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

  19. Purification of peroxidase from red cabbage (Brassica oleracea var. capitata f. rubra) by affinity chromatography.

    PubMed

    Somtürk, Burcu; Kalın, Ramazan; Özdemir, Nalan

    2014-08-01

    Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9% from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K m and V max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC50 and K i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702±0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.

  20. Necator americanus secretory acetylcholinesterase and its purification from excretory-secretory products by affinity chromatography.

    PubMed

    Pritchard, D I; Leggett, K V; Rogan, M T; McKean, P G; Brown, A

    1991-03-01

    Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship. PMID:2052405

  1. Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

    PubMed

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

    We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535

  2. Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.

    PubMed

    Tan, Lik Chern Melvin; Chua, Anthony Jin Shun; Goh, Li Shan Liza; Pua, Shu Min; Cheong, Yuen Kuen; Ng, Mah Lee

    2010-11-01

    Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1-4 and WNV - New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI-TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus.

  3. Engineering foot-and-mouth disease virus serotype O IND R2/1975 for one-step purification by immobilized metal affinity chromatography.

    PubMed

    Biswal, Jitendra K; Bisht, Punam; Subramaniam, Saravanan; Ranjan, Rajeev; Sharma, Gaurav K; Pattnaik, Bramhadev

    2015-09-01

    Immobilized metal affinity chromatography (IMAC) allows for the efficient protein purification via metal affinity tag such as hexa-histidine (His6) sequence. To develop a new chromatography strategy for the purification and concentration of foot-and-mouth disease virus (FMDV) particles, we inserted the His6-tag at the earlier reported site in the VP1 G-H loop of the FMD virus serotype O vaccine strain IND R2/1975. Display of the His6-tag on the capsid surface, endowed the virus with an increased affinity for immobilized nickel ions. We demonstrated that the His6-tagged FMDV could be produced to high titre and purified from the infected BHK-21 cell lysates by IMAC efficiently. Further, a 1150-fold reduction in protein contaminant level and an 8400-fold reduction in DNA contaminant level were achieved in the IMAC purification of His6-tagged FMDV. Through various functional assays it has been found that the tagged virus retains its functionality and infectivity similar to the non-tagged virus. The affinity purification of the His6-tagged FMDV may offer a feasible, alternative approach to the current methods of FMDV antigen purification, concentration and process scalability. PMID:26123433

  4. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 2: Experimental Study

    PubMed Central

    2015-01-01

    We present an experimental study of coupling of isotachophoresis (ITP) and affinity chromatography (AC) to effect rapid, selective purification with high column utilization and high resolution. We provide a detailed protocol for performing ITP-AC and describe the design of a buffer system to perform sequence specific separation of nucleic acids. We describe the synthesis and functionalization of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM). This substrate allows easy immobilization of affinity probes, is nonsieving (even to macromolecules), and exhibits negligible nonspecific binding. We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal dynamics using epifluorescence imaging. We make qualitative and quantitative comparisons between these data and the model presented in the first part of this two-paper series. We vary the target concentration from 1 pg μL–1 to 100 pg μL–1 and ITP velocity over the range of 10–50 μm s–1, and thereby explore over 4 orders of magnitude of scaled target amount. We observe very good agreement between predictions and experimental data for the spatiotemporal behavior of the coupled ITP and affinity process, and for key figures of merit, including scaled capture length and maximum capture efficiency. Lastly, we demonstrate that the resolution of ITP-AC increases linearly with time and purify 25 nt target DNA from 10 000-fold higher abundance background (contaminating) genomic fish sperm DNA. We perform this capture from 200 μL of sample in under 1 mm column length and within <10 min. PMID:24937777

  5. The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

    Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form. PMID:26695022

  6. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  7. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    PubMed

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

  8. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  9. Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

    PubMed Central

    Brehm, R D; Tranter, H S; Hambleton, P; Melling, J

    1990-01-01

    A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production. Images PMID:2339869

  10. Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

    PubMed

    Efremenko, E; Votchitseva, Y; Plieva, F; Galaev, I; Mattiasson, B

    2006-05-01

    Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.

  11. Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

    PubMed

    Efremenko, E; Votchitseva, Y; Plieva, F; Galaev, I; Mattiasson, B

    2006-05-01

    Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration. PMID:16088350

  12. Transient conformational modification of immunoglobulin G during purification by protein A affinity chromatography.

    PubMed

    Gagnon, Pete; Nian, Rui; Leong, Denise; Hoi, Aina

    2015-05-22

    Exposure of three native IgG1 monoclonal antibodies to 100mM acetate, pH 3.5 had no significant effect on their hydrodynamic size (11.5±0.5nm), while elution from protein A with the same buffer created a conformation of 5.5±1.0nm. Formation of the reduced-size conformation was preceded by the known destabilization of the second constant domain of the heavy chain (Cγ2) by contact with protein A, then compounded by exposure to low pH, creating extended flexibility in the hinge-Cγ2 region and allowing the Fab region to fold over the Fc region. The reduced-size conformation was necessary for complete elution. It persisted unchanged for at least 7 days under elution conditions. Physiological conditions restored native size, and it was maintained on re-exposure to 100mM acetate, pH 3.5. Protein A-mediated destabilization and subsequent restoration of native size did not create aggregates, but the reduced-size conformation was more susceptible to aggregation by secondary stress than native antibody. Protein A-mediated formation of the reduced-size conformation is probably universal during purification of human IgG1 antibodies, and may occur with other subclasses and IgG from other species, as well as Fc-fusion proteins. PMID:25882588

  13. Purification of human immunoglobulins A, G and M from Cohn fraction II/III by small peptide affinity chromatography.

    PubMed

    Liu, Zhuo; Gurgel, Patrick V; Carbonell, Ruben G

    2012-11-01

    This work describes attempts to purify human IgG, IgA and IgM from Cohn fraction II/III using HWRGWV affinity peptide resin. The effects of peptide density and different elution additives on recovery of the three antibodies were investigated. At low peptide density, salting-in salts such as magnesium chloride and calcium chloride facilitated antibody elution. Ethylene glycol, urea and arginine also facilitated elution because of their ability to decrease hydrophobic interactions, hydrogen bonding and electrostatic interactions. However, at high peptide density, no recovery improvements were observed because of increased non-specific hydrophobic interactions. The final elution conditions for each antibody were chosen based on the resulting yields and purities when a 10:2:1mg/mL mixture of human IgG, IgA and IgM was used as starting material. Different pretreatment methods were employed in order to improve the purity of antibodies from Cohn fraction II/III. After pretreatment with caprylic acid precipitation or combination of caprylic acid and polyethylene glycol precipitation, purities over 95% and yields of about 60% were obtained for hIgG, which are comparable to current chromatographic purification methods involving two chromatography steps when hIgG is isolated from plasma fractions. A hIgA-enriched fraction with 42% hIgA and 56% hIgG, as well as a hIgM enriched fraction with 46% hIgM, 28% hIgA and 24% hIgG, were obtained as the by-products. PMID:23026261

  14. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  15. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

    Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody. PMID:26160561

  16. The purification of human enterokinase by affinity chromatography and immunoadsorption. Some observations on its molecular characteristics and comparisons with the pig enzyme.

    PubMed Central

    Grant, D A; Hermon-Taylor, J

    1976-01-01

    A method is described for the purification of human enterokinase from accumulated duodenal fluid by affinity chromatography using p-aminobenzamidine as the ligand. Resolution was greatest when glycylglycine was substituted as the spacer arm. Purification was not a one-step procedure, and some contamination, principally by the alpha-glucosidases, remained. Their removal was completed by immunoadsorption using antisera raised to enterokinase-free material containing these enzymes, prepared as a by-product of the purification procedure. The final preparation had an activity of 4260 nmol of trypsin/min per mg and was free of other enzymic activity tested. Amino acid and sugar analyses of the highly purified enzyme indicated an acidic glycoprotein containing 57% sugar (neutral sugars 47%, amino sugars 10%). The apparent mol.wts. and Stokes radii of human and pig enterokinase were 296 000 and 316 000, and 5.65 and 5.78 nm respectively. Two isoenzymes were identified for human enterokinase and three for the pig enzyme. Human enterokinase demonstrated a resistance to reduction of disulphide linkages and to sodium dodecyl sulphate binding, which may be related to the need for it to retain its integrity in the digestive environment of the upper small intestine. Antisera to highly purified pig and human enterokinases specifically inhibited enterokinase activity. Immuno-inhibition of intestinal aminopeptidase, maltase and glucoamylase by homologous antisera was not observed. Images PLATE 1 PMID:945736

  17. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  18. Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

    PubMed Central

    Gounaris, A D; Brown, M A; Barrett, A J

    1984-01-01

    Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated. Images Fig. 2. Fig. 3. Fig. 4. PMID:6548132

  19. Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

    PubMed

    Arimitsu, Hideyuki; Sasaki, Keiko; Kojima, Hiroe; Yanaka, Tadashi; Tsuji, Takao

    2013-01-01

    Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease. PMID:24340102

  20. Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group

    PubMed Central

    Arimitsu, Hideyuki; Sasaki, Keiko; Kojima, Hiroe; Yanaka, Tadashi; Tsuji, Takao

    2013-01-01

    Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease. PMID:24340102

  1. Immobilized iminodiacetic acid (IDA)-type Cu2+ -chelating membrane affinity chromatography for purification of bovine liver catalase.

    PubMed

    Yang, L; Jia, L; Zou, H; Zhang, Y

    1999-05-01

    A metal ion chelating membrane medium based on iminodiacetate-substituted modified short cotton cellulose was examined for the purification of bovine liver catalase (BLC). The effect of buffer pH, chelator surface density, initial concentration of crude enzyme and flow rate on BLC binding efficiency to the copper ion chelating membrane adsorbent were examined. Under the chromatographic conditions chosen, 67.7% recovery of BLC was attained with an overall 4.2-fold increase in specific activity in a single step. After performance of BLC purification, the chelating membrane adsorbent can be easily regenerated by imidazole or EDTA buffer with higher reviving effectiveness with the latter. PMID:10375124

  2. Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa.

    PubMed Central

    Yerima, A; Safi, A; Gastin, I; Michalski, J C; Saunier, M; Gueant, J L

    1996-01-01

    We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor. PMID:8573109

  3. Molecular insight in the purification of immunoglobulin by pseudobiospecific ligand l-histidine and histidyl moieties in histidine ligand affinity chromatography (HLAC) by molecular docking.

    PubMed

    Savane, Tushar S; Kumar, Sanjit; Janakiraman, Vignesh Narasimhan; Kamalanathan, Agamudi S; Vijayalakshmi, Mookambeswaran A

    2016-05-15

    Pseudobiospecific ligand l-histidine is an inexpensive, highly stable, non-toxic ligand explored successfully over the last twenty years for the purification of immunoglobulins in immobilised histidine ligand affinity chromatography. It is of great interest to know the molecular recognition sites of IgG to immobilized l-histidine. Here, we have used an in silico approach to explore the molecular recognition of l-histidine by IgG. We have assessed the feasible binding modes of histidine and its moieties at different sites of IgG and considered only those binding conformations which are exhibited via the imidazole ring NH group or any other OH donating group apart from the ones which are terminally conjugated with the support matrix. We categorised binding site into two categories; category I: inner binding groove and category II: surface binding groove and observed that the hinge region of IgG has most favourable binding pocket for l-histidine and histidyl moieties. Ser and Tyr residues on the hinge region make several significant interactions with l-histidine and histidyl moieties. In case of Fc region of IgG, l-histidine and histidyl moieties closely resemble the binding modes of Protein A, biomimetic ligand 22/8 and B domain of SpA to IgG. In addition to these we have also observed a significant binding site for l-histidine and histidyl moieties at Fab region of IgG. PMID:26476866

  4. Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

    PubMed

    Huangfu, Chaoji; Ma, Yuyuan; Lv, Maomin; Jia, Junting; Zhao, Xiong; Zhang, Jingang

    2016-07-01

    As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource. PMID:27214605

  5. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  6. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  7. Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay.

    PubMed Central

    Armstrong, Michael; Liu, Andrew H.; Harbeck, Ronald; Reisdorph, Rick; Rabinovitch, Nathan; Reisdorph, Nichole

    2009-01-01

    A new analytical method suitable for high throughput measurements of LTE4 in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/ tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500 pg/mL in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE4 from healthy adults and children are reported. PMID:19726242

  8. Affinity Purification of Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Kadonaga, James T.; Tjian, Robert

    1986-08-01

    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.

  9. Affinity chromatography with an immobilized RNA enzyme.

    PubMed Central

    Vioque, A; Altman, S

    1986-01-01

    M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid. Images PMID:3526344

  10. Improving affinity chromatography resin efficiency using semi-continuous chromatography.

    PubMed

    Mahajan, Ekta; George, Anupa; Wolk, Bradley

    2012-03-01

    Protein A affinity chromatography is widely used for purification of monoclonal antibodies (MAbs) from harvested cell culture fluid (HCCF). At the manufacturing scale, the HCCF is typically loaded on a single Protein A affinity chromatography column in cycles until all of the HCCF is processed. Protein A resin costs are significant, comprising a substantial portion of the raw material costs in MAb manufacturing. Cost can be reduced by operating the process continuously using multiple smaller columns to a higher binding capacity in lieu of one industrial scale column. In this study, a series of experiments were performed using three 1-ml Hi-Trap™ MabSelect SuRe™ columns on a modified ÄKTA™ system operated according to the three Column Periodic Counter Current Chromatography (3C PCC) principle. The columns were loaded individually at different times until the 70% breakthrough point was achieved. The HCCF with unbound protein from the column was then loaded onto the next column to capture the MAb, preventing any protein loss. At any given point, all three columns were in operation, either loading or washing, enabling a reduction in processing time. The product yield and quality were evaluated and compared with a batch process to determine the effect of using the three column continuous process. The continuous operation shows the potential to reduce both resin volume and buffer consumption by ∼40%, however the system hardware and the process is more complex than the batch process. Alternative methods using a single standard affinity column, such as recycling load effluent back to the tank or increasing residence time, were also evaluated to improve Protein A resin efficiency. These alternative methods showed similar cost benefits but required longer processing time. PMID:22265178

  11. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. PMID:26830537

  12. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

  13. FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody.

    PubMed

    Zhao, Wei-Wei; Shi, Qing-Hong; Sun, Yan

    2014-08-15

    This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles. PMID:24947889

  14. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  15. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  16. Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography ▿ † ‡

    PubMed Central

    Robichon, Carine; Luo, Jianying; Causey, Thomas B.; Benner, Jack S.; Samuelson, James C.

    2011-01-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein. PMID:21602383

  17. Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography.

    PubMed

    Akuta, Teruo; Kikuchi-Ueda, Takane; Imaizumi, Keitaro; Oshikane, Hiroyuki; Nakaki, Toshio; Okada, Yoko; Sultana, Sara; Kobayashi, Kenichiro; Kiyokawa, Nobutaka; Ono, Yasuo

    2015-01-01

    Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.

  18. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

  19. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes. PMID:25764651

  20. Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography.

    PubMed

    Bansal, Vibha; Roychoudhury, Pradip K; Mattiasson, Bo; Kumar, Ashok

    2006-01-01

    An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(II)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different elution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography. PMID:16761300

  1. A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode.

    PubMed

    Shamashkin, Michael; Godavarti, Ranga; Iskra, Timothy; Coffman, Jon

    2013-10-01

    A significant consequence of scaling up production of high titer monoclonal antibody (mAb) processes in existing facilities is the generation of in-process pools that exceed the capacity of storage vessels. A semi-continuous downstream process where columns and filters are linked and operated in tandem would eliminate the need for intermediate holding tanks. This study is a bench-scale demonstration of the feasibility of a tandem process for the purification of mAbs employing an affinity Protein A capture step, followed by a flow-through anion-exchange (AEX) step with the possibility of adding an in-line virus filtration step (VF). All three steps were linked sequentially and operated as one continuous process using an ÄKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX column was operated in a weak partitioning (WP) mode enabled by a precise in-line titration of Protein A effluent. In order to avoid complex control schemes and facilitate validation, quality and robustness were built into the system through selection of buffers based on thermodynamic and empirical models. The tandem system utilized the simplest possible combination of valves, pumps, controls, and automation, so that it could easily be implemented in a clinical or commercial production facility. Linking the purification steps in a tandem process is expected to generate savings in time and production costs and also reduce the size of quality systems due to reduced documentation requirements, microbial sampling, and elimination of hold time validation. PMID:23633385

  2. Functional characterization of the kinase activation loop in nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) using tandem affinity purification and liquid chromatography-mass spectrometry.

    PubMed

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >or=1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

  3. Studies on ram acrosin. Activation of proacrosin accompanying the isolation of acrosin from spermatozoa, and purification of the enzyme by affinity chromatography.

    PubMed Central

    Brown, C R; Hartree, E F

    1978-01-01

    1. A previously described, freeze-dried, partially purified ram acrosin preparation was fractionated on a column of Sepharose linked to the acrosin inhibitor p-(p'-aminophenoxypropoxy)benzamidine. Two acrosin fractions were obtained. 2. beta-Acrosin was homogeneous, quite stable at low pH and very stable when freeze-dried. Its molecular weight is about 38000, and it contains about six sugar residues per molecule, but no sialic acid. psi-Acrosin consisted of at least three unstable forms of acrosin. 3. When the entire purification process, starting from collection of semen, was carried out as rapidly as possible, the yield of beta-acrosin was increased and very little psi-acrosin was obtained. 4. In fresh ram semen the acrosin is present as the intra-acrosomal zymogen, proacrosin. After its extraction from spermatozoa autoproteolytic reactions convert proacrosin into beta-acrosin; psi-acrosin appears to be breakdown products of beta-acrosin. 5. When beta-acrosin was passed through a column of Sepharose linked to the non-inhibitory deamidinated analogue of the inhibitor it behaved as a hydrophobic protein. This is consistent with our view that acrosin (as zymogen) occurs in spermatozoa as a membrane-bound protein. 6. Success in the isolation of pure acrosin in high yield calls for an affinity adsorbent with the appropriate subsidiary hydrophobic properties. PMID:736895

  4. Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry*

    PubMed Central

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C.

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of ≥1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK. PMID:19887368

  5. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  6. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  7. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  8. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  9. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  10. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  11. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  12. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  13. A modular approach to multifunctional polypeptide/ceramic fluorapatite-based self-assembled system in affinity chromatography for the purification of human Immunoglobulin G.

    PubMed

    Islam, Tuhidul; Fernández-Lahore, Marcelo

    2015-03-01

    The multifunctional bone sialoprotein/apatite (AP) self-assembled systems in the mineralized tissues show a pathway for the noncovalent immobilization of ligands on the AP chromatographic matrix. A model approach is presented here regarding the physical immobilization of ligands on the ceramic fluorapatite (CFT) matrix for the purification of human Immunoglobulin G (hIgG). The peptide pIC, HWRGWV-KPRSVSG, composed of a hIgG-specific peptide, HWRGWV (pLI), and a CFT-specific peptide, KPRSVSG (pTC), was synthesized and subjected to physicochemical characterization. A circular dichroism study showed that pIC possesses a flexible structural feature, which is significant in terms of its multifunctional activities. With the current approach, hIgG will be retained selectively by the self-assembled pIC/CFT column, while other biomolecules will pass through the column without being interacted. Therefore, the chromatographic conditions that are the key factors for the successful implementation of this technique were optimized as a function of the composition and pH of the mobile phase. Here, 115 mM sodium chloride (NaCl) in 20 mM sodium phosphate, pH 7.4, was used as the binding buffer, and the elution was performed with 225 mM NaCl in 20 mM sodium phosphate containing 0.3% w/v sodium acetate at pH 6. The binding capacity of the pIC/CFT column was 21.5 mg hIgG/ml matrix with a ligand density of 18.8 µmol/ml, and the binding capacity of the column increased with the increment of ligand density. Afterward, the applicability of a spacer arm between pLI and pTC was also verified. The hIgG-binding capacity of the column decreased with the increment in size of the spacer. In conclusion, the peptide-mediated self-assembled biomimetic system can be used as an alternative to the chemical immobilization of ligands in order to prevent unwanted consequences that result from some of the conventional ligand coupling chemistry.

  14. A modular approach to multifunctional polypeptide/ceramic fluorapatite-based self-assembled system in affinity chromatography for the purification of human Immunoglobulin G.

    PubMed

    Islam, Tuhidul; Fernández-Lahore, Marcelo

    2015-03-01

    The multifunctional bone sialoprotein/apatite (AP) self-assembled systems in the mineralized tissues show a pathway for the noncovalent immobilization of ligands on the AP chromatographic matrix. A model approach is presented here regarding the physical immobilization of ligands on the ceramic fluorapatite (CFT) matrix for the purification of human Immunoglobulin G (hIgG). The peptide pIC, HWRGWV-KPRSVSG, composed of a hIgG-specific peptide, HWRGWV (pLI), and a CFT-specific peptide, KPRSVSG (pTC), was synthesized and subjected to physicochemical characterization. A circular dichroism study showed that pIC possesses a flexible structural feature, which is significant in terms of its multifunctional activities. With the current approach, hIgG will be retained selectively by the self-assembled pIC/CFT column, while other biomolecules will pass through the column without being interacted. Therefore, the chromatographic conditions that are the key factors for the successful implementation of this technique were optimized as a function of the composition and pH of the mobile phase. Here, 115 mM sodium chloride (NaCl) in 20 mM sodium phosphate, pH 7.4, was used as the binding buffer, and the elution was performed with 225 mM NaCl in 20 mM sodium phosphate containing 0.3% w/v sodium acetate at pH 6. The binding capacity of the pIC/CFT column was 21.5 mg hIgG/ml matrix with a ligand density of 18.8 µmol/ml, and the binding capacity of the column increased with the increment of ligand density. Afterward, the applicability of a spacer arm between pLI and pTC was also verified. The hIgG-binding capacity of the column decreased with the increment in size of the spacer. In conclusion, the peptide-mediated self-assembled biomimetic system can be used as an alternative to the chemical immobilization of ligands in order to prevent unwanted consequences that result from some of the conventional ligand coupling chemistry. PMID:25663265

  15. Affinity purification of a siderophore that exhibits an antagonistic effect against soft rot bacterium.

    PubMed

    Helmy, Mohamed; Baddar, Doa; El'Masry, Mohamed Hisham

    2008-07-01

    Bacterial colonies were isolated from different Egyptian soil samples. From these isolates, one bacterial species was found to produce siderophore. Using classical and biochemical identification methods, the siderophore producing isolate was identified as Pseudomonas fluorescens. Based on the affinity of siderophores for metal ions, an affinity chromatography system was designed for the purification of the siderophore in one step. It was possible to isolate 25 mg siderophore per liter of culture media. The purified siderophore was found to exist in two forms of approximately 30 and 90 kD. They are believed to be polymers of several siderophore molecules. Both forms were found to be active against the pathogen Erwinia carotovora var. carotovora, the causal bacteria of soft rot disease on potato tubers. The advantage of this method over other purification methods is that it uses metal ion so it can be applied for the purification of the known types of siderophores. Moreover, the purification is based on affinity chromatography, so the siderophore purity state permits several biotechnological applications without further treatments. PMID:18707585

  16. Specific recognition of supercoiled plasmid DNA by affinity chromatography using a synthetic aromatic ligand.

    PubMed

    Caramelo-Nunes, Catarina; Tomaz, Cândida T

    2015-01-01

    Liquid chromatography is the method of choice for the purification of plasmid DNA (pDNA), since it is simple, robust, versatile, and highly reproducible. The most important features of a chromatographic procedure are the use of suitable stationary phases and ligands. As conventional purification protocols are being replaced by more sophisticated and selective procedures, the focus changes toward designing and selecting ligands of high affinity and specificity. In fact, the chemical composition of the chromatographic supports determines the interactions established with the target molecules, allowing their preferential retention over the undesirable ones. Here it is described the selective recognition and purification of supercoiled pDNA by affinity chromatography, using an intercalative molecule (3,8-diamino-6-phenylphenanthridine) as ligand. PMID:25749945

  17. Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

    PubMed

    Roque, Ana C A; Silva, Cláudia S O; Taipa, M Angela

    2007-08-10

    Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

  18. The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

    NASA Astrophysics Data System (ADS)

    Giannone, Richard J.; Liu, Yie; Wang, Yisong

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  19. Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

    PubMed

    Roque, Ana C A; Silva, Cláudia S O; Taipa, M Angela

    2007-08-10

    Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography. PMID:17618635

  20. The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs

    SciTech Connect

    Giannone, Richard J; Liu, Yie; Wang, Yisong

    2009-01-01

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter we describe a comprehensive methodology that utilizes our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we have demonstrated the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  1. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  2. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

    PubMed

    Sainsbury, Frank; Jutras, Philippe V; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

  3. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants

    PubMed Central

    Sainsbury, Frank; Jutras, Philippe V.; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

  4. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  5. Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, María del Mar; Alaiz, Manuel; Girón-Calle, Julio; Millan, Francisco; Vioque, Javier

    2006-09-20

    A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.

  6. Bimolecular affinity purification: a variation of TAP with multiple applications.

    PubMed

    Starokadomskyy, Petro; Burstein, Ezra

    2014-01-01

    The identification of true interacting partners of any given bait can be plagued by the nonspecific purification of irrelevant proteins. To avoid this problem, Tandem Affinity Purification (TAP) is a widely used procedure in molecular biology as this reduces the chance of nonspecific proteins being present in the final preparation. In this approach, two different affinity tags are fused to the protein bait. Herein, we review in detail a variation on the TAP procedure that we have previously developed, where the affinity moieties are placed on two different proteins that form a complex in vivo. This variation, which we refer to as Bimolecular Affinity Purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known proteins. We have utilized BAP for characterization of molecular complexes and evaluation of proteins interaction. Another application of BAP is the isolation of ubiquitin-like proteins (UBL)-modified fractions of a given protein and characterization of the lysine-acceptor site and structure of UBL-chains. PMID:24943324

  7. Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

    PubMed

    Mulcahy, P; Griffin, T; O'Carra, P

    1997-02-01

    The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata). PMID:9116492

  8. A fullerene C60-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

    NASA Astrophysics Data System (ADS)

    Amirshakhi, N.; Alyautdin, R. N.; Orlov, A. P.; Poloznikov, A. A.; Kuznetsov, D. A.

    2008-11-01

    A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.

  9. Multimodal charge-induction chromatography for antibody purification.

    PubMed

    Tong, Hong-Fei; Lin, Dong-Qiang; Chu, Wen-Ning; Zhang, Qi-Lei; Gao, Dong; Wang, Rong-Zhu; Yao, Shan-Jing

    2016-01-15

    Hydrophobic charge-induction chromatography (HCIC) has advantages of high capacity, salt-tolerance and convenient pH-controlled elution. However, the binding specificity might be improved with multimodal molecular interactions. New ligand W-ABI that combining tryptophan and 5-amino-benzimidazole was designed with the concept of mutimodal charge-induction chromatography (MCIC). The indole and benzimidazole groups of the ligand could provide orientated mutimodal binding to target IgG under neutral pH, while the imidazole groups could induce the electrostatic repulsion forces for efficient elution under acidic pH. W-ABI ligand was coupled successfully onto agarose gel, and IgG adsorption behaviors were investigated. High affinity to IgG was found with the saturated adsorption capacity of 70.4 mg/ml at pH 7, and the flow rate of mobile phase showed little impact on the dynamic binding capacity. In addition, efficient elution could be achieved at mild acidic pH with high recovery. Two separation cases (IgG separation from albumin containing feedstock and monoclonal antibody purification from cell culture supernatant) were verified with high purity and recovery. In general, MCIC with the specially-designed ligand is an expanding of HCIC with improved adsorption selectivity, which would be a potential alternative to Protein A-based capture for the cost-effective purification of antibodies.

  10. Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein.

    PubMed

    Jiang, Xuemei; Diraviyam, Thirumalai; Zhang, Xiaoying

    2016-02-15

    Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.

  11. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  12. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

    PubMed

    Coyle, Brandon L; Baneyx, François

    2014-10-01

    We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

  13. Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

    PubMed

    Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

    2014-01-01

    Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

  14. Chromatography purification of canine adenoviral vectors.

    PubMed

    Segura, María Mercedes; Puig, Meritxell; Monfar, Mercè; Chillón, Miguel

    2012-06-01

    Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks. PMID:22799886

  15. Mining the soluble chloroplast proteome by affinity chromatography

    PubMed Central

    Bayer, Roman G; Stael, Simon; Csaszar, Edina; Teige, Markus

    2011-01-01

    Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO2, they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions. PMID:21365755

  16. Frontal affinity chromatography (FAC): theory and basic aspects.

    PubMed

    Kasai, Ken-ichi

    2014-01-01

    Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory. PMID:25117240

  17. Dimerization Capacities of FGF2 Purified with or without Heparin-Affinity Chromatography

    PubMed Central

    Chiu, Liang-Yuan; Taouji, Said; Moroni, Elisabetta; Colombo, Giorgio; Chevet, Eric; Sue, Shih-Che; Bikfalvi, Andreas

    2014-01-01

    Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well. PMID:25299071

  18. Yeast 3',5'-bisphosphate nucleotidase: an affinity tag for protein purification.

    PubMed

    Yang, Yang; Ma, Jianhui; Yang, Yilin; Zhang, Xiao; Wang, Yanxing; Yang, Ling; Sun, Meihao

    2014-05-01

    Affinity chromatography is one of the most popular methods for protein purification. Each tag method has its advantages and disadvantages, and combination of different tags and developing of new tags had been proposed and performed. Yeast 3',5'-bisphosphate nucleotidase, also known as HAL2, hydrolyzes 3'-phosphoadenosine 5'-phosphate (PAP) with submicromolar Km, which indicated the tight interactions between HAL2 and PAP. In order to explore the feasibility of HAL2 as a protein purification affinity tag, HAL2 was further characterized with PAP as substrate. Results demonstrated that KmPAP and kcatPAP were ∼0.3μM and ∼11s(-)(1), respectively. Kd for PAP was 0.008μM in the presence of Ca(2+). pH was also found to affect interactions between HAL2 and PAP, with tightest binding (Kd∼8nM) at pH 7.5 and 8. The purification protocol was rationally designed based on nanomolar affinity to PAP agarose in the presence of Ca(2+), which could satisfy the metal requirement for PAP binding, prevent hydrolysis of immobilized PAP and could be chelated by ethylene glycol tetraacetic acid (EGTA) for elution. A series of expression vectors were further constructed and Escherichia coli adenosine 5'-phosphosulfate kinase (APSK) was prokaryotically expressed, purified and characterized. Ready to use expression vector with eight commonly used restriction enzyme recognition sites in multiple cloning site was subsequently constructed. By comparing with current popular tags, HAL2 was found to be an efficient and economical tag for prokaryotic protein expression and purification. PMID:24613729

  19. Short cut of protein purification by integration of cell-disrupture and affinity extraction.

    PubMed

    Schuster, M; Wasserbauer, E; Ortner, C; Graumann, K; Jungbauer, A; Hammerschmid, F; Werner, G

    2000-01-01

    Screening strategies based on functional genomics require the isolation of gene products of several hundred cDNA clones in a fast and versatile manner. Conventional purification strategies will fail to accomplish this goal within a reasonable time frame. In order to short-cut these procedures, we have developed a combination of cell disintegration and affinity technique for rapid isolation and purification. For our purpose, tagged proteins have been produced in yeast by fusing the FLAG-sequence adjacent to the 5' end of cDNAs coding for the respective protein. The example of an over-expressed FLAG-tagged fusion protein, human serum albumin (HSA), was released into the cytoplasm. Detection and purification of the FLAG-fusion protein were carried out by using a mouse monoclonal antibody directed against the FLAG-peptide. For purification purposes, the antibody was immobilized on PROSEP magnetic glass beads. These magnetic glass beads with 500 microns diameter have been investigated for disintegration of yeast and simultaneous capturing of the target protein. After 60 s, 90% of the maximal disintegration level was achieved when a ratio of 20 microliters yeast cell suspension and 100 microliters glass are vortexed. After a wash step, the FLAG-fusion proteins have been eluted with chelating agents such as EDTA. The short-cut procedure has been compared to a conventional purification strategy using an affinity chromatography process. Due to the highly favorable binding characteristics of the applied immunoaffinity sorbent the yield observed in batch operation was 90% and purity in the range of 70-80%.

  20. Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography

    PubMed Central

    Kanakaraj, Indhu; Jewell, David L.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2011-01-01

    Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and “histidine tags” genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu2+-iminodiacetic acid (IDA) agarose spin column, 94–99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu2+-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs. PMID:21264292

  1. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  2. Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.

    PubMed

    Velkov, Tony; Jones, Alun; Lim, Maria L R

    2008-01-01

    A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor. PMID:18800304

  3. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  4. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  5. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

    PubMed Central

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-01-01

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

  6. Identification of protein interacting partners using tandem affinity purification.

    PubMed

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-01-01

    A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous

  7. Scoring Large Scale Affinity Purification Mass Spectrometry Datasets with MIST

    PubMed Central

    Verschueren, Erik; Von Dollen, John; Cimermancic, Peter; Gulbahce, Natali; Sali, Andrej; Krogan, Nevan

    2015-01-01

    High-throughput Affinity Purification Mass Spectrometry (AP-MS) experiments can identify a large number of protein interactions but only a fraction of these interactions are biologically relevant. Here, we describe a comprehensive computational strategy to process raw AP-MS data, perform quality controls and prioritize biologically relevant bait-prey pairs in a set of replicated AP-MS experiments with Mass spectrometry interaction STatistics (MiST). The MiST score is a linear combination of prey quantity (abundance), abundance invariability across repeated experiments (reproducibility), and prey uniqueness relative to other baits (specificity); We describe how to run the full MiST analysis pipeline in an R environment and discuss a number of configurable options that allow the lay user to convert any large-scale AP-MS data into an interpretable, biologically relevant protein-protein interaction network. PMID:25754993

  8. Partial purification of the 5-hydroxytryptophan-reuptake system from human blood platelets using a citalopram-derived affinity resin

    SciTech Connect

    Biessen, E.A.L; Horn, A.S.; Robillard, G.T. )

    1990-04-03

    This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific ({sup 3}H) imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 {mu}M citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after {sup 125}I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of ({sup 3}H) imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and ({sup 3}H)imipramine binding activity.

  9. SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications.

    PubMed

    Haney, Paul J; Draveling, Connie; Durski, Wendy; Romanowich, Kathryn; Qoronfleh, M Walid

    2003-04-01

    Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry. PMID:12699691

  10. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    PubMed

    Müller, Egbert; Vajda, Judith

    2016-05-15

    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected. PMID:26830536

  11. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  12. Synthesis and characterization of pseudo-affinity ligand for penicillin acylase purification.

    PubMed

    Keçili, Rüstem; Say, Ridvan; Yavuz, Handan

    2006-11-15

    The aim of this work was to test a chromatographic affinity support containing methacryloyl antipyrine (MAAP) for penicillin acylase (PA) purification by using pure penicillin acylase and crude extract. First, MAAP as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Polymer beads (average size diameter: 40-120 micro m) were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAAP. This approach for the preparation of adsorbent has several advantages over conventional preparation protocols. An expensive and time consuming step in the preparation of adsorbent is immobilization of a ligand to the adsorption matrix. In this procedure, affinity ligand MAAP acts as comonomer without further modification steps. Poly(EGDMA-MAAP) beads were characterized by FTIR, NMR and screen analysis. Elemental analysis of MAAP for nitrogen was estimated as 89.3 micro mol/g. The prepared adsorbent was then used for the capture of penicillin acylase in batch system. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) beads was found to be 82.2 mg/g at pH 5.0. Chromatography with crude feedstock resulted in 23.2-fold purification and 93% recovery with 1.0 M NaOH.

  13. Purification of glucagon by subunit exchange chromatography.

    PubMed

    Carrea, G; Pasta, P; Antonini, E

    1985-05-01

    Glucagon was immobilized onto Sepharose matrices activated with CNBr or tresyl chloride, as a function of several parameters including pH of coupling, concentration of added polypeptide, and presence or absence of urea. The hormone was linked to the matrix through a single point per molecule, namely, the epsilon -amino group of Lys(12) when the coupling was carried out at alkaline pH, or the imidazole group of His(1) when the coupling was carried out at acidic pH. Glucagon immobilized at alkaline pH interacted specifically with soluble glucogon. The extent of self-association was similar to that of free glucagon, which exists in solution in a monomer-trimer equilibrium whose association constant is highly dependent on the characteristics of the buffer (pH, ionic strength, and nature of anions). The immobilized hormone proved to be suitable for the purification of the free one from a pancreatic extract. After a preliminary treatment with charcoal-dextran, the extract was percolated on a glucagon-Sepharose column under associating conditions (high concentrations of salting out anions and alkaline pH) and then, following a washing to remove extraneous compounds, the specifically bound hormone was eluted under dissociating conditions (low ionic strength). The subunit exchange chromatography of the extract gave a ca. 90% pure product. The overall recovery of the process was ca. 66%. The leakage of immobilized hormone was 40% in the case of CNBr activation of Sepharose and 15% in the case of tresyl chloride activation, after an eight-day treatment under working conditions.

  14. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  15. Challenges and recent advances in affinity purification of tag-free proteins.

    PubMed

    Guan, Dongli; Chen, Zhilei

    2014-07-01

    There is currently no generic, simple, lowcost method for affinity chromatographic purification of proteins in which the purified product is free of appended tags. Existing approaches for the purification of tagless proteins fall into two broad categories: (1) direct affinity-based capture of tag-free proteins that utilize affinity ligands specific to the target protein or class of target protein, and (2) removal of an appended affinity tag following tag-mediated protein capture. This paper reviews current state-of-the-art approaches for tagless protein purification in both categories, including specific examples of affinity ligands used for the capture of different classes of proteins and cleavage systems for affinity tag removal following chromatographic capture. A particular focus of this review is on recent developments in affinity tag removal systems utilizing split inteins. PMID:24658742

  16. Specific capture of uranyl protein targets by metal affinity chromatography.

    PubMed

    Basset, Christian; Dedieu, Alain; Guérin, Philippe; Quéméneur, Eric; Meyer, Daniel; Vidaud, Claude

    2008-03-28

    To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis. PMID:18308325

  17. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  18. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  19. Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification.

    PubMed

    Wu, Ming-Chang; Lin, Guan-Hui; Wang, Yuh-Tai; Jiang, Chii-Ming; Chang, Hung-Min

    2005-10-01

    Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).

  20. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  1. Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

    PubMed

    Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

    1993-12-01

    A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

  2. Cell-Type-Specific mRNA Purification by Translating Ribosome Affinity Purification (TRAP)

    PubMed Central

    Heiman, Myriam; Kulicke, Ruth; Fenster, Robert J.; Greengard, Paul; Heintz, Nathaniel

    2014-01-01

    Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian central nervous system (CNS) at a molecular level. To address this problem, we recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell-type-specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell-type-specific mRNA profiles of any genetically defined cell type, and has been successfully used to date in organisms ranging from D. melanogaster to mice and human cultured cells. Unlike other methodologies that rely upon micro-dissection, cell panning, or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and potential artifacts these treatments introduce), and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implementing the TRAP methodology, which takes two days to complete once all materials are in hand. PMID:24810037

  3. Affinity chromatography of human leukocyte and diploid cell interferons on sepharose-bound antibodies.

    PubMed

    Berg, K; Ogburn, C A; Paucker, K; Mogensen, K E; Cantell, K

    1975-02-01

    Interferons produced in human peripheral leukocytes (LE) and foreskin fibroblast (FS-4) cells were subjected to affinity chromatography on Sepharose-bound globulins from rabbits immunized with these interferons. Anti-LE interferon sera neutralized both interferons, but titers against FS-4 interferon were consistently lower than those against LE interferon. Anti-FS-4 interferon sera neutralized only FS-4 but not LE interferon. Accordingly, affinity columns constructed with anti-FS-4 globulin excluded LE but not FS-4 interferon, whereas those prepared with anti-LE interferon globulin bound and eluted both LE and FS-4 interferons. Purification of native interferons of both types on anti-LE interferon-Sepharose ranged from 680- to 3,600-fold and recoveries from 72 to 126%. Specific activities of eluate pools varied from 4 to 30 times 10-6 reference (B, 69/19) units per milligram protien.

  4. Magnetic particles as affinity matrix for purification of antithrombin

    NASA Astrophysics Data System (ADS)

    Mercês, A. A. D.; Maciel, J. C.; Carvalho Júnior, L. B.

    2015-11-01

    Immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials. It provides advantages: enhanced stability and easy separation. In this work two different magnetic composites were synthesized (MAG-PANI-HS and mDAC-HS) to human antithrombin purification. The magnetic particles (MAG) were obtained by co-precipitation method of iron salts II and III and subsequently coated with polyaniline (MAG-PANI particles). Dacron (polyethylene terephthalate) suffered a hydrazinolysis reaction to obtain a powder (Dacron hydrazide) which was subsequently magnetized (mDAC particles) also by co-precipitation method. Heparan sulfate (HS) was immobilized to MAG-PANI and mDAC retained respectively 35μg and 38.6μg per of support. The magnetic composite containing HS immobilized (MAG-PANI-HS and mDAC-HS) was incubated with human blood plasma (1mL) and then washed with NaCl gradients. Electrophoresis of proteins present in eluates showed bands of antithrombin (58kDa). A reduction in the antithrombin activity was detected in plasma that were incubated in the composites magnetic with HS immobilized, suggesting that the antithrombin was removed of the human blood plasma and then purified. Therefore, the above results suggest that both preparations: MAG-PANI-HS and mDAC-HS are able to affinity purify antithrombin, an important component of blood coagulation.

  5. Predicting direct protein interactions from affinity purification mass spectrometry data

    PubMed Central

    2010-01-01

    Background Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) in vivo. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest. Results We first propose a simple probabilistic model for the interactions captured by AP-MS experiments, under which the problem of separating direct interactions from indirect ones is formulated. Then, given idealized quantitative AP-MS data, we study the problem of identifying the most likely set of direct interactions that produced the observed data. We address this challenging graph theoretical problem by first characterizing signatures that can identify weakly connected nodes as well as dense regions of the network. The rest of the direct PPI network is then inferred using a genetic algorithm. Our algorithm shows good performance on both simulated and biological networks with very high sensitivity and specificity. Then the algorithm is used to predict direct interactions from a set of AP-MS PPI data from yeast, and its performance is measured against a high-quality interaction dataset. Conclusions As the sensitivity of AP-MS pipeline improves, the fraction of indirect interactions detected will also increase, thereby making the ability to distinguish them even more desirable. Despite the simplicity of our model for indirect interactions, our method provides a good performance on the test networks. PMID:21034440

  6. Dual-tagging system for the affinity purification of mammalian protein complexes

    SciTech Connect

    Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory {Greg} B; Huang, Ying; Wu, Jun; Liu, Yie; Wang, Yisong

    2007-01-01

    Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway{reg_sign}-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10{sup 7} cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.

  7. Use and application of hydrophobic interaction chromatography for protein purification.

    PubMed

    McCue, Justin T

    2014-01-01

    The objective of this section is to provide the reader with guidelines and background on the use and experimental application of Hydrophobic Interaction chromatography (HIC) for the purification of proteins. The section will give step by step instructions on how to use HIC in the laboratory to purify proteins. General guidelines and relevant background information is also provided.

  8. Mixed-bed affinity chromatography: principles and methods.

    PubMed

    Boschetti, Egisto; Righetti, Pier Giorgio

    2015-01-01

    Mixed-bed chromatography is far from being a well-established technology within the panoply of bioseparation tools. Composed of an assembly of distinct sorbents that are mixed in a single bed, they have been mostly developed in the last decade for the reduction of dynamic concentration range where they allowed discovering many low-copy proteins within very complex proteomes. Other interesting preparative applications of mixed-bed chromatography have since been developed. In this chapter the basic concepts first and then detailed application recipes are described for (1) the reduction of protein dynamic concentration range, (2) the removal of impurity traces at the last stage of a biopurification process, and (3) the selection and use of sorbents as mixed bed in protein purification. PMID:25749952

  9. [Progresses in screening active compounds from herbal medicine by affinity chromatography].

    PubMed

    Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

    2015-03-01

    Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening. PMID:26226740

  10. Purification of beta-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1991-08-01

    The purification of rat liver beta-glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300,000 and 150,000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3-7.4. The structural assessment of the carbohydrate chains of lysosomal and microsomal beta-glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction with Lens culinaris agglutinin, Pisum sativum agglutinin and Lotus tetragonolobus lectin revealed that part of the glycans contained a fucose alpha(1-6)-linked to the N-acetylglucosamine attached to asparagine. The presence of terminal beta(1-4)-galactose residues was detected with Ricinus communis agglutinin I. PMID:1841676

  11. Affinity Purification of a Recombinant Protein Expressed as a Fusion with the Maltose-Binding Protein (MBP) Tag

    PubMed Central

    Duong-Ly, Krisna C.; Gabelli, Sandra B.

    2015-01-01

    Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag. The protocol that follows was designed by the authors as a first step in the purification of a recombinant protein fused with MBP, using fast protein liquid chromatography (FPLC). Cells should have been thawed, resuspended in binding buffer, and lysed by sonication or microfluidization before mixing with the amylose resin or loading on the column. Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment. PMID:26096500

  12. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  13. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  14. Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

    PubMed

    Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

    2015-01-01

    Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels. PMID:25749956

  15. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  16. Kinetic analysis of drug-protein interactions by affinity chromatography.

    PubMed

    Bi, Cong; Beeram, Sandya; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-10-01

    Information on the kinetics of drug-protein interactions is of crucial importance in drug discovery and development. Several methods based on affinity chromatography have been developed in recent years to examine the association and dissociation rates of these processes. These techniques include band-broadening measurements, the peak decay method, peak fitting methods, the split-peak method, and free fraction analysis. This review will examine the general principles and applications of these approaches and discuss their use in the characterization, screening and analysis of drug-protein interactions in the body. PMID:26724332

  17. Enrichment of Phosphopeptides via Immobilized Metal Affinity Chromatography.

    PubMed

    Swaney, Danielle L; Villén, Judit

    2016-03-01

    Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved. PMID:26933247

  18. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed. Images Fig. 4. Fig. 7. PMID:597251

  19. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories. PMID:25749949

  20. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

    PubMed Central

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-01-01

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. PMID:26389898

  1. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1.

    PubMed

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-09-16

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3' end of dengue virus (DENV) 5'-3' UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

  2. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    PubMed

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  3. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

    PubMed Central

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  4. Comparison of Inlet Geometry in Microfluidic Cell Affinity Chromatography

    PubMed Central

    Li, Peng; Tian, Yu; Pappas, Dimitri

    2011-01-01

    Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design has not been extensively described or studied. The most common inlets—used to connect the microfluidic chip to pumps, tubing, etc—are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performance of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than the remaining channel, while for parallel inlet chips cell density at the inlet area was similar to the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices. PMID:21207967

  5. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme. PMID:26644295

  6. Separation of recombinant human protein C from transgenic animal milk using immobilized metal affinity chromatography.

    PubMed

    Dalton, J C; Bruley, D F; Kang, K A; Drohan, W N

    1997-01-01

    Protein C is an important serine protease due to its ability to proteolytically cleave activated Factors V and VIII. Excess coagulation and blood agglutination can lead to plugged capillaries, thereby reducing oxygen transport to interstitial tissues. To treat patients with hereditary and acquired protein C deficiency would require a greater amount of Protein C than that available from human plasma. However, the potential demand for this protein could be met by the production of human protein C from transgenic animal mammary glands. Thus, research into inexpensive, efficient methods to purify proteins from transgenic animal milk will be a critical area of study for the large scale production of protein C. Immobilized metal affinity chromatography (IMAC) is a novel method for the purification of protein C. A proposed method of purification is to take advantage of protein C's strong metal ion binding characteristics with IMAC to assist in the separation from transgenic animal milk. The separation procedure is benchmarked against current systems in use by the American Red Cross for purification of Protein C from transgenic porcine milk. Common problems in developing separation schemes for new therapeutics are the initial availability of the product (protein), and time-to-market concerns. Extensive experimental tests for scaleable purification schemes are often cost and time prohibitive. In order to optimize an IMAC protocol with minimal waste of time and resources, total quality management tools have been adopted. Initial experiments were designed to choose buffer conditions, eluents, immobilized valence metals, and flow rates using Taguchi experimental design, which is a total quality management (TQM) tool. One of the values of Taguchi methods lies in the use of Latin orthogonal sets. Through the use of the orthogonal sets, the total number of experiments may be reduced, shortening the focus time on optimal conditions.

  7. Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation

    PubMed Central

    Wang, Jianlong; Cantor, Alan B.; Orkin, Stuart H.

    2009-01-01

    In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification. PMID:19306258

  8. Identification of IgG alloantibodies in patients with high-titer IgM cold agglutinins by serum/plasma affinity chromatography.

    PubMed

    Stahl, D; Kreft, H; Hack, H; Schraven, B; Roelcke, D

    1997-01-01

    The detection of IgG alloantibodies in the presence of high-titer cold autoagglutinins (CAs) can be extremely difficult, especially under pressure of time when transfusion of red blood cells is urgently needed. Here we demonstrate that IgG alloantibodies in the presence of high-titer IgM CAs can be easily detected by quantitative IgG purification from serum or plasma by affinity chromatography. In comparison with the routinely used methods for IgG alloantibody identification, affinity chromatography shows better or identical results and is the method leading to results most rapidly.

  9. Purification of L-( sup 3 H) Nicotine eliminates low affinity binding

    SciTech Connect

    Romm, E.; Marks, M.J.; Collins, A.C. ); Lippiello, P.M. )

    1990-01-01

    Some studies of L-({sup 3}H) nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicated that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-({sup 3}H)nicotine eliminates the low affinity site.

  10. Search for Amyloid-Binding Proteins by Affinity Chromatography

    PubMed Central

    Calero, Miguel; Rostagno, Agueda; Ghiso, Jorge

    2013-01-01

    ‘Amyloid binging proteins’ is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer’s amyloid β (Aβ) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Aβ-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors. PMID:22528093

  11. A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids.

    PubMed

    Maeda, Kenji; Poletto, Mattia; Chiapparino, Antonella; Gavin, Anne-Claude

    2014-09-01

    Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo-assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30-50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography-mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms.

  12. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

  13. Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins.

    PubMed

    Demishtein, Alik; Karpol, Alon; Barak, Yoav; Lamed, Raphael; Bayer, Edward A

    2010-01-01

    Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca(2+)-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K(A)) of 7.7 × 10(8)M(-1), calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca(2+)-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. PMID:21038354

  14. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. PMID:24375581

  15. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

    PubMed Central

    Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

    2011-01-01

    The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure. PMID:21904040

  16. Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.

    PubMed

    Campden, Rhiannon; Pétrin, Darlaine; Robitaille, Mélanie; Audet, Nicolas; Gora, Sarah; Angers, Stéphane; Hébert, Terence E

    2015-01-01

    It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

  17. Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

    PubMed Central

    Lackmann, M; Bucci, T; Mann, R J; Kravets, L A; Viney, E; Smith, F; Moritz, R L; Carter, W; Simpson, R J; Nicola, N A; Mackwell, K; Nice, E C; Wilks, A F; Boyd, A W

    1996-01-01

    Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor. Images Fig. 2 Fig. 3 PMID:8637907

  18. The Purification of Natural and Recombinant Peptide Antibodies by Affinity Chromatographic Strategies.

    PubMed

    Ma, Hui; O'Kennedy, Richard

    2015-01-01

    The purification of peptide antibodies (e.g., IgG, IgY, scFv, and Fab) are described in this chapter. Affinity chromatographic purification, a very convenient and effective antibody purification strategy, is used to isolate peptide antibodies based on specific binding, i.e., binding of the antibody to a column on which its specific ligand is immobilized with subsequent elution of the purified antibody. In addition, the application of purification methods based on the use of proteins A, G, and L, each of which bind to specific domains on an antibody/fragment, or the use of specific tags (e.g., histidine and biotin) attached to antibodies or antigens are also described.

  19. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

  20. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection. PMID:25271333

  1. [Isolation and purification of enhanced green fluorescent protein using chromatography].

    PubMed

    Hou, Qinghua; Song, Shuliang; Liang, Hao; Wang, Weili; Ji, Aiguo

    2013-02-01

    Enhanced green fluorescent protein (EGFP) is a common biological marker. In this research, on the foundation of successful clone and expression of EGFP, a two-step chromatographic method was established to separate and purify EGFP, which includes the use of HisTrap HP immobilized metal affinity chromatography (IMAC) and Sephadex G-10 HR size exclusion chromatography in sequence. Sephacryl S-300 HR size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to check out the purity of EGFP. At last, it was found that EGFP still had fluorescent activity using fluorescence spectrophotometric detection and Native-PAGE detection. This method can effectively separate the active EGFP. The purity of the obtained EGFP was more than 98%.

  2. Automated hydrophobic interaction chromatography column selection for use in protein purification.

    PubMed

    Murphy, Patrick J M; Stone, Orrin J; Anderson, Michelle E

    2011-01-01

    In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein (1). The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH(4;))(2;)SO(4;)). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) (2). As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter (3). Automated column scouting allows for an efficient approach for determining

  3. [Hydrophobic membrane chromatography for fast purification of biological macromolecules].

    PubMed

    Yang, L; Chen, X; Jia, L; Zou, H; Zhang, Y

    1999-07-01

    Cellulose membrane bonded with four commonly used hydrophobic groups, octyl, butyl, phenyl and polyethylene glycol was first investigated for their binding and purification characteristics of protein and enzyme with octyl- and phenyl-Sepharose CL-4 B as controls. Hydrophobic membranes bound BSA effectively by hydrophobic interaction in high salt solution. Their binding capacities were not notably affected by significantly increasing the flow mass rate or decreasing the mass concentration of protein solution, but were much lower than those of octyl- and phenyl-Sepharose CL-4B. 11.8 fold of purification with an approximately 100% recovery of bovine liver catalase was achieved by step gradient elution on the phenyl cellulose membrane cartridge in a single step in only ten mins or a little more. Increase of the flow mass rate had no effect on the purification of catalase, however, the processing time was shortened greatly. Hydrophobic membrane chromatography here reported exibits a potential of fast processing of the protein solution in large volume with low mass concentration of the target protein, such as genetic engineering culture solution. PMID:12552843

  4. Purification of organic acids by chromatography with strong anionic resins: Investigation of uptake mechanisms.

    PubMed

    Lemaire, Julien; Blanc, Claire-Line; Lutin, Florence; Théoleyre, Marc-André; Stambouli, Moncef; Pareau, Dominique

    2016-08-01

    Bio-based organic acids are promising renewable carbon sources for the chemical industry. However energy-consuming purification processes are used, like distillation or crystallization, to reach high purities required in some applications. That is why preparative chromatography was studied as an alternative separation technique. In a previous work dealing with the purification of lactic, succinic and citric acids, the Langmuir model was insufficient to explain the elution profiles obtained with a strong anionic resin. Consequently the Langmuir model was coupled with a usual ion-exchange model to take into account the retention of their conjugate bases (<2%), which are commonly neglected at low pH (<1.5). Elution simulations with both uptake mechanisms fitted very well with experimental pulse tests. Only two parameters were optimized (equilibrium constant of acid uptake and ion-exchange selectivity coefficient of conjugate base) and their value were coherent with experimental and resin suppliers' data. These results confirmed that the singular tailing and apparent delay observed with succinic and citric acids can be explained by the high affinity of succinate and citrate for resin cationic sites. The model was implemented in a preparative chromatography simulation program in order to optimize operating parameters of our pilot-scale ISMB unit (Improved Simulated Moving Bed). The comparison with experimental ISMB profiles was conclusive. PMID:27373374

  5. Purification of organic acids by chromatography with strong anionic resins: Investigation of uptake mechanisms.

    PubMed

    Lemaire, Julien; Blanc, Claire-Line; Lutin, Florence; Théoleyre, Marc-André; Stambouli, Moncef; Pareau, Dominique

    2016-08-01

    Bio-based organic acids are promising renewable carbon sources for the chemical industry. However energy-consuming purification processes are used, like distillation or crystallization, to reach high purities required in some applications. That is why preparative chromatography was studied as an alternative separation technique. In a previous work dealing with the purification of lactic, succinic and citric acids, the Langmuir model was insufficient to explain the elution profiles obtained with a strong anionic resin. Consequently the Langmuir model was coupled with a usual ion-exchange model to take into account the retention of their conjugate bases (<2%), which are commonly neglected at low pH (<1.5). Elution simulations with both uptake mechanisms fitted very well with experimental pulse tests. Only two parameters were optimized (equilibrium constant of acid uptake and ion-exchange selectivity coefficient of conjugate base) and their value were coherent with experimental and resin suppliers' data. These results confirmed that the singular tailing and apparent delay observed with succinic and citric acids can be explained by the high affinity of succinate and citrate for resin cationic sites. The model was implemented in a preparative chromatography simulation program in order to optimize operating parameters of our pilot-scale ISMB unit (Improved Simulated Moving Bed). The comparison with experimental ISMB profiles was conclusive.

  6. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.

  7. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. PMID:25277090

  8. Aptamer stationary phase for protein capture in affinity capillary chromatography.

    PubMed

    Connor, Adam C; McGown, Linda B

    2006-04-14

    The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.

  9. NiCoMnO4: A Bifunctional Affinity Probe for His-Tagged Protein Purification and Phosphorylation Sites Recognition.

    PubMed

    Qi, Xiaoyue; Chen, Long; Zhang, Chaoqun; Xu, Xinyuan; Zhang, Yiding; Bai, Yu; Liu, Huwei

    2016-07-27

    A bifunctional affinity probe NiCoMnO4 was designed and prepared with controllable morphology and size using facile methods. It was observed that the probe could be applied in His-tagged proteins purification and phosphopeptides enrichment simply through the buffer modulation. NiCoMnO4 particles showed satisfactory cycling performance for His-tagged proteins purification and broad pH-tolerance of loading buffer for phosphopeptides affinity. Therefore, a high-throughput, cost-effective, and efficient protein/peptide purification method was developed within 10 min based on the novel bifunctional affinity probe. PMID:27381638

  10. A novel affinity disks for bovine serum albumin purification.

    PubMed

    Tuzmen, Nalan; Kalburcu, Tülden; Uygun, Deniz Aktaş; Akgol, Sinan; Denizli, Adil

    2015-01-01

    The adsorption characteristics of bovine serum albumin (BSA) onto the supermacroporous poly(hydroxyethylmethacrylate)-Reactive Green 19 [p(HEMA)-RG] cryogel disks have been investigated in this paper. p(HEMA) cryogel disks were prepared by radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Reactive Green (RG) 19 was covalently attached to the p(HEMA) cryogel disks. These disks were used in BSA adsorption studies to interrogate the effects of pH, initial protein concentration, ionic strength, and temperature. BSA adsorption capacity of the p(HEMA)-RG cryogel disk was significantly improved after the incorporation of RG. Adsorption capacity reached a plateau value at about 0.8 mg/mL at pH 4.0. The amount of adsorbed BSA decreased from 37.7 to 13.9 mg/g with increasing NaCl concentration. The enthalpy of BSA adsorption onto the p(HEMA)-RG cryogel disk was calculated as -58.4 kJ/mol. The adsorption equilibrium isotherm was fitted well by the Freundlich model. BSA was desorbed from cryogel disks (over 90 %) using 0.5 M NaSCN, and the purity of desorbed BSA was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The experimental results showed that the p(HEMA)-RG cryogel disks have potential for the quick protein separation and purification process. PMID:25308615

  11. Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

    PubMed

    Ball, D J; Nishimura, J S

    1980-11-25

    Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits. PMID:7430155

  12. Selection of ceramic fluorapatite-binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography.

    PubMed

    Islam, Tuhidul; Bibi, Noor Shad; Vennapusa, Rami Reddy; Fernandez-Lahore, Marcelo

    2013-08-01

    Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.

  13. Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography.

    PubMed

    Kölln, Johanna; Braren, Ingke; Bredehorst, Reinhard; Spillner, Edzard

    2007-01-01

    The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart. PMID:17584174

  14. Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography.

    PubMed

    Kölln, Johanna; Braren, Ingke; Bredehorst, Reinhard; Spillner, Edzard

    2007-01-01

    The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.

  15. Cyclic peptide ligand with high binding capacity for affinity purification of immunoglobulin G.

    PubMed

    Kang, Hyo Jin; Choe, Weonu; Min, Jeong-Ki; Lee, Young-Mi; Kim, B Moon; Chung, Sang J

    2016-09-30

    The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has necessitated the development of antibody production and purification technologies for both academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the present study we describe the development of a reusable high affinity column for antibody purification utilizing immobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins, packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Ser one showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab). The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than was required for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swine human sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in the present study.

  16. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

    PubMed Central

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

    Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

  17. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

    PubMed Central

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-01-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  18. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-15

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

  19. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  20. Affinity Purification of O-Acetylserine(thiol)lyase from Chlorella sorokiniana by Recombinant Proteins from Arabidopsis thaliana.

    PubMed

    Salbitani, Giovanna; Wirtz, Markus; Hell, Rüdiger; Carfagna, Simona

    2014-01-01

    In the unicellular green alga Chlorella sorokiniana (211/8 k), the protein O-acetylserine(thiol)lyase (OASTL), representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S) deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h) cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32-34 kDa and are thus slightly larger compared to those found in Arabidopsis thaliana and other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species. PMID:25093930

  1. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

    SciTech Connect

    James, W.M.; Emerick, M.C.; Agnew, W.S. )

    1989-07-11

    The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

  2. Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine.

    PubMed Central

    Smith, G D; Roberts, D V; Daday, A

    1977-01-01

    Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo. PMID:889568

  3. High speed immuno-affinity chromatography on supports with gigapores and porous glass.

    PubMed

    Schuste, M; Wasserbauer, E; Neubauer, A; Jungbauer, A

    2000-01-01

    Immuno-affinity chromatography exploiting the Ca2+ dependent interaction of the anti-Flag antibody and Flag-tagged proteins has been investigated. The antibody has been immobilized on porous glass beads (Prosep) containing gigapores and on a monolith, the polymethacrylate based Convective Interactive Media (CIM) column at a ligand density of 2 mg/g and 10 mg/ml respectively. The performance of the columns was assessed by applying clarified yeast culture supernatant containing overexpressed Flag-human serum albumin. Dynamic binding capacity and purity was checked at various flow rates ranging from 100 cm/h to 800 cm/h. 95% purity could be obtained. Anti Flag-CIM columns showed a higher unspecific adsorption, requiring a longer wash cycle to obtain the same purity compared to the Prosep column. Anti Flag-CIM columns showed a flow independent performance, which is explained by its monolithic structure. A decreasing dynamic binding capacity with flow was observed with anti-Flag-Prosep columns. Both columns are suited to purify milligrams of protein out of a yeast culture supernatant within a few minutes. We considered them as promising candidates for high throughput screening, where fast purification is a necessity.

  4. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  5. Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry.

    PubMed

    Aryal, Uma K; Ross, Andrew R S

    2010-01-01

    Titanium dioxide metal oxide affinity chromatography (TiO(2)-MOAC) is widely regarded as being more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO(2)-MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO(2)-MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO(2)-MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO(2)-MOAC showed higher specificity than immobilized gallium (Ga(3+)), immobilized iron (Fe(3+)), or zirconium dioxide (ZrO(2)) affinity chromatography for phosphopeptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was more efficient for smaller, singly phosphorylated peptides. PMID:20014058

  6. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

    PubMed

    Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Dedecker, Maarten; Verkest, Aurine; Vandepoele, Klaas; Martens, Lennart; Witters, Erwin; Gevaert, Kris; De Jaeger, Geert

    2015-01-01

    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

  7. The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions

    PubMed Central

    Di Tomasso, Geneviève; Lampron, Philipe; Dagenais, Pierre; Omichinski, James G.; Legault, Pascale

    2011-01-01

    Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications. PMID:21071425

  8. The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions.

    PubMed

    Di Tomasso, Geneviève; Lampron, Philipe; Dagenais, Pierre; Omichinski, James G; Legault, Pascale

    2011-02-01

    Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications. PMID:21071425

  9. The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data

    PubMed Central

    Mellacheruvu, Dattatreya; Wright, Zachary; Couzens, Amber L.; Lambert, Jean-Philippe; St-Denis, Nicole; Li, Tuo; Miteva, Yana V.; Hauri, Simon; Sardiu, Mihaela E.; Low, Teck Yew; Halim, Vincentius A.; Bagshaw, Richard D.; Hubner, Nina C.; al-Hakim, Abdallah; Bouchard, Annie; Faubert, Denis; Fermin, Damian; Dunham, Wade H.; Goudreault, Marilyn; Lin, Zhen-Yuan; Badillo, Beatriz Gonzalez; Pawson, Tony; Durocher, Daniel; Coulombe, Benoit; Aebersold, Ruedi; Superti-Furga, Giulio; Colinge, Jacques; Heck, Albert J. R.; Choi, Hyungwon; Gstaiger, Matthias; Mohammed, Shabaz; Cristea, Ileana M.; Bennett, Keiryn L.; Washburn, Mike P.; Raught, Brian; Ewing, Rob M.; Gingras, Anne-Claude; Nesvizhskii, Alexey I.

    2013-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is now a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (e.g. proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. While the standard approach is to identify nonspecific interactions using one or more negative controls, most small-scale AP-MS studies do not capture a complete, accurate background protein set. Fortunately, negative controls are largely bait-independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the Contaminant Repository for Affinity Purification (the CRAPome) and describe the use of this resource to score protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely available online at www.crapome.org. PMID:23921808

  10. Unravelling plant molecular machineries through affinity purification coupled to mass spectrometry.

    PubMed

    Dedecker, Maarten; Van Leene, Jelle; De Jaeger, Geert

    2015-04-01

    Rather than functioning independently, proteins tend to work in concert with each other and with other macromolecules to form macromolecular complexes. Affinity purification coupled to mass spectrometry (AP-MS) can lead to a better understanding of the cellular functions of these complexes. With the development of easy purification protocols and ultra-sensitive MS, AP-MS is currently widely used for screening co-complex membership in plants. Studying complexes in their developmental context through the isolation of specific organs and tissues has now become feasible. Besides, the tagged protein can be employed for probing other interactions like protein-DNA and protein-RNA interactions. With the tools at hand, protein-centred interaction studies will greatly improve our knowledge of how plant cells wire their functional components in relation to their function. PMID:25603557

  11. Frontal affinity chromatography: A unique research tool for biospecific interaction that promotes glycobiology

    PubMed Central

    KASAI, Kenichi

    2014-01-01

    Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

  12. Frontal affinity chromatography: a unique research tool for biospecific interaction that promotes glycobiology.

    PubMed

    Kasai, Kenichi

    2014-01-01

    Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

  13. The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

    PubMed

    Amarasinghe, Chinthaka; Jin, Jian-Ping

    2015-01-01

    Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement. PMID:26216265

  14. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417]. PMID:23108

  15. A mutant trypsin-like enzyme from Streptomyces fradiae, created by site-directed mutagenesis, improves affinity chromatography for protein trypsin inhibitors.

    PubMed

    Katoh, T; Kikuchi, N; Nagata, K; Yoshida, N

    1996-08-01

    The Ser-170 residue of a trypsin-like enzyme from Streptomyces fradiae (SFT), which is considered to be the active-site serine, was replaced with alanine by site-directed mutagenesis to improve the affinity chromatography step for a Kazal-type trypsin inhibitor pancreatic secretory trypsin inhibitor (PSTI). The resulting mutant SFT, designated as [S170A]SFT, was expressed in Streptomyces lividans and purified to homogeneity. [S170A]SFT was catalytically inactive, but still had the ability to bind tightly to PSTI and to soybean trypsin inhibitor with dissociation constants of 3.1 x 10(-7) M and 1.9 x 10(-8) M respectively. We further demonstrated that recombinant human PSTI secreted into Saccharomyces cerevisiae culture broth could be purified to homogeneity with a one-step [S170A]SFT-affinity column. The purified PSTI contained no molecules intramolecularly cleaved by active trypsin, which are found when trypsin-affinity chromatography is used for the purification. This eliminated the need for further separation of intact PSTI from intramolecularly cleaved PSTI by high-performance liquid chromatography, thus simplifying and improving its purification process.

  16. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies.

    PubMed

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  17. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  18. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  19. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, Huamin; Smith, Lloyd M.

    1997-01-01

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

  20. Large-scale monoclonal antibody purification by continuous chromatography, from process design to scale-up.

    PubMed

    Girard, Valérie; Hilbold, Nicolas-Julian; Ng, Candy K S; Pegon, Laurence; Chahim, Wael; Rousset, Fabien; Monchois, Vincent

    2015-11-10

    The development and optimization of a purification process of monoclonal antibodies based on two continuous chromatography steps for capture and intermediate purification are presented. The two chromatography steps were individually optimized using either batch chromatography or sequential multicolumn chromatography (SMCC). Proprietary simulation software was used to optimize SMCC and to evaluate the potential gains compared with batch chromatography. The SMCC recipes provided by the simulation software were evaluated experimentally. A good correlation was found between the simulated results and experimental observations. Significant gains were observed on the productivity, buffer consumption and the volume of resin required for SMCC over batch chromatography. Based on these results, a chained process from the capture to polishing steps was implemented. This chained process demonstrated significantly better performance compared with the batch equivalent while satisfying the specifications. The expected positive impact provided by implementing continuous chromatography is also discussed.

  1. Large-scale monoclonal antibody purification by continuous chromatography, from process design to scale-up.

    PubMed

    Girard, Valérie; Hilbold, Nicolas-Julian; Ng, Candy K S; Pegon, Laurence; Chahim, Wael; Rousset, Fabien; Monchois, Vincent

    2015-11-10

    The development and optimization of a purification process of monoclonal antibodies based on two continuous chromatography steps for capture and intermediate purification are presented. The two chromatography steps were individually optimized using either batch chromatography or sequential multicolumn chromatography (SMCC). Proprietary simulation software was used to optimize SMCC and to evaluate the potential gains compared with batch chromatography. The SMCC recipes provided by the simulation software were evaluated experimentally. A good correlation was found between the simulated results and experimental observations. Significant gains were observed on the productivity, buffer consumption and the volume of resin required for SMCC over batch chromatography. Based on these results, a chained process from the capture to polishing steps was implemented. This chained process demonstrated significantly better performance compared with the batch equivalent while satisfying the specifications. The expected positive impact provided by implementing continuous chromatography is also discussed. PMID:25962790

  2. Cell type-specific affinity purification of nuclei for chromatin profiling in whole animals.

    PubMed

    Steiner, Florian A; Henikoff, Steven

    2015-01-01

    Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

  3. Fermium purification using TEVA{trademark} resin extraction chromatography

    SciTech Connect

    Porter, C.E.; Riley, F.D. Jr.; Vandergrift, R.D.; Felker, L.K.

    1997-01-01

    The Radiochemical Engineering Development Center at Oak Ridge National Laboratory processes irradiated targets to recover the transplutonium actinides for research and industrial users. In a typical processing campaign, dekagram quantities of curium are recovered for recycle into targets for subsequent irradiation and processing; decigram quantities of californium are recovered for fabrication into neutron sources; and milligram quantities of einsteinium and berkelium as well as picogram quantities of fermium are recovered for distribution to the research community. The transcurium actinides are separated in a series of chromatographic elutions using a cation-exchange resin and ammonium {alpha}-hydroxyisobutyrate as the eluant. The fermium fraction from these final purification runs still contains significant amounts of rare earth fission products, such as yttrium, dysprosium, and holmium. In the most recent campaign, a process using a TEVA{trademark} resin extraction chromatography column was developed and tested to determine its effectiveness in providing a fermium product free of rare earth fission products. Gamma spectroscopy indicated that dysprosium and holmium were reduced to levels less than minimum detectable limits and that only 0.07 pg of {sup 91}Y remained in the final fermium product, which contained 0.5 pg of {sup 257}Fm. An overall decontamination factor of {approximately}10{sup 3} was achieved for fission product removal.

  4. A hydrophobic interaction chromatography strategy for purification of inactivated foot-and-mouth disease virus.

    PubMed

    Li, Hao; Yang, Yanli; Zhang, Yan; Zhang, Songping; Zhao, Qizu; Zhu, Yuanyuan; Zou, Xingqi; Yu, Mengran; Ma, Guanghui; Su, Zhiguo

    2015-09-01

    A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold purification, with 92% recovery. The columns were coupled through an intermediate ultrafiltration unit to concentrate the virus. The entire process was completed in about 3.5h, with 75% final FMDV recovery, and 247-fold purification. The final product had purity above 98%, with over 99.5% of host cell DNA removed. High-performance size exclusion chromatography (HPSEC), Western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM) indicated that the purified virus contained the required antigen, and was structurally intact with a spherical shape and a particle size of 28 nm.

  5. Purification of small interfering RNA using nondenaturing anion-exchange chromatography.

    PubMed

    Noll, Bernhard; Seiffert, Stephan; Hertel, Frank; Debelak, Harald; Hadwiger, Philipp; Vornlocher, Hans-Peter; Roehl, Ingo

    2011-12-01

    A manufacturing and purification process for duplex oligonucleotides was established, which shortens and simplifies currently used procedures, yielding a product of higher purity. The reported procedure is based on nondenaturing anion-exchange (AEX) chromatography, which is performed on the annealed duplex rather than the individual single strands. The duplex is formed early in the process by annealing of the crude single strands directly after solid-phase synthesis. Two 30 μmol manufacturing runs using duplex purification were performed on 2 different AEX resins and compared with a manufacturing run of the same scale using conventional single-strand chromatography. The same pooling strategy was employed for all purifications. Content of optimal duplex (duplex exclusively comprising full-length single strands) was 90.5% and 90.2% for the batches obtained by duplex purification and 86.1% for the batch obtained by single-strand purification. Maximum chromatographic recoveries were 67% for the duplex purification and 68% for the single-strand purification. Hence, the manufacture of small interfering RNA (siRNA) using duplex purification was simpler and faster than conventional single-strand purification and provided better purity and similar yield of final siRNA.

  6. A simple and efficient purification platform for monoclonal antibody production based on chromatin-directed cell culture clarification integrated with precipitation and void-exclusion anion exchange chromatography.

    PubMed

    Chen, Quan; Abdul Latiff, Sarah Maria; Toh, Phyllicia; Peng, Xinying; Hoi, Aina; Xian, Mo; Zhang, Haibo; Nian, Rui; Zhang, Wei; Gagnon, Pete

    2016-10-20

    Protein A affinity chromatography, featured by its robustness and high-specificity, is still dominant as a first capture step for the purification of immunoglobulin G monoclonal antibodies (IgG mAbs). However, the material and operational costs of protein A are universally recognized as high, and its productivity is also limited as column mode. In order to overcome these limitations, industry is increasingly considering the use of non-protein A-based processes for IgG purification. In this study, sodium citrate precipitation (SCP) was developed as the primary purification step, and chromatin-directed cell culture clarification was demonstrated to significantly elevate the purification capability. Additional 0.05% (w/v) of Tween 20 was shown to effectively reduce the residual free antibody light chain (LC) during precipitation. The resuspended IgG was further polished by void-exclusion anion exchange chromatography (VEAX), which supported protein loading without buffer adjustment. The non-histone host cell protein (nh-HCP) content in the final product was about 5ppm and histone HCP below limit of detection (LOD). DNA was reduced to less than 1ppb, and aggregates/free LC less than 0.1%. The overall IgG recovery was 87.2%. A simple and efficient purification platform with only one-column step was therefore established, providing a more promising alternative to the current prevailing protein A-based purification platforms. PMID:27568167

  7. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.

    PubMed

    Banks, Charles A S; Kong, Stephanie E; Washburn, Michael P

    2012-12-01

    Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.

  8. A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

    PubMed

    Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

    2014-01-01

    Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

  9. A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

    PubMed Central

    Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

    2014-01-01

    Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

  10. A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

    PubMed

    Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

    2014-01-01

    Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes.

  11. Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

    PubMed

    Yuan, Jie; Zhou, Huifang; Yang, Yicong; Li, Weimin; Wan, Yi; Wang, Lili

    2015-05-01

    Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.

  12. Site-specific DNA-affinity chromatography of the lac repressor.

    PubMed Central

    Herrick, G

    1980-01-01

    To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity. PMID:7001362

  13. Negative Enrichment of Target Cells by Microfluidic Affinity Chromatography

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2011-01-01

    A three-dimensional microfluidic channel was developed for high purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of non-target cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled, and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multi-step processes in microfluidic systems. In previous work, we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity, ease of fabrication and use, is suitable for cell separations when subsequent analysis of target cells is required. PMID:21939198

  14. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    PubMed

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-01

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166

  15. Carnosol purification. Scaling-up centrifugal partition chromatography separations.

    PubMed

    Bouju, Elodie; Berthod, Alain; Faure, Karine

    2016-09-30

    This paper illustrates the application of a recently proposed protocol allowing the scale-up prediction on hydrostatic countercurrent chromatography columns (centrifugal partition chromatographs or CPC). A commercial extract of rosemary (Rosmarinus officinalis L.) was used as the starting material containing 0.48% of carnosol, an active pharmaceutical ingredient with great potential. After a rapid method development on a small-scale 35-mL CPC instrument that allowed for the determination of the solvent system and maximum sample concentration and volume, the purification was transferred on two larger instruments using the "free space between peaks" method. The method takes into account the technical limitations of the larger instruments, such as pressure and/or maximum centrifugal field, and allows, by simply running an analytical-sized injection on the large scale rotor, to give an accurate prediction of the maximum sample load and best throughput. The 0.27g of rosemary extract maximum load on a 35-mL CPC was transferred as a 1.9g load on a 254-mL medium size CPC and 9g load on a 812-mL CPC. The maximum process efficiency of 3.1mg of carnosol per hour obtained on the small 35-mL column was transferred on the 254-mL CPC giving 8.3mg/h and, on the larger 812-mL column 49.4mg of carnosol could be obtained per hour. If the scaling-up in CPC instruments is not directly homothetic, it can be highly predictable through a few simple experiments. PMID:27590084

  16. Purification of selenoprotein P from human plasma using immunoaffinity chromatography

    SciTech Connect

    Aakesson, B.; Bellew, T.; Burk, R.F. )

    1991-03-11

    Selenoprotein P was purified from rat plasma using immunoaffinity chromatography. The same approach was used with human plasma. HepG2 cells were labeled with {sup 75}Se. The labeled medium, containing proteins secreted by the cells, was added to human plasma and the {sup 75}Se was used as a marker for {gt}1,000-fold purification of the major {sup 75}Se-containing protein. This material was used to produce 2 monoclonal antibodies. In a competitive assay, human plasma, but not plasma from 5 other species, inhibited binding of {sup 75}Se by these 2 antibodies. The antibodies were coupled to agarose and columns were made. Human plasma was processed in 2 steps. Step 1 was an antibody column and step 2 was a heparin-agarose column. SDS-PAGE demonstrated bands at 61 and 55 kDa. Both bands stained with PAS. Amino acid analysis of carboxymethylated material indicated that selenocysteine was {gt}1% of the total amino acids. N-terminal sequencing revealed a strong similarity to rat selenoprotein P. Immunodepleted human plasma and control plasma were chromatographed on Sephacryl S200 and selenium was measured in the eluted fractions. Immunodepletion removed one-third of the selenium. The elution pattern of control plasma revealed a broad peak of selenium just ahead of and including the albumin peak. Most of this peak was absent from the immunodepleted serum and a graph of the difference between the 2 chromatograms was a single peak of selenium well separated from the albumin peak.

  17. Carnosol purification. Scaling-up centrifugal partition chromatography separations.

    PubMed

    Bouju, Elodie; Berthod, Alain; Faure, Karine

    2016-09-30

    This paper illustrates the application of a recently proposed protocol allowing the scale-up prediction on hydrostatic countercurrent chromatography columns (centrifugal partition chromatographs or CPC). A commercial extract of rosemary (Rosmarinus officinalis L.) was used as the starting material containing 0.48% of carnosol, an active pharmaceutical ingredient with great potential. After a rapid method development on a small-scale 35-mL CPC instrument that allowed for the determination of the solvent system and maximum sample concentration and volume, the purification was transferred on two larger instruments using the "free space between peaks" method. The method takes into account the technical limitations of the larger instruments, such as pressure and/or maximum centrifugal field, and allows, by simply running an analytical-sized injection on the large scale rotor, to give an accurate prediction of the maximum sample load and best throughput. The 0.27g of rosemary extract maximum load on a 35-mL CPC was transferred as a 1.9g load on a 254-mL medium size CPC and 9g load on a 812-mL CPC. The maximum process efficiency of 3.1mg of carnosol per hour obtained on the small 35-mL column was transferred on the 254-mL CPC giving 8.3mg/h and, on the larger 812-mL column 49.4mg of carnosol could be obtained per hour. If the scaling-up in CPC instruments is not directly homothetic, it can be highly predictable through a few simple experiments.

  18. Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-06-01

    The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with (/sup 125/I)ovine PRL ((/sup 125/I)oPRL). PRL receptors were solubilized from mammary microsomes with 3-((3-cholamidopropyl)dimethylammonio)1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with /sup 125/I using the chloramine-T method. Covalent labeling of PRL receptors with (/sup 125/I)oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of (/sup 125/I)oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five (/sup 125/I)oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit.

  19. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

  20. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario. PMID:25748537

  1. Affinity monolith chromatography: A review of principles and recent analytical applications

    PubMed Central

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  2. A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags.

    PubMed

    Islam, Tuhidul; Aguilar-Yañez, José Manuel; Simental-Martínez, Jesús; Ortiz-Alcaraz, Cesar Ivan; Rito-Palomares, Marco; Fernandez-Lahore, Marcelo

    2014-04-25

    In recent years, affinity fusion-tag systems have become a popular technique for the purification of recombinant proteins from crude extracts. However, several drawbacks including the high expense and low stability of ligands, their leakage during operation, and difficulties in immobilization, make it important to further develop the method. The present work is concerned with the utilization of a ceramic fluorapatite (CFT)-based chromatographic matrix to overcome these drawbacks. A heptapeptide library exhibiting a range of properties have been synthesized and subjected to ceramic fluorapatite (CFT) chromatography to characterize their retention behavior as a function of pH and composition of the binding buffer. The specific binding and elution behavior demonstrates the possible application of CFT-binding peptides as tags for enhancing the selective recovery of proteins by CFT chromatography. To materialize this strategy, a phage-derived CFT-specific sequence KPRSVSG (Tag1) with/without a consecutive hexalysine sequence, KKKKKKKPRSVSG (Tag2), were fused at the C-terminus of an enhanced green fluorescent protein (eGFP). The resulting gene constructs H-eGFP, H-eGFP-Tag1 and H-eGFP-Tag2 were expressed in Escherichia coli strain BL-21, and the clarified cell lysate was applied to the CFT column equilibrated with binding buffer (20-50mM sodium phosphate, pH 6-8.4). Sodium phosphate (500mM) or 1M NaCl in the respective binding buffer was used to elute the fused proteins, and the chromatographic fractions were analyzed by gel electrophoresis. Both the yield and purity were over 90%, demonstrating the potential application of the present strategy.

  3. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media.

  4. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media. PMID:27524303

  5. Purification of rat liver arylsulfatase A and its microheterogeneity assayed by crossed affinity-immunoelectrophoresis.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1992-01-01

    Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A.

  6. Purification of rat liver arylsulfatase A and its microheterogeneity assayed by crossed affinity-immunoelectrophoresis.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1992-01-01

    Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A. PMID:1363453

  7. 3'-Amino thymidine affinity matrix for the purification of herpes simplex virus thymidine kinase.

    PubMed Central

    Tung, P. P.; Respass, J.; Summers, W. C.

    1996-01-01

    A simple procedure for preparation of an affinity resin with 3'-amino thymidine linked to the carboxyl residues on 6-amino-hexanoic agarose is described. We have used this column for a rapid and simple purification of the thymidine kinase encoded by the herpes simplex virus type 1 genome. This resin has two major advantages over the most widely use used resin made with thymidine-p-nitrophenyl phosphate: first it is easily obtainable, and second, it is not subject to destruction by phosphodiesterases. The two resins are very similar in behavior and the resin made with amino thymidine has allowed us to prepare large quantities of highly purified HSV TK for crystallization studies. Images Figure 3 Figure 4 PMID:9436293

  8. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  9. Affinity chromatography using 2' fluoro-substituted RNAs for detection of RNA-protein interactions in RNase-rich or RNase-treated extracts.

    PubMed

    Hovhannisyan, Ruben; Carstens, Russ

    2009-02-01

    Use of RNA affinity chromatography is commonly used to identify RNA binding proteins that interact with specific RNA cis-elements that function in post-transcriptional gene regulation. These purifications can be complicated by residual RNase activity in cellular extracts that can degrade the RNAs on these affinity columns. Furthermore, some proteins may associate indirectly with the column as a component of multi-protein complexes that are "tethered" through the binding of cellular RNAs. We present a protocol for an RNA affinity procedure that can be used in conjunction with RNase-rich or RNase-treated extracts by using RNAs synthesized with 2' fluoro-substituted cytidine triphosphate (CTP) and uridine triphosphate (UTP). The resulting RNAs are shown to be RNase A-resistant and capable of direct coupling to adipic acid dihydrazide agarose beads. Using an RNA cis-element previously shown to bind hnRNP M, we demonstrated that the substituted RNAs preserve binding capability by a common class of RNA binding proteins. Our results provide a method that may be used more generally for RNA affinity purification or as a validation step to verify more direct binding of a given RNA binding protein to a target RNA. PMID:19317654

  10. Glycan-specific whole cell affinity chromatography: a versatile microbial adhesion platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a C-glycoside ketohydrazide affinity chromatography resin that interacts with viable whole-cell microbial populations with biologically appropriate stereo-specificity in a carbohydrate-defined manner. It readily allows for the quantification, selection, and manipulation of target...

  11. Selective retention of basic compounds by metal aquo-ion affinity chromatography.

    PubMed

    Asakawa, Yoshiki; Yamamoto, Eiichi; Asakawa, Naoki

    2014-10-01

    A novel metal aquo-ion affinity chromatography has been developed for the analysis of basic compounds using heat-treated silica gel containing hydrated metal cations (metal aquo-ions) as the packing material. The packing materials of the metal aquo-ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo-ions to present cation-exchange ability for basic analytes and the cation-exchange ability for basic analytes depends on pKa of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo-ion affinity chromatography, the on-line solid-phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo-ion affinity chromatography for basic analytes with sufficient capacity. PMID:25044622

  12. Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry*

    PubMed Central

    Shen, Zhouxin; Kay, Steve A.

    2016-01-01

    Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling

  13. Affinity purification of in vitro transcribed RNA with homogeneous ends using a 3'-ARiBo tag.

    PubMed

    Di Tomasso, Geneviève; Salvail-Lacoste, Alix; Bouvette, Jonathan; Omichinski, James G; Legault, Pascale

    2014-01-01

    Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5'- and 3'-ends. Thus, native affinity purification strategies that incorporate 5' and 3' trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3'-ARiBo tag, which yield RNAs with a homogeneous 3'-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29-614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5'-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5'-CRISPR tag; or (3) self-cleavage of a 5'-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5'-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest. PMID:25432744

  14. Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain.

    PubMed

    Mahmood, Rubab

    2016-01-01

    Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.

  15. The quest for affinity chromatography ligands: are the molecular libraries the right source?

    PubMed

    Perret, Gérald; Santambien, Patrick; Boschetti, Egisto

    2015-08-01

    Affinity chromatography separations of proteins call for highly specific ligands. Antibodies are the most obvious approach; however, except for specific situations, technical and economic reasons are arguments against this choice especially for preparative purposes. With this in mind, the rationale is to select the most appropriate ligands from collections of pre-established molecules. To reach the objective of having a large structural coverage, combinatorial libraries have been proposed. These are classified according to their nature and origin. This review presents and discusses the most common affinity ligand libraries along with the most appropriate screening methods for the identification of the right affinity chromatography selective structure according to the type of library; a side-by-side comparison is also presented. PMID:26033846

  16. A Highly Selective Hsp90 Affinity Chromatography Resin with a Cleavable Linker

    PubMed Central

    Hughes, Philip F; Barrott, Jared J; Carlson, David A; Loiselle, David R; Speer, Brittany L; Bodoor, Khaldon; Rund, Lauretta A; Haystead, Timothy A J

    2012-01-01

    Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media. PMID:22520629

  17. Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

    PubMed Central

    Zlatic, Stephanie A.; Ryder, Pearl V.; Salazar, Gloria; Faundez, Victor

    2010-01-01

    The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1. Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work. PMID:20216526

  18. Metal chelate affinity precipitation: purification of BSA using poly(N-vinylcaprolactam-co-methacrylic acid) copolymers.

    PubMed

    Ling, Yuan-Qing; Nie, Hua-Li; Brandford-White, Christopher; Williams, Gareth R; Zhu, Li-Min

    2012-06-01

    This investigation involves the metal chelate affinity precipitation of bovine serum albumin (BSA) using a copper ion loaded thermo-sensitive copolymer. The copolymer of N-vinylcaprolactam with methacrylic acid PNVCL-co-MAA was synthesized by free radical polymerization in aqueous solution, and Cu(II) ions were attached to provide affinity properties for BSA. A maximum loading of 48.1mg Cu(2+) per gram of polymer was attained. The influence of pH, temperature, BSA and NaCl concentrations on BSA precipitation and of pH, ethylenediaminetetraacetic acid (EDTA) and NaCl concentrations on elution were systematically probed. The optimum conditions for BSA precipitation occurred when pH, temperature and BSA concentration were 6.0, 10°C and 1.0 mg/ml, respectively and the most favorable elution conditions were at pH 4.0, with 0.2M NaCl and 0.06 M EDTA. The maximum amounts of BSA precipitation and elution were 37.5 and 33.7 mg BSA/g polymer, respectively. It proved possible to perform multiple precipitation/elution cycles with a minimal loss of polymer efficacy. The results show that PNVCL-co-MAA is a suitable matrix for the purification of target proteins from unfractionated materials.

  19. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    PubMed

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R

    2016-03-01

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  20. Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

    PubMed

    Zhao, Wei-Wei; Liu, Fu-Feng; Shi, Qing-Hong; Sun, Yan

    2014-09-12

    In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. PMID:25064536

  1. Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases.

    PubMed

    Nakamura, Koji; Suzuki, Takao; Hasegawa, Masazumi; Kato, Yoshio; Sasaki, Hiroo; Inouye, Kuniyo

    2003-08-15

    An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C. PMID:13677653

  2. Improved purification of immunoglobulin G from plasma by mixed-mode chromatography.

    PubMed

    Chai, Dong-Sheng; Sun, Yan; Wang, Xiao-Ning; Shi, Qing-Hong

    2014-12-01

    Efficient loading of immunoglobulin G in mixed-mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed-mode ligand, 4-(1H-imidazol-1-yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15-64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification.

  3. Preparative purification of antibodies with protein A—an alternative to conventional chromatography

    NASA Astrophysics Data System (ADS)

    Holschuh, Karl; Schwämmle, Achim

    2005-05-01

    Protein A coated magnetic particles are used for the preparative purification of antibodies from up to 100 L cell culture supernatant. The comparison with conventional column and expanded bed chromatography results in similar yield and purity of the product but much faster separation times.

  4. The derivatization of oxidized polysaccharides for protein immobilization and affinity chromatography.

    PubMed

    Junowicz, E; Charm, S E

    1976-03-25

    The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5'-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography. PMID:1260016

  5. Identification of Protein Partners in Mycobacteria Using a Single-Step Affinity Purification Method

    PubMed Central

    Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein–protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein–protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  6. Identification of protein partners in mycobacteria using a single-step affinity purification method.

    PubMed

    Płociński, Przemysław; Laubitz, Daniel; Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  7. Affinity chromatography of nicotinamide-adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide.

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems

  8. A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.

    PubMed

    Kaya, Mustafa Oguzhan; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-01-01

    In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. L-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-L-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-L-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) values obtained from the Lineweaver-Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH. PMID:25373501

  9. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

    PubMed Central

    Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

    2009-01-01

    The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

  10. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach. PMID:25935261

  11. Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.

    PubMed

    Kennedy, Jacob J; Yan, Ping; Zhao, Lei; Ivey, Richard G; Voytovich, Uliana J; Moore, Heather D; Lin, Chenwei; Pogosova-Agadjanyan, Era L; Stirewalt, Derek L; Reding, Kerryn W; Whiteaker, Jeffrey R; Paulovich, Amanda G

    2016-02-01

    A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

  12. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose

    PubMed Central

    Jia, Yinshan; Jarrett, Harry W.

    2015-01-01

    The uses of a method of coupling DNA is investigated for trapping and purifying transcription factors. Using the GFP-C/EBP fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry utilized is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA-binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose which couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes including E2A, c-myc, and myo-D were also purified but myogenenin and NFκB were not. Therfore, this approach proved valuable for both affinity chromatography and for the trapping approach. PMID:25935261

  13. IgA-affinity purification and characterization of the lectin jacalin.

    PubMed

    Roque-Barreira, M C; Praz, F; Halbwachs-Mecarelli, L; Greene, L J; Campos-Neto, A

    1986-01-01

    We describe the use of IgA-Sepharose 4B affinity chromatography to purify the lectin jacalin from saline extracts of Artocarpus integrifolia L. seeds. Elution with 0.8 M D-galactose provides 10-15 mg lectin/50 mg seed protein. Jacalin behaved like a single component on immunoelectrophoresis and a single, somewhat diffuse band was obtained by polyacrylamide gel electrophoresis (PAGE) at pH 4.5. A single peak corresponding to an apparent molecular weight of 43 kDa was obtained by gel filtration on Sephadex G-75 (10 mM phosphate buffered saline (PBS), pH 7.4). On SDS-PAGE +/- 2-mercaptoethanol two bands of apparent molecular weights 11.8 and 14.7 kDa were detected. Jacalin behaved like a protein of apparent molecular weight of 13-14 kDa on Sephadex G-50 eluted with PBS containing 0.2% SDS. These data indicate that the jacalin molecule consists of 3-4 non-identical polypeptide subunits not connected by disulfide bridges. The amino acid composition of IgA affinity-purified jacalin (mol/405 mol amino acids) is Lys (24), His (5), Arg (4), Trp (6), Asx (36), Thr (35), Ser (48), Glx (31), Pro (18), Gly (53), Ala (13), Val (25), Met (3), Ile (23), Leu (25), Tyr (30), Phe (26), which corresponds to a molecular weight of 44.163 kDa.

  14. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

  15. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

    PubMed Central

    Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

    2013-01-01

    The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

  16. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  17. Protecting group-free immobilization of glycans for affinity chromatography using glycosylsulfonohydrazide donors.

    PubMed

    Hernandez Armada, Daniel; Santos, Jobette T; Richards, Michele R; Cairo, Christopher W

    2015-11-19

    A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are known, they may provide certain advantages or disadvantages in how the sugar is attached to the affinity matrix. Additionally, the products of some methods may be hard to characterize chemically due to non-specific reactions. The lack of specificity in standard immobilization reactions makes affinity chromatography with expensive oligosaccharides challenging. As a result, methods for specific and efficient immobilization of oligosaccharides remain of interest. Herein, we present a method for the immobilization of saccharides using N'-glycosylsulfonohydrazide (GSH) carbohydrate donors. We have compared GSH immobilization to known strategies, including the use of divinyl sulfone (DVS) and cyanuric chloride (CC), for the generation of affinity matrices. We compared immobilization methods by determining their immobilization efficiency, based on a comparison of the mass of immobilized carbohydrate and the concentration of active binding sites (determined using lectins). Our results indicate that immobilization using GSH donors can provide comparable amounts of carbohydrate epitopes on solid support while consuming almost half of the material required for DVS immobilization. The lectin binding capacity observed for these two methods suggests that GSH immobilization is more efficient. We propose that this method of oligosaccharide immobilization will be an important tool for glycobiologists working with precious glycan samples purified from biological sources. PMID:26454791

  18. Purification process of recombinant monoclonal antibodies with mixed mode chromatography.

    PubMed

    Maria, Sophie; Joucla, Gilles; Garbay, Bertrand; Dieryck, Wilfrid; Lomenech, Anne-Marie; Santarelli, Xavier; Cabanne, Charlotte

    2015-05-01

    An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs.

  19. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.

  20. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification.

    PubMed

    Nie, Minghua; Vashisht, Ajay A; Wohlschlegel, James A; Boddy, Michael N

    2015-09-25

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function.

  1. Optimisation of Downscaled Tandem Affinity Purifications to Identify Core Protein Complexes

    PubMed Central

    Haura, Eric B.; Sacco, Roberto; Li, Jiannong; Müller, André C.; Grebien, Florian; Superti-Furga, Giulio; Bennett, Keiryn L.

    2013-01-01

    In this study we show that via stable, retroviral-expression of tagged EGFR del (L747-S752 deletion mutant) in the PC9 lung cancer cell line and stable doxycycline-inducible expression of tagged Grb2 using a Flp-mediated recombination HEK293 cell system, the SH-TAP can be downscaled to 5 to 12.5 mg total protein input (equivalent to 0.5 - 1 × 15 cm culture plate or 4 - 8 × 106 cells). The major constituents of the EGFR del complex (USB3B, GRB2, ERRFI, HSP7C, GRP78, HSP71) and the Grb2 complex (ARHG5, SOS1, ARG35, CBL, CBLB, PTPRA, SOS2, DYN2, WIPF2, IRS4) were identified. Adjustment of the quantity of digested protein injected into the mass spectrometer reveals that optimisation is required as high quantities of material led to a decrease in protein sequence coverage and the loss of some interacting proteins. This investigation should aid other researchers in performing tandem affinity purifications in general, and in particular, from low quantities of input material. PMID:24077984

  2. Reverse immunoaffinity chromatography: application to the purification of the 60- to 90-kDa gastric parietal cell autoantigen associated with autoimmune gastritis.

    PubMed

    Goldkorn, I; Gleeson, P A; Toh, B H

    1991-05-01

    Previous attempts to purify autoantigens by affinity chromatography using the total IgG from autoantibody-positive sera have not been very successful. We describe here a novel method using parietal cell autoantibodies, isolated on a crude autoantigen immunoabsorbent column, for the purification of the 60- to 90-kDa gastric parietal cell antigen targeted in autoimmune gastritis. The 60- to 90-kDa parietal cell antigen was first enriched 2.5-fold from a 0.5% Triton X-100 extract of total pig gastric mucosal membranes by DEAE-Sepharose 4B chromatography. The fraction containing the 60- to 90-kDa antigen was directly coupled to CNBr-activated Sepharose 4B to produce an immunoadsorbent column for the "reverse" purification of the specific parietal cell autoantibodies from pooled gastric parietal cell autoantibody-positive human sera. A 0.5% Triton X-100 extract of pig gastric mucosal membranes was initially precleared by sequential passage through Protein A-Sepharose 4B and normal human Ig-Sepharose 4B columns. The 60- to 90-kDa gastric parietal cell antigen (approximately 10 micrograms) was then purified from this precleared Triton X-100 membrane extract by affinity chromatography on a 1-ml column of purified autoantibody-Sepharose 4B, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophonesis. This method, incorporating a "reverse" immunoaffinity chromatography step, has general applicability for the purification of tissue autoantigens using highly specific autoantibodies present in the sera of humans and experimental animals with a variety of other autoimmune diseases.

  3. Structural assessment of beta-glucuronidase carbohydrate chains by lectin affinity chromatography.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1993-04-01

    Rat liver beta-glucuronidase was studied by sequential lectin affinity chromatography. beta-Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography. Ulex europaeus agglutinin-agarose chromatography revealed the presence of alpha(1-3)linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin, Ricinus communis agglutinin and Phaseolus vulgaris erythroagglutinin. PMID:8400827

  4. Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography.

    PubMed

    Jin, Xin; Liu, Mingyan; Chen, Zaixing; Mao, Ruikun; Xiao, Qinghuan; Gao, Hua; Wei, Minjie

    2015-10-01

    Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40-50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications.

  5. Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography.

    PubMed

    Jin, Xin; Liu, Mingyan; Chen, Zaixing; Mao, Ruikun; Xiao, Qinghuan; Gao, Hua; Wei, Minjie

    2015-10-01

    Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40-50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications. PMID:26319304

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

    PubMed

    Kery, Vladimir; Savage, Justin R; Widjaja, Kartika; Blake, B Kelly; Conklin, David R; Ho, Yew-Seng J; Long, Xinghua; von Rechenberg, Moritz; Zarembinski, Thomas I; Boniface, J Jay

    2003-06-15

    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.

  7. Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae.

    PubMed

    Trahan, Christian; Aguilar, Lisbeth-Carolina; Oeffinger, Marlene

    2016-01-01

    Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems. In the last decade, affinity purification coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their assemblies. However, the study of these interactomes has been hampered by severe methodological limitations. In particular, the affinity purification of intact complexes from cell lysates suffers from protein and RNA degradation, loss of transient interactors, and poor overall yields. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol described here has been optimized for the yeast S. cerevisiae.

  8. Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

    NASA Astrophysics Data System (ADS)

    Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

    2016-10-01

    Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

  9. The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly.

    PubMed

    Hartmann, B M; Kaar, W; Yoo, I K; Lua, L H L; Falconer, R J; Middelberg, A P J

    2009-12-01

    One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P(11)-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P(11)-2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P(11)-2 from partitioning to the precipitate. This process structure gave recombinant P(11)-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials.

  10. Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps.

    PubMed

    Chandler, D P; Stults, J R; Cebula, S; Schuck, B L; Weaver, D W; Anderson, K K; Egholm, M; Brockman, F J

    2000-08-01

    Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe

  11. Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography

    SciTech Connect

    Jang, H.D.; Swaisgood, H.E. )

    1990-12-01

    Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.

  12. Simultaneous concentration and purification through gradient deformation chromatography

    NASA Technical Reports Server (NTRS)

    Velayudhan, A.; Hendrickson, R. L.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

    1995-01-01

    Mobile-phase additives, commonly used to modulate absorbate retention in gradient elution chromatography, are usually assumed to be either linearly retained or unretained. Previous theoretical work from our laboratory has shown that these modulators, such as salts in ion-exchange and hydrophobic interaction chromatography and organic modifiers in reversed-phase chromatography, can absorb nonlinearly, giving rise to gradient deformation. Consequently, adsorbate peaks that elute in the vicinity of the head of the deformed gradient may exhibit unusual shapes, form shoulders, and/or be concentrated. These effects for a reversed-phase sorbent with aqueous acetonitrile (ACN) as the modulator are verified experimentally. Gradient deformation is demonstrated experimentally and agrees with simulations based on ACN isotherm parameters that are independently determined from batch equilibrium studies using the layer model. Unusual absorbate peak shapes were found experimentally for single-component injections of phenylalanine, similar to those calculated by the simulations. A binary mixture of tryptophan and phenylalanine is used to demonstrate simultaneous concentration and separation, again in agreement with simulations. The possibility of gradient deformation in ion-exchange and hydrophobic interaction chromatography is discussed.

  13. Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

    PubMed

    Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

    2016-09-01

    Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27091327

  14. Comparison of preparative reversed phase liquid chromatography and countercurrent chromatography for the kilogram scale purification of crude spinetoram insecticide.

    PubMed

    DeAmicis, Carl; Edwards, Neil A; Giles, Michael B; Harris, Guy H; Hewitson, Peter; Janaway, Lee; Ignatova, Svetlana

    2011-09-01

    Reversed phase HPLC (RP-HPLC) and high performance countercurrent chromatography (HPCCC) were compared for the pilot scale purification of two semi-synthetic spinosyns, spinetoram-J and spinetoram-L, the major components of the commercial insecticide spinetoram. Two, independently performed, 1 kg, purification campaigns were compared. Each method resulted in the isolation of both components at a purity of >97% and yields for spinetoram-J and spinetoram-L of >93% and ≥ 63% of theoretical, respectively. The HPCCC process produced a 2-fold higher throughput and consumed approximately 70% less solvent than preparative scale RP-HPLC, the volume of product containing fractions from HPCCC amounted to 7% of that produced by HPLC and so required much less post-run processing.

  15. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  16. Centrifugal partition chromatography as a tool for preparative purification of pea albumin with enhanced yields.

    PubMed

    Bérot, Serge; Le Goff, Elisabeth; Foucault, Alain; Quillien, Laurence

    2007-01-15

    A new procedure including the use of centrifugal partition chromatography (CPC) is proposed to purify PA1b and its isoforms. These pea (Pisum sativum L.) seed proteins are toxic against weevils and can be used as an environment-friendly insecticide. CPC was applied to a whole albumin fraction prepared from pea flour. The butanol:aqueous TFA system used in CPC allowed the separation of PA1b from other albumins and a degree of purification above 95%. Compared to analytical procedures based on methanol extraction, anion exchange and then reversed-phase chromatography (RPC), CPC recovered PA1b in much better yield, which is indispensable for large-scale purification of a biodegradable insecticide.

  17. Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

    PubMed Central

    2010-01-01

    Background Poly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions. Results PARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1. Conclusions This study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism. PMID:20388209

  18. Column chromatography as a useful step in purification of diatom pigments.

    PubMed

    Tokarek, Wiktor; Listwan, Stanisław; Pagacz, Joanna; Leśniak, Piotr; Latowski, Dariusz

    2016-01-01

    Fucoxanthin, diadinoxanthin and diatoxanthin are carotenoids found in brown algae and most other heterokonts. These pigments are involved in photosynthetic and photoprotective reactions, and they have many potential health benefits. They can be extracted from diatom Phaeodactylum tricornutum by sonication, extraction with chloroform : methanol and preparative thin layer chromatography. We assessed the utility of an additional column chromatography step in purification of these pigments. This novel addition to the isolation protocol increased the purity of fucoxanthin and allowed for concentration of diadinoxanthin and diatoxanthin before HPLC separation. The enhanced protocol is useful for obtaining high purity pigments for biochemical studies. PMID:27486920

  19. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification

    PubMed Central

    Paradina Fernández, Lesly; Calvo, Loany; Viña, Lisel

    2014-01-01

    Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product. PMID:27379284

  20. Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Edwards, Katarina; Eriksson, Jonny; Ohlson, Sten; Ying, Janet To Yiu; Torres, Jaume; Hernández, Víctor Agmo

    2016-02-01

    Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures. PMID:26673836

  1. Crosslinked glass fiber affinity membrane chromatography and its application to fibronectin separation.

    PubMed

    Guo, Wei; Ruckenstein, Eli

    2003-09-25

    Macroporous glass membranes were prepared from glass fiber filters via chemical crosslinking and modification, and used for the membrane affinity chromatography of fibronectin from human blood plasma. The filters were first treated with a piranha solution (a concentrated solution of H2SO4 + H2O2 in water), and then crosslinked with bifunctional organosilanes and modified to introduce amino or aniline moieties. Ligand immobilizations via diazotization and glutaraldehyde pathways were carried out and compared. Characterization of the membranes was performed using bovine serum albumin and trypsin as test ligands. By using a cartridge containing gelatin immobilized affinity membranes followed by another cartridge containing heparin immobilized membranes, fibronectin from human blood plasma could be separated.

  2. Anacardium occidentale bark lectin: purification, immobilization as an affinity model and influence in the uptake of technetium-99M by rat adipocytes.

    PubMed

    Maciel, Maria Inês Sucupira; de Mendonça Cavalcanti, Maria do Socorro; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; de Almeida Catanho, Maria Teresa Jansem; Coelho, Luana Cassandra Breitenbach Barroso

    2012-10-01

    Lectins, proteins that recognize carbohydrates, have been immobilized on inert supports and used in the screening or purification of glycoproteins. Anacardium occidentale bark infusion has been used as a hypoglycemic agent in Brazil. The toxicity of natural products may be evaluated determining their capability to alter the biodistribution of technetium-99M ((99m)Tc). This work reports the isolation and characterization of a lectin from A. occidentale bark (AnocBL), its evaluation as an affinity support for glycoprotein isolation and lectin effect on the uptake of (99m)Tc by rat adipocytes. AnocBL was isolated from 80 % ammonium sulphate supernatant by affinity chromatography on fetuin-agarose. SDS-PAGE showed a single protein band of 47 kDa. The monossacharide L-arabinose and the glycoproteins fetuin, asialofetuin, ovomucoid, casein, thyroglobulin, peroxidase, fetal bovine serum and IgG inhibited the activity. The lectin activity was stable until 70 °C and at a pH range of 3.0-7.5. AnocBL-Sepharose column bound fetuin indicating that the lectin matrix may be used to obtain glycoconjugates of biotechnological interest. In vitro assay revealed that glucose and insulin increase (99m)Tc uptake by rat adipocytes. AnocBL decreases (99m)Tc uptake, and this effect was not detected in the presence of glucose. Fetuin inhibited AnocBL effect in all insulin concentrations.

  3. Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin.

    PubMed

    Narahari, Akkaladevi; Swamy, Musti J

    2010-04-21

    The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% alpha-helix, 35.8% beta-sheet, 22.5% beta-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4-70 degrees C, but a sharp decrease was seen between 75 and 85 degrees C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 degrees C, indicating that it is a relatively stable protein.

  4. Isolation and partial characterization of Bromelia hemisphaerica protease by affinity chromatography.

    PubMed

    Ochoa, N; Agundis, C; Córdoba, F

    1987-01-01

    Hemisphaericin, the protease from Bromelia hemisphaerica fruit juice was isolated by affinity chromatography in one step, using a mercurial sepharose derivative. The enzyme behaves as a single component in immunodifussion, immunoelectrophoresis and polyacrylamide electrophoresis in the presence of SDS and 2-mercaptoethanol. Association and dissociation of active components were evidenced in electrophoresis at pH 3.6 and at pH 8.6. Immunoelectrophoresis analyses also disclosed a certain degree of internal immunological heterogeneity. The results are explained by the presence of an enzyme subunit, of about 8000 daltons, endowed with polymeric properties induced by the pH and oxidative environment.

  5. Procedure for rapid isolation of photosynthetic reaction centers using cytochrome c affinity chromatography

    SciTech Connect

    Brudvig, G.W.; Worland, S.T.; Sauer, K.

    1983-02-01

    Horse heart cytochrome c linked to Sepharose 4B is used to purify reaction centers from Rhodopseudomonas sphaeroides R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (A/sub 280//A/sub 802/ < 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type Rps. sphaeroides and Rps. capsulata also bind to a cytochrome c column. Cytochrome c affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.

  6. Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography

    SciTech Connect

    Boyer, R.F.; Allen, T.L.; Dykema, P.A.

    1987-02-05

    Eight chemically modified cellulose supports were tested for their ability to adsorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography. 22 references.

  7. Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

    PubMed

    Yatsukawa, Yoh-Ichi; Ito, Hironobu; Matsuda, Takahiro; Nakamura, Munetomo; Watai, Masatoshi; Fujita, Kazuhiro

    2011-01-01

    A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.

  8. Plasmid DNA purification using a multimodal chromatography resin.

    PubMed

    Matos, Tiago; Queiroz, João A; Bülow, Leif

    2014-04-01

    Multimodal chromatography is widely used for isolation of proteins because it often results in improved selectivity compared to conventional separation resins. The binding potential and chromatographic behavior of plasmid DNA have here been examined on a Capto Adhere resin. Capto Adhere is a recent multimodal chromatography material allowing molecular recognition between the ligand and target molecule, which is based on combined ionic and aromatic interactions. Capto Adhere proved to offer a very strong binding of nucleic acids. This property could be used to isolate plasmid DNA from a crude Escherichia coli extract. Using a stepwise NaCl gradient, pure plasmid DNA could be obtained without protein and endotoxin contaminations. The RNA fraction bound most strongly to the resin and could be eluted only at very high salt concentrations (2.0 M NaCl). The chromatographic separation behavior was very robust between pH values 6 and 9, and the dynamic binding capacity was estimated to 60 µg/ml resin.

  9. Preparative two-dimensional liquid chromatography/mass spectrometry for the purification of complex pharmaceutical samples.

    PubMed

    Zhang, Yinong; Zeng, Lu; Pham, Catherine; Xu, Rongda

    2014-01-10

    A new preparative two-dimensional liquid chromatography/mass spectrometry system (2D LC-LC/MS) has been designed and implemented to enhance capability and resolving power for the separation and purification of pharmaceutical samples. The system was constructed by modifications of a conventional preparative LC/MS instrument with the addition of a set of switching valves and a sample loop, as well as interfacing a custom software program with MassLynx. The system integrates two chromatographic separations from the first and second dimensions into a single automated run to perform the purification of a target compound from a complex mixture without intermediate steps of sample preparation. The chromatography in the first dimension, operated in the heart-cutting mode, separates the target compound from the impurities by mass-triggered fractionation based on its molecular weight. This purified fraction from the first dimension is stored in the sample loop, and then gets transferred to the second column by using at-column dilution. A control software program, coined Prep 2D LCMS, was designed to integrate with MassLynx to retrieve data acquisition status. All of the chromatographic hardware components used in this preparative 2D LC-LC/MS system are from the original open access preparative LC/MS system, which has high level of robustness and affords easy and user-friendly operation. The new system is very versatile and capable of collecting multiple fractions with different masses under various purification modes as configured in the methods, such as conventional one-dimensional (1D) purification and/or 2D purification. This new preparative 2D LC-LC/MS system is therefore the ideal tool for medicinal chemistry lab in drug discovery environment. PMID:24309715

  10. Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies.

    PubMed

    Chollangi, Srinivas; Parker, Ray; Singh, Nripen; Li, Yi; Borys, Michael; Li, Zhengjian

    2015-11-01

    To be administered to patients, therapeutic monoclonal antibodies must have very high purity, with process related impurities like host-cell proteins (HCPs) and DNA reduced to <100 ppm and <10 ppb, respectively, relative to desired product. Traditionally, Protein-A chromatography as a capture step has been the work horse for clearing a large proportion of these impurities. However, remaining levels of process and product related impurities still present significant challenges on the development of polishing steps further downstream. In this study, we have incorporated high throughput screening to evaluate three areas of separation: (i) Harvest treatment; (ii) Protein-A Chromatography; and (iii) Low pH Viral Inactivation. Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein-protein interaction resulted in further and more pronounced reduction in impurity levels. In addition, our study also demonstrate that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities. When applied over multiple mAbs, this optimization method proved to be very robust and the strategy provides a new and improved purification process that reduces process related impurities like HCPs and DNA to drug substance specifications with just one chromatography column and open avenues for significant decrease in operating costs in monoclonal antibody purification.

  11. [PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma.

    PubMed

    Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay; Elkak, Assem; Denizli, Adil

    2016-04-01

    The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PMID:26838913

  12. Characterization of Murine Brain Membrane Glycoproteins by Detergent Assisted Lectin Affinity Chromatography (DALAC)

    PubMed Central

    Wei, Xin; Dulberger, Charles; Li, Lingjun

    2010-01-01

    Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization has been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that under certain concentrations, detergents can minimize the nonspecific bindings and facilitate the elution of hydrophobic glycoproteins. With the Detergent Assisted Lectin Affinity Chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. 699 proteins were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome. PMID:20700909

  13. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high-performance affinity chromatography.

    PubMed

    Zhang, Jiwen; Li, Haiyan; Sun, Lixin; Wang, Caifen

    2015-01-01

    The kinetics of the association and dissociation are fundamental kinetic processes for the host-guest interactions (such as the drug-target and drug-excipient interactions) and the in vivo performance of supramolecules. With advantages of rapid speed, high precision and ease of automation, the high-performance affinity chromatography (HPAC) is one of the best techniques to measure the interaction kinetics of weak to moderate affinities, such as the typical host-guest interactions of drug and cyclodextrins by using a cyclodextrin-immobilized column. The measurement involves the equilibration of the cyclodextrin column, the upload and elution of the samples (non-retained substances and retained solutes) at different flow rates on the cyclodextrin and control column, and data analysis. It has been indicated that cyclodextrin-immobilized chromatography is a cost-efficient high-throughput tool for the measurement of (small molecule) drug-cyclodextrin interactions as well as the dissociation of other supramolecules with relatively weak, fast, and extensive interactions. PMID:25749964

  14. Affinity chromatography of branched oligosaccharides in rat liver beta-glucuronidase.

    PubMed

    Hoja-Lukowicz, D; Lityńska, A; Wójczyk, B S

    2001-05-01

    Rat liver microsomal and lysosomal beta-glucuronidase-derived glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. These glycopeptides were studied by sequential chromatography on lectin-affinity columns such as concanavalin A, lentil lectin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin I, Triticum vulgaris agglutinin, Glycine max agglutinin and Ulex europaeus agglutinin. Using serial lectin affinity chromatography approach combined with neuraminidase treatment allowed us to show the unexpected presence of complex tri- and/or tetraantennary type glycans (40.8 and 17.0% for microsomal and lysosomal enzyme, respectively). Moreover, the application of neuraminidase treatment revealed that complex biantennary type glycans, present on lysosomal beta-glucuronidase, are almost fully sialylated while the same type of glycans present on microsomal enzyme do not contain sialic acid. Furthermore, the results obtained confirmed that microsomal and lysosomal beta-glucuronidases possess high mannose and/or hybrid type glycans (19.6 and 36.6%, respectively), and complex biantennary type glycans (38.9 and 46.4%, respectively). PMID:11393703

  15. Purification of tracer for somatomedin C radioimmunoassay by hydrophobic interaction chromatography

    SciTech Connect

    Baxter, R.C.; Brown, A.S.

    1982-03-01

    Researcherse have purified tracer for use in the somatomedin C radioimmunoassay by hydrophobic interaction chromatography. Material showing greatest immunoreactivity binds to Octyl Sepharose CL-4B (Pharmacia) in a buffer mixture consisting of 130 mL of acetonitrile and 870 mL of 0.1 mol/L NH4HCO3, pH 7.8, but is eluted by increasing the acetonitrile content to 180 mL/L. As compared with tracer purified by binding to specific antiserum in liquid phase, precipitating the complex with second antibody, and then dissociating by gel chromatography at acid pH, this tracer shows equal immunoreactivity against specific somatomedin C antiserum. Either preparation allows excellent discrimination between extracts of normal, acromegalic, and hypopituitary plasma samples; thus either is suitable for use in the somatomedin C radioimmunoassay. Tracer purification by hydrophobic interaction chromatography is rapid and inexpensive. It may be useful in preparing highly immunoreactive tracers for other peptide radioimmunoassays.

  16. Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags

    PubMed Central

    Salvail-Lacoste, Alix; Di Tomasso, Geneviève; Piette, Benjamin L.; Legault, Pascale

    2013-01-01

    Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3′ homogeneity. Here, we explored strategies to also ensure 5′ homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5′ heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II ϕ2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5′-sequence heterogeneity in several cases, significant levels of 5′ heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5′ homogeneity, we tested the suitability of using a small CRISPR RNA stem–loop at the 5′ end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5′-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR–SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5′-CRISPR-based strategy can be combined with affinity purification using the 3′-ARiBo tag for quick purification of RNA with both 5′ and 3′ homogeneity. PMID:23657939

  17. Synthesis and purification of iodoaziridines involving quantitative selection of the optimal stationary phase for chromatography.

    PubMed

    Boultwood, Tom; Affron, Dominic P; Bull, James A

    2014-05-16

    The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 °C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 °C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control. Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by (1)H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by (1)H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to

  18. Recombinant expression and affinity purification of snake venom gland parvalbumin in Escherichia coli.

    PubMed

    Jia, Ying; Pérez, John C

    2009-07-01

    Parvalbumins (PV) are small, acidic, water soluble and calcium-binding proteins generally present in muscular and nervous tissues. In the present study, we identified and characterized a cDNA clone encoding PV, named AplPV, from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. AplPV belongs to EF-hand proteins with six alpha-helices constituting three EF-hand domains. The deduced amino acid sequence of AplPV is 91% and 68% identical to the previously characterized PVs of Boa constrictor and Cyprinus carpio, respectively. The full-length cDNA was subcloned into the expression vector pGEX and transformed into Escherichia coli (E.coli) to produce recombinant protein. The bacterially expressed GST-AplPV fusion protein was highly expressed, and effectively purified by Glutathione-Sepharose affinity chromatography. A high concentration of thrombin protease specifically cleaved and removed the GST tag from fusion protein, and further purified by Benzamidine column for removal of thrombin protease. As a result, the 12 kDa AplPV recombinant protein alone was purified. To investigate the tissue-specific biological occurrence of AplPV, a polyclonal antibody (anti-AplPV-antibody) was raised against GST-AplPV fusion protein in rabbit. Western blot analysis revealed that immunoreactive bands were exhibited in both recombinant protein and samples of venom glands, but not in any crude venom. This specific occurrence indicates a specialized function of AplPV in snake venom glands.

  19. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  20. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  1. ANALYSIS OF DRUG INTERACTIONS WITH HIGH DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Sike; Sobansky, Matthew R.; Hage, David S.

    2009-01-01

    Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography. This approach was evaluated by using it to examine the binding of high density lipoprotein (HDL) to the drugs propranolol or verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPLC columns with reproducible binding to propranolol over four to five days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interactions were occurring between R/S-propranolol and HDL at 37°C: saturable binding with an association equilibrium constant (Ka) of 1.1–1.9 × 105 M−1, and non-saturable binding with an overall affinity constant (n Ka) of 3.7–4.1 × 104 M−1. Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a Ka of 6.0 × 104 M−1 at 37°C for the saturable sites and a n Ka value for the non-saturable sites of 2.5 × 104 M−1. These measured affinities gave good agreement with solution-phase values. The results indicated HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body. PMID:19833090

  2. Weak affinity chromatography as a new approach for fragment screening in drug discovery.

    PubMed

    Duong-Thi, Minh-Dao; Meiby, Elinor; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2011-07-01

    Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM-μM in dissociation constant (K(D))) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM-10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC-MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening. PMID:21352794

  3. Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

    PubMed

    Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

    2012-11-01

    Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential. PMID:22918538

  4. Isolation and purification of monosialotetrahexosylgangliosides from pig brain by extraction and liquid chromatography.

    PubMed

    Bian, Liujiao; Yang, Jianting; Sun, Yu

    2015-10-01

    Monosialotetrahexosylganglioside (GM1), one of glycosphingolipids containing sialic acid, plays particularly important role in fighting against paralysis, dementia and other diseases caused by brain and nerve damage. In this work, a simple and highly efficient method with high yield was developed for isolation and purification of GM1 from pig brain. The method consisted of an extraction by chloroform-methanol-water and a two-step chromatographic separation by DEAE-Sepharose Fast Flow anion-exchange medium and Sephacryl S-100 HR size-exclusion medium. The purified GM1 was proved to be homogeneous and had a purity of >98.0% by high-performance anion-exchange and size-exclusion chromatography. The molecular weight was 30.0 kDa by high-performance size-exclusion chromatography and 1546.9 Da by electrospray ionization mass spectrometry. The chromogenic reaction by resorcinol-hydrochloric acid solution indicated that the purified GM1 showed a specific chromogenic reaction of sialic acid. Through this isolation and purification program, ~1.0 mg of pure GM1 could be captured from 500 g wet pig brain tissue and the yield of GM1 was around 0.022%, which was higher than the yields by other methods. The method may provide an alternative for isolation and purification of GM1 in other biological tissues.

  5. A simple one pot purification of bacterial amylase from fermented broth based on affinity toward starch-functionalized magnetic nanoparticle.

    PubMed

    Paul, Tanima; Chatterjee, Saptarshi; Bandyopadhyay, Arghya; Chattopadhyay, Dwiptirtha; Basu, Semanti; Sarkar, Keka

    2015-08-18

    Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet-visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.

  6. Reduced surface area chromatography for flow-through purification of viruses and virus like particles.

    PubMed

    Iyer, Ganesh; Ramaswamy, Senthilkumar; Asher, Damon; Mehta, Ushma; Leahy, Anne; Chung, Franklin; Cheng, Kwok-Shun

    2011-07-01

    A method for flow-through purification of viruses and virus like nano-particles using a combination of binding and size-exclusion chromatography was developed. This technique relies on minimizing the external surface area per unit volume available for virus binding by increasing the mean diameter of the beads used in the column. At the same time the impurity binding capacity of the column is maximized by utilizing beads with multiple functionalities of the optimum size. Purification of different types of viruses and virus-like-particles could be achieved using this technique. Flow-through purification of influenza virus using this technique yielded virus recoveries greater than 70-80% coupled with impurity removal greater than 80%. Finally an approach to optimize and facilitate process development using this technology is presented. Since the impurity binding occurs via a non-specific mechanism and virus recovery is achieved through reduced surface area, the technique is not limited to specific types of viruses and offers the potential as a universal purification tool.

  7. Single-step purification of chitosanases from Bacillus cereus using expanded bed chromatography.

    PubMed

    de Araújo, Nathália Kelly; Pagnoncelli, Maria Giovana Binder; Pimentel, Vanessa Carvalho; Xavier, Maria Luiza Oliveira; Padilha, Carlos Eduardo Araújo; de Macedo, Gorete Ribeiro; Dos Santos, Everaldo Silvino

    2016-01-01

    A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01. The purification and characterization of two chitosanases were studied. The purification assay was accomplished by ion exchange expanded-bed chromatography. Experiments were carried out in the presence and in the absence of cells through different expansion degree to evaluate the process performance. The adsorption experiments demonstrated that the biomass does not affect substantially the adsorption capacity of the matrix. The enzyme bound to the resin with the same extent using clarified and unclarified broth (0.32 and 0.30 U/g adsorbent, respectively). The fraction recovered exhibited 31% of the yield with a 1.26-fold increase on the specific activity concerned to the initial broth. Two chitosanases from different elution steps were recovery. Chit A and Chit B were stable at 30-60°C, pH 5.5-8.0 and 5.5-7.5, respectively. The highest activity was found at 55°C, pH 5.5 to Chit A and 50°C, pH 6.5 to Chit B. The ions Cu(2+), Fe(2+) and Zn(2+) indicated inhibitory effect on chitosanases activities that were significantly activated by Mn(2+). The methodology applied in this study enables the partial purification of a stable chitosanase using a feedstock without any pre-treatment using a single-step purification.

  8. Selective isolation of β-glucan from corn pericarp hemicelluloses by affinity chromatography on cellulose column.

    PubMed

    Yoshida, Tomoki; Honda, Yoichi; Tsujimoto, Takashi; Uyama, Hiroshi; Azuma, Jun-ichi

    2014-10-13

    A combination of anion-exchange chromatography and affinity chromatography on a cellulose column was found to be effective for the isolation of β-(1,3;1,4)-glucan (BG) from corn pericarp hemicelluloses (CPHs). CPHs containing 6.6% BG were extracted from corn pericarp with 6M urea-2 wt% NaOH solution and initially fractionated into neutral and acidic parts by anion exchange chromatography to remove acidic arabinoxylan consisting of arabinose (35.6%) and xylose (50.9%). The neutral fraction (yield; 10.1% on the basis of CPHs) consisting of 1.0% arabinose, 10.1% xylose and 80.3% glucose containing 28.4% BG was then applied to a cellulose column of Whatman CF-11. BG could be recovered from the adsorbed fraction on the cellulose column by elution with 2% NaOH in a yield of 2.6% on the basis of CPHs with a purity of 84.7%. The chemical structure of the isolated corn pericarp BG was confirmed by (13)C NMR spectroscopic, methylation and lichenase treatment analyses. The results indicate that the ratios of (1,4)/(1,3) linkage and cellotriosyl/cellotetraosyl segments of the BG were 2.60 and 2.5, respectively.

  9. Challenges and opportunities in the purification of recombinant tagged proteins.

    PubMed

    Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

    2014-01-01

    The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. PMID:24334194

  10. Affinity chromatography of alpha/sub 2/-adrenergic receptors (. cap alpha. /sub 2/AR) from pig cerebral cortex

    SciTech Connect

    Repaske, M.G.; Limbird, L.E.

    1986-03-01

    A high capacity, ..cap alpha../sub 2/AR-selective affinity resin (YOH. ag) has been prepared by coupling yohimbinic acid to diaminodipropylamine agarose with 1,3 dicyclohexylcarbodiimide. Unreacted amino groups on the agarose matrix are blocked subsequently by acetylation. One volume of YOH. ag adsorbs 75% of the ..cap alpha../sub 2/AR from 50 volumes of digitonin-solubilized preparation containing 0.2 pmol ..cap alpha../sub 2/AR/mg protein. Digitonin-solubilized preparations are derived from cholate extracts of porcine cerebral cortex containing approx. 0.075 pmol ..cap alpha../sub 2/AR/mg protein. Adsorption of ..cap alpha../sub 2/AR to YOH. ag is selective and thus is blocked by the ..cap alpha..-adrenergic antagonist phentolamine. Adsorbed ..cap alpha../sub 2/AR are eluted with 10 ..mu..M phentolamine (20% yield) after removal of non-related proteins with NaCl gradients. Following hydroxylapatite chromatography to concentrate ..cap alpha..''AR and to remove phentolamine, the ..cap alpha..AR is present at 200-400 pmol/mg protein, assayed using sub-saturating concentrations of (/sup 3/H)-yohimbine. (It is estimated that the specific activity of a homogeneous ..cap alpha../sub 2/AR preparation would be 12,000-16,000 pmol/mg protein.) The availability of large quantities of cortical ..cap alpha../sub 2/AR and a resin easily prepared from commercially-supplied reagents suggests that purification of quantities of ..cap alpha../sub 2/AR sufficient for subsequent biochemical studies is feasible.

  11. Oriented covalent immobilization of antibodies onto heterofunctional agarose supports: a highly efficient immuno-affinity chromatography platform.

    PubMed

    Batalla, Pilar; Bolívar, Juan M; Lopez-Gallego, Fernando; Guisan, Jose M

    2012-11-01

    The development of new bioconjugates formed by one antibody optimally bound (through its Fc region) to fairly inert solid surfaces is of primary relevance in immuno-affinity chromatography. Immunoglobulins G (IgG) have a Fc region very rich in histidine (His) residues. In this way, immobilization of IgGs on heterofunctional metal chelate-glyoxyl supports (Ag-Me(2+)/G) takes place in two steps: firstly the antibodies are conjugated to the support via His-metal coordination bonds. Secondly, their incubation under alkaline condition promotes an intramolecular covalent attachment between lysine residues at the Fc region and glyoxyl groups on the support surface. The IgG that recognizes as antigen the HRP (antiHRP-IgG) has been conjugated to Ag-Me(2+)/G supports. The resulting bioconjugate is highly inert and able to specifically bind the antigen (HRP) without significant unspecific binding of any other proteins, resulting in an excellent HRP purification platform. The binding activity of this bioconjugate has been optimized by controlling the antibody distribution throughout the bead's surface in order to avoid high antibody densities that led to a low binding activity of the antibodies. The optimal antibody distribution has been achieved when these proteins were slowly immobilized on Ag-Cu(2+)/G in presence of imidazole. This bioconjugate was able to bind up to 1.5 moles of antigen per mole of antibody, only 1.3-fold less than the antibody in solution. Hence, we have been able to develop an optimal protocol to prepare bioconjugated composites in an oriented and irreversible fashion which results in highly efficient and specific surfaces for the exclusive biological recognition. PMID:23021645

  12. Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography

    PubMed Central

    Moldt, Brian; Saye-Francisco, Karen; Schultz, Niccole; Burton, Dennis R.; Hessell, Ann J.

    2013-01-01

    The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiply roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1M Citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6 mg of SIgA2 b12 was achieved from the combination of 20 mg of purified dIgA2 b12 and 2 L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a

  13. Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

    PubMed

    Tang, Weizhuo; Sun, Baoshan; Zhao, Yuqing

    2014-02-01

    In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management.

  14. Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

    PubMed

    Tang, Weizhuo; Sun, Baoshan; Zhao, Yuqing

    2014-02-01

    In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management. PMID:24381020

  15. Separation and purification of five alkaloids from Aconitum duclouxii by counter-current chromatography.

    PubMed

    Wang, Yarong; Cai, Shining; Chen, Yang; Deng, Liang; Zhou, Xumei; Liu, Jia; Xu, Xin; Xia, Qiang; Lin, Mao; Zhang, Jili; Huang, Weili; Wang, Wenjun; Xiang, Canhui; Cui, Guozhen; Du, Lianfeng; He, Huan; Qi, Baohui

    2015-07-01

    C19 -diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter-current chromatography. In the process of separation, the critical conditions of counter-current chromatography were optimized. The two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1, 24.1 mg of 2, 250.6 mg of 3, 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra-high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation.

  16. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction.

    PubMed

    Karpol, Alon; Kantorovich, Lia; Demishtein, Alik; Barak, Yoav; Morag, Ely; Lamed, Raphael; Bayer, Edward A

    2009-01-01

    Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. PMID:18979459

  17. Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers.

    PubMed

    Croucher, David R; Iconomou, Mary; Hastings, Jordan F; Kennedy, Sean P; Han, Jeremy Z R; Shearer, Robert F; McKenna, Jessie; Wan, Adrian; Lau, Joseph; Aparicio, Samuel; Saunders, Darren N

    2016-01-01

    The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells. PMID:27405979

  18. Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method.

    PubMed

    Fan, Yunpeng; Fu, Yanhui; Fu, Qing; Cai, Jianfeng; Xin, Huaxia; Dai, Mei; Jin, Yu

    2016-07-01

    An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.

  19. Salt-independent hydrophobic displacement chromatography for antibody purification using cyclodextrin as supermolecular displacer.

    PubMed

    Ren, Jun; Yao, Peng; Chen, Jingjing; Jia, Lingyun

    2014-11-21

    Hydrophobic interaction chromatography (HIC) offers an orthogonal selectivity to ion exchange chromatography and the combination of the two processes can provide a potential cost-effective alternative to protein A chromatography in industrial antibody purification. However, the application of HIC is limited by its close dependence on high concentrations of kosmotropic salts to achieve desired separation. These salts can cause antibody precipitation and induce the corrosion of manufacturing facilities. Here, we report a new strategy of salt-independent HIC, which can capture antibody at the physiological salt concentration and allow the recovery of bound proteins through cyclodextrin (CD)-based displacement elution. Hydrophobicity-intensified HIC media with different coupling amount of phenyl ligands were prepared and assessed for their antibody binding capacity and selectivity. β-CD was investigated for its supermolecular interaction with phenyl ligands and elution capacity as a displacer. The results clarified a nearly linear correlation between binding capacity of human immunoglobulin G (IgG) and phenyl coupling density in the range of 44-159 μmol/mL. The host-guest interaction between β-CD and the phenyl ligands revealed a modest binding strength (Ka=4.1×10(3) M(-1)), and 15 mM β-CD solution showed a general effectiveness as displacement eluent for these HIC media, with IgG recovery varying with the ligand density. This strategy allowed the direct purification of human IgG from serum with satisfactory purity. The whole procedure of this method, including loading and elution, can be performed under physiological conditions. We expect such a salt-independent mode of HIC could be used as a capture or intermediate step in industrial antibody purification.

  20. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  1. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.

    PubMed

    Babu, Mohan; Kagan, Olga; Guo, Hongbo; Greenblatt, Jack; Emili, Andrew

    2012-01-01

    Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria(1-6). In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes(1, 2, 7). Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast(8, 9), and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system(10). Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large

  2. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    SciTech Connect

    Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J.

    2012-02-07

    Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

  3. Purification of dinosterol for hydrogen isotopic analysis using high-performance liquid chromatography-mass spectrometry.

    PubMed

    Smittenberg, Rienk H; Sachs, Julian P

    2007-10-26

    A semi-preparative normal-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS) method is presented for the purification of various alcohol fractions from total lipid extracts derived from sediments, for the purpose of hydrogen isotopic measurement by gas chromatography-isotope ratio mass spectrometry (GC-IRMS). 4-methylsterols, including the dinoflagellate-specific marker dinosterol (4,23,24-trimethylcholestan-22-en-3beta-ol), were successfully separated from notoriously co-eluting plant-derived pentacyclic triterpenoid alcohols and alkyl alcohols. We find that substantial hydrogen isotope fractionation occurs during chromatographic separation, demonstrating the importance of recovering the entire peak when subsequent hydrogen isotope analyses are to be performed. This is the first report of such hydrogen isotopic fractionation for a natural unlabelled compound.

  4. High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

    PubMed

    Li, Zhao; Beeram, Sandya R; Bi, Cong; Suresh, D; Zheng, Xiwei; Hage, David S

    2016-01-01

    The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples. PMID:26827600

  5. ANALYSIS OF DRUG INTERACTIONS WITH VERY LOW DENSITY LIPOPROTEIN BY HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2014-01-01

    High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein's non-polar core. This partitioning was described by overall affinity constants of 1.2 (± 0.3) × 106 M-1 for R-propranolol and 2.4 (± 0.6) × 106 M-1 for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (± 2.3) × 104 M-1 for R-propranolol and 9.6 (± 2.2) × 104 M-1 for S-propranolol. Comparable results were obtained at 20 °C and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes. PMID:25103529

  6. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  7. DETECTION OF HETEROGENEOUS DRUG-PROTEIN BINDING BY FRONTAL ANALYSIS AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Tong, Zenghan; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding. PMID:21612784

  8. Glycan-specific whole cell affinity chromatography: A versatile microbial adhesion platform

    PubMed Central

    Van Tassell, Maxwell L.; Price, Neil P.J.; Miller, Michael J.

    2014-01-01

    We have sought a universal platform for elucidating and exploiting specificity of glycan-mediated adhesion by potentially uncharacterized microorganisms. Several techniques exist to explore microbial interactions with carbohydrate structures. Many are unsuitable for investigating specific mechanisms or uncharacterized organisms, requiring pure cultures, labeling techniques, expensive equipment, or other limitations such as questionable stability, stereospecificity, or scalability. We have adapted an affinity chromatography resin as a model to overcome these drawbacks, among others. It readily allows for the quantification, selection, and manipulation of target organisms based on interactions with glycan ligands. To maximize its utility as a selective screening method, we have constructed the tool such that it:•Promotes whole-cell interactions using viable, unaltered cells.•Provides robust spatial interactions with target glycans, presented with controlled stereo-specificity, for high affinity/avidity interactions that reflect a complex in vivo matrix.•Has the ability to utilize any reducing glycan, is quick, efficient, safe, and affordable to construct, and is scalable and reusable for multiple applications. PMID:26150959

  9. CHARACTERIZATION OF THE BINDING OF SULFONYLUREA DRUGS TO HSA BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions. PMID:20435530

  10. Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part I: Polymer permeation-immobilized metal ion affinity chromatography separation adsorbents with polyethylene glycol and immobilized metal ions.

    PubMed

    González-Ortega, Omar; Porath, Jerker; Guzmán, Roberto

    2012-03-01

    Despite the many efforts to develop efficient protein purification techniques, the isolation of peptides and small proteins on a larger than analytical scale remains a significant challenge. Recovery of small biomolecules from diluted complex biological mixtures, such as human serum, employing porous adsorbents is a difficult task mainly due to the presence of concentrated large biomolecules that can add undesired effects in the system such as blocking of adsorbent pores, impairing diffusion of small molecules, or competition for adsorption sites. Adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide matrix, have been developed and explored in this work to overcome such effects and to preferentially adsorb small molecules while rejecting large ones. In the first part of this work, adsorption studies were performed with small peptides and proteins from synthetic mixtures using controlled access polymer permeation adsorption (CAPPA) media created by effectively grafting PEG on an immobilized metal affinity chromatography (IMAC) agarose resin, where chelating agents and immobilized metal ions were used as the primary affinity binding sites. Synthetic mixtures consisted of bovine serum albumin (BSA) with small proteins, peptides, amino acids (such as histidine or Val⁴-Angiotensin III), and small molecules-spiked human serum. The synthesized hybrid adsorbent consisted of agarose beads modified with iminodiacetic (IDA) groups, loaded with immobilized Cu(II) ions, and PEG. These CAPPA media with grafted PEG on the interior and exterior surfaces of the agarose matrix were effective in rejecting high molecular weight proteins. Different PEG grafting densities and PEG of different molecular weight were tested to determine their effect in rejecting and controlling adsorbent permeation properties. Low grafting density of high molecular weight PEG was found to be as

  11. Recovery and purification of a Kluyvermyces lactis β-galactosidase by Mixed Mode Chromatography.

    PubMed

    Lima, Micael de Andrade; de Freitas, Maria de Fátima Matos; Gonçalves, Luciana Rocha Barros; da Silva Junior, Ivanildo José

    2016-03-15

    Mixed Mode Chromatography (MMC) is a potential separation technique that allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. The aim of this work was to assess the recovery and purification of a Kluyveromyces lactis β-galactosidase employing MMC. Protein precipitation and dialysis were performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium. The best conditions for both adsorption and desorption were attained by a non-factorial Central Composite Experimental Design and employed in the chromatographic runs with resin CAPTO MMC. Fermentation yielded mean values of total enzyme concentration of 0.44 mg/mL, enzymatic activity (employing lactose as a substrate) of 74 U/mL and specific activity of 168 U/mg. The Purification Factor (PF) obtained was of 1.17. After precipitation and dialysis, the subsequent chromatographic run resulted in recovery values ​​of 41.0 and 48.2% of total protein concentration and enzymatic activity, respectively. SDS-PAGE electrophoresis confirmed the purification evolution throughout the unit operations employed, attesting the feasibility of the technique to obtain enzymes with not only considerable degree of purity but also possessing high-added value.

  12. Efficient purification of antiproliferative polysaccharides from Hypsizigus marmoreus with radial flow chromatography.

    PubMed

    Yan, Pei-Sheng; Cao, Li-Xin; Zhang, Bing-Zhao

    2014-01-01

    The increasing commercial significance of natural polysaccharides for use in medicinal products is stimulating the development of efficient and easy scale-up techniques for polysaccharide purification. In this research, the crude polysaccharides from submerged cultivation broth of Hypsizigus marmoreus were purified using radial flow chromatography (RFC), and the antiproliferative activity of the purified fractions was evaluated in vitro. DEAE Sepharose CL-6B was selected to be packed in the RFC column based on its good resolution, physical stability, and low cost. Compared with axial flow chromatography (AFC), an efficient chromatographic process with significantly less time and buffer consumption but yielding higher polysaccharide recovery and resolution was established in RFC, which could clearly purify the crude polysaccharides into different fractions. An acceptable linear scale-up effect of RFC from 100 to 500 mL was successfully achieved without loss of resolution and enhancement of time consumption. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in cell cultures indicated that the purified polysaccharide fractions possess moderate antiproliferative activities in three different human cancer cell lines, but have significantly lower cytotoxicity in normal human cell lines in vitro. Among the polysaccharide fractions, the main purified acidic fraction W-I could be considered as a novel potential antitumor agent candidate for several tumors, especially for human alveolar epithelial tumors. This research confirmed for the first time that RFC would be a new fast and efficient tool for purification of polysaccharides into different fractions, both at laboratory and commercial scales.

  13. Alternative separation steps for monoclonal antibody purification: combination of centrifugal partitioning chromatography and precipitation.

    PubMed

    Oelmeier, Stefan A; Ladd-Effio, Christopher; Hubbuch, Jürgen

    2013-12-01

    Protein drugs continue to grow both in medicinal importance as in scale of their production. This furthers the interest in separation technologies that have the potential to replace chromatographic steps in a protein purification process. Two such unit operations that are employed in large scale in the chemical industry are extraction and precipitation. Their usefulness for the purification of proteins has been demonstrated, but the integration of such unit operations in a way that generate an output stream of high protein concentration and low process related impurities was missing. In this work, we employ centrifugal partitioning chromatography ('CPC') in combination with precipitation of the protein of interest to purify a cell culture supernatant of a monoclonal antibody producing cell line. Centrifugal partitioning chromatography was used as means of multi-step extraction using aqueous two-phase systems and was able to remove up to 88.2% of host cell protein ('HCP'). The following PEG driven precipitation and resolubilization of the protein of interest was use to condition the CPC output stream to suit subsequent chromatographic steps, to increase mAb concentration, remove the phase forming polymer, further improve HCP clearance, and integrate a low pH hold step for viral clearance. The entire process reduced HCP content by 99.4% while recovering 93% of the protein of interest. High throughput screening techniques were extensively employed during the development of the process.

  14. Purification of Active Myrosinase from Plants by Aqueous Two-Phase Counter-Current Chromatography

    PubMed Central

    Wade, Kristina L.; Ito, Yoichiro; Ramarathnam, Aarthi; Holtzclaw, W. David; Fahey, Jed W.

    2014-01-01

    Introduction Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (frombroccoli) and 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (β-thioglucoside N-hydroxysulphate) precursors. Objective To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. Methods A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). Results Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. Conclusion Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable. PMID:25130502

  15. Alternative separation steps for monoclonal antibody purification: combination of centrifugal partitioning chromatography and precipitation.

    PubMed

    Oelmeier, Stefan A; Ladd-Effio, Christopher; Hubbuch, Jürgen

    2013-12-01

    Protein drugs continue to grow both in medicinal importance as in scale of their production. This furthers the interest in separation technologies that have the potential to replace chromatographic steps in a protein purification process. Two such unit operations that are employed in large scale in the chemical industry are extraction and precipitation. Their usefulness for the purification of proteins has been demonstrated, but the integration of such unit operations in a way that generate an output stream of high protein concentration and low process related impurities was missing. In this work, we employ centrifugal partitioning chromatography ('CPC') in combination with precipitation of the protein of interest to purify a cell culture supernatant of a monoclonal antibody producing cell line. Centrifugal partitioning chromatography was used as means of multi-step extraction using aqueous two-phase systems and was able to remove up to 88.2% of host cell protein ('HCP'). The following PEG driven precipitation and resolubilization of the protein of interest was use to condition the CPC output stream to suit subsequent chromatographic steps, to increase mAb concentration, remove the phase forming polymer, further improve HCP clearance, and integrate a low pH hold step for viral clearance. The entire process reduced HCP content by 99.4% while recovering 93% of the protein of interest. High throughput screening techniques were extensively employed during the development of the process. PMID:24182866

  16. Characterization of Extracellular Proteins in Tomato Fruit using Lectin Affinity Chromatography and LC-MALDI-MS/MS analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The large-scale isolation and analysis of glycoproteins by lectin affinity chromatography coupled with mass spectrometry has become a powerful tool to monitor changes in the “glycoproteome” of mammalian cells. Thus far, however, this approach has not been used extensively for the analysis of plant g...

  17. Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

    PubMed

    Chen, Bin; Li, Rong; Li, Shiyu; Chen, Xiaoli; Yang, Kaidi; Chen, Guoliang; Ma, Xiaoxun

    2016-02-01

    The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors. PMID:26632098

  18. Affinity purification and characterisation of zinc chelating peptides from rapeseed protein hydrolysates: possible contribution of characteristic amino acid residues.

    PubMed

    Xie, Ningning; Huang, Jingjing; Li, Bo; Cheng, Jianghua; Wang, Zhuochen; Yin, Junfeng; Yan, Xiaoming

    2015-04-15

    Zinc is an essential trace element for human growth and development. In this work, zinc-chelating peptides from rapeseed protein hydrolysates produced with alcalase were investigated by affinity chromatography with immobilized zinc and Sephadex G-25 gel filtration. Four small peptides, namely, Ala-Arg, Asn-Ser-Met (NSM), Gly-Lys-Arg, and Glu-Pro-Ser-His, were obtained and identified by reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The zinc-chelating ability of the four peptides was further validated by inductively coupled plasma atomic emission spectrometry (ICP-AES). NSM was found to exhibit the highest zinc-chelating rate, which was better than that of reduced glutathione. We speculated that the Asn residue at the amino-terminus might facilitate this zinc-chelating ability. Therefore, utilizing small peptides from rapeseed protein as novel carriers for zinc supplement was feasible.

  19. Isolation and purification of immunoglobulin G from bovine colostrums by hydrophobic charge-induction chromatography.

    PubMed

    Wu, Mianbin; Zhang, Feifei; Liang, Yafei; Wang, Rutao; Chen, Zhengjie; Lin, Jianping; Yang, Lirong

    2015-05-01

    Hydrophobic charge-induction chromatography, a novel chromatographic technique for bioseparation, was developed to isolate and purify bovine IgG with high purity. In this work, the raw IgG solution, a precipitate from bovine colostrum powder solution with 40% (wt/vol) ammonium sulfate, was dissolved in 50mM phosphate buffer and used as loading solution for investigating chromatographic conditions on a mercapto-ethyl-pyridine (MEP) HyperCel (Pall Corp., Port Washington, NY) sorbent. The initial IgG concentration had no effect on the dynamic binding capacity of MEP HyperCel resin, but the linear velocity of loading solution had an obvious effect on the dynamic IgG binding capacity and IgG recovery. The maximum linear velocity was optimized as 0.4cm/min of loading solution, and 90% recovery of IgG was achieved. Under these optimized binding conditions, the pH and ionic strength for the elution process were selected as pH 4.5 and 0.5 M NaCl, respectively. Subsequently, hydrophobic charge-induction chromatography was performed on a MEP HyperCel sorbent to isolate IgG using bovine colostrum whey as the loading solution. Under the optimized operation conditions, a remarkable process improvement in IgG purification was received, which includes a yield of 91.5%, a purity of 93.9% (wt/wt), and a purification factor of 6.8. The results indicated that MEP HyperCel chromatography offers an efficient means to purify IgG from bovine colostrums.

  20. Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins.

    PubMed

    Ito, Y; Yamasaki, Y; Seno, N; Matsumoto, I

    1986-04-01

    Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin. PMID:3711062

  1. A new method of quantitative affinity chromatography and its application to the study of myosin.

    PubMed Central

    Bottomley, R C; Storer, A C; Trayer, I P

    1976-01-01

    A new method of quantifying the interactions between two or three components of an interacting system, one of which is insoluble, is described. The method differs from those previously applied to affinity chromatography systems in that it does not require that elution volumes be measured, but is instead dependent on measurements of the quantity of affinity-bound material. Theoretical expressions are derived for systems in which the acceptor is immobilized. Examples presented to illustrate the validity of the theory are of the latter type and are from studies on the myosin-adenosine nucleotide-PPi system. With Sepharose-myosin columns (myosin covalently coupled to CNBr-activated Sepharose) a dissociation constant of 1.8 muM for ATP4- was found. Data were also obtained under conditions that closely approximate to those found in vivo, i.e. on columns packed with a slurry of Sephadex G-50 and precipitated myosin filaments formed at low ionic strength. The binding of MgATP2-, MgADP-, ATP4- and MgPPi2- to "filamentous" myosin in both two- (myosin and nucleotide) and three- (myosin, nucleotide and PPi) component systems at different temperatures was studied and the dissociation constants obtained agreed well with previously published values. Except for the binding of ATP4- to filamentous myosin at 4 degrees when 85% of the protein was interacting with the nucleotide, much lower values for the number of available sites occupied by the nucleotides were as a routine found in this system. Although this apparent discrepancy is difficult to explain, it is not an anomaly of the theoretical approach and may reflect the present state of understanding of the myosin system. PMID:1008824

  2. Comparing multistep immobilized metal affinity chromatography and multistep TiO2 methods for phosphopeptide enrichment.

    PubMed

    Yue, Xiaoshan; Schunter, Alissa; Hummon, Amanda B

    2015-09-01

    Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multistep enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multiphosphopeptides as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multistep enrichment. PMID:26237447

  3. BIOINTERACTION ANALYSIS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: KINETIC STUDIES OF IMMOBILIZED ANTIBODIES

    PubMed Central

    Nelson, Mary Anne; Moser, Annette; Hage, David S.

    2009-01-01

    A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7–12 × 106 M−1 at pH 7.0 and 25°C. Split-peak analysis gave association rate constants of 1.4–12 × 105 M−1s−1 and calculated dissociation rate constants of 0.01–0.4 s−1 under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056–0.17 s−1. A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4 × 10−4 s−1. This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports. PMID:19394281

  4. Monosize poly(glycidyl methacrylate) beads for dye-affinity purification of lysozyme.

    PubMed

    Altintaş, Evrim Banu; Denizli, Adil

    2006-03-30

    Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for purification of lysozyme from chicken egg white. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by a dispersion polymerization technique. The content of epoxy groups on the surface of the poly(GMA) sample determined by the HCl-pyridine method (3.8 mmol/g). Cibacron Blue F3GA loading was 1.73 mmol/g. The monosize beads were characterized by elemental analysis, FTIR and SEM. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature and ionic strength). Maximum lysozyme adsorption amount of poly(GMA) and poly(GMA)-Cibacron Blue F3GA beads were 1.6 and 591.7 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, poly(GMA)-Cibacron Blue F3GA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg-white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the eluted lysozyme was analyzed by SDS-PAGE and found to be 88% with recovery about 79%. The specific activity of the eluted lysozyme was high as 43,600 U/mg.

  5. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  6. Affinity purifications of aldose reductase and xylitol dehydrogenase from the xylose-fermenting yeast Pachysolen tannophilus

    SciTech Connect

    Bolen, P.L.; Roth, K.A.; Freer, S.N.

    1986-10-01

    Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.

  7. Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry.

    PubMed

    Ding, Xi-Qin; Matveev, Alexander; Singh, Anil; Komori, Naoka; Matsumoto, Hiroyuki

    2014-01-01

    Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations.

  8. One-pot preparation of a sulfamethoxazole functionalized affinity monolithic column for selective isolation and purification of trypsin.

    PubMed

    Xiao, Yuan; Guo, Jialiang; Ran, Danni; Duan, Qianqian; Crommen, Jacques; Jiang, Zhengjin

    2015-06-26

    A facile and efficient "one-pot" copolymerization strategy was used for the preparation of sulfonamide drug (SA) functionalized monolithic columns. Two novel SA-immobilized methacrylate monolithic columns, i.e. poly(GMA-SMX-co-EDMA) and poly(GMA-SAA-co-EDMA) were prepared by one-pot in situ copolymerization of the drug ligand (sulfamethoxazole (SMX) or sulfanilamide (SAA)), the monomer (glycidyl methacrylate, GMA) and the cross-linker (ethylene dimethacrylate, EDMA) within 100 μm i.d. capillaries under optimized polymerization conditions. The physicochemical properties and column performance of the fabricated monolithic columns were characterized by elemental analysis, scanning electron microscopy and micro-HPLC. Satisfactory column permeability, efficiency and separation performance were obtained on the optimized poly(GMA-SMX-co-EDMA) monolithic column for small molecules, such as a standard test mixture and eight aromatic ketones. Notably, it was found that the poly(GMA-SMX-co-EDMA) monolith showed a selective affinity to trypsin, while the poly(GMA-SAA-co-EDMA) monolith containing sulfanilamide did not exhibit such affinity at all. This research not only provides a novel monolith for the selective isolation and purification of trypsin, but it also offers the possibility to easily prepare novel drug functionalized methacrylate monoliths through a one-pot copolymerization strategy.

  9. Spotlite: web application and augmented algorithms for predicting co-complexed proteins from affinity purification--mass spectrometry data.

    PubMed

    Goldfarb, Dennis; Hast, Bridgid E; Wang, Wei; Major, Michael B

    2014-12-01

    Protein-protein interactions defined by affinity purification and mass spectrometry (APMS) suffer from high false discovery rates. Consequently, lists of potential interactions must be pruned of contaminants before network construction and interpretation, historically an expensive, time-intensive, and error-prone task. In recent years, numerous computational methods were developed to identify genuine interactions from the hundreds of candidates. Here, comparative analysis of three popular algorithms, HGSCore, CompPASS, and SAINT, revealed complementarity in their classification accuracies, which is supported by their divergent scoring strategies. We improved each algorithm by an average area under a receiver operating characteristics curve increase of 16% by integrating a variety of indirect data known to correlate with established protein-protein interactions, including mRNA coexpression, gene ontologies, domain-domain binding affinities, and homologous protein interactions. Each APMS scoring approach was incorporated into a separate logistic regression model along with the indirect features; the resulting three classifiers demonstrate improved performance on five diverse APMS data sets. To facilitate APMS data scoring within the scientific community, we created Spotlite, a user-friendly and fast web application. Within Spotlite, data can be scored with the augmented classifiers, annotated, and visualized ( http://cancer.unc.edu/majorlab/software.php ). The utility of the Spotlite platform to reveal physical, functional, and disease-relevant characteristics within APMS data is established through a focused analysis of the KEAP1 E3 ubiquitin ligase.

  10. Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA.

    PubMed

    Stark, B C; Chooi, W Y

    1985-02-20

    The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species. PMID:3923010

  11. Analysis of the Glycoproteome of Toxoplasma gondii using Lectin Affinity Chromatography and Tandem Mass Spectrometry

    PubMed Central

    Luo, Qilie; Upadhya, Rajendra; Zhang, Hong; Madrid-Aliste, Carlos; Nieves, Edward; Kim, Kami; Angeletti, Ruth Hogue; Weiss, Louis M.

    2011-01-01

    Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1 to 5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This is data provides a large scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates. PMID:21920448

  12. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography

    PubMed Central

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S.

    2015-01-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  13. MEASUREMENT OF DRUG-PROTEIN DISSOCIATION RATES BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Schiel, John E.; Ohnmacht, Corey M.; Hage, David S.

    2012-01-01

    The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus non-retained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (± 0.2) s−1 was obtained by this approach at pH 7.4 and 37°C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates. PMID:19422253

  14. Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

    PubMed Central

    Liang, Aiye; Thakkar, Jay N.; Hindle, Michael; Desai, Umesh R.

    2013-01-01

    Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I35, I55 and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I35 possessed good anticoagulation potential (APTT = 4.2 μM; PT = 6.8 μM), while I55 and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4 m/z, which most probably arise from variably functionalized (β-O4—β-β-linked trimers and/or a β-O4—β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants. PMID:23122400

  15. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  16. Analysis of Lidocaine Interactions with Serum Proteins Using High-Performance Affinity Chromatography

    PubMed Central

    Soman, Sony; Yoo, Michelle J.; Jang, Yoon Jeong; Hage, David S.

    2010-01-01

    High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α1–acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (Ka) of 1.1-1.7 × 105 M-1 at 37 °C and pH 7.4. Lidocaine had weak-to-moderate binding to HSA, with a Ka in the range of 103 to 104 M-1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes. PMID:20138813

  17. Characterization of glycoproteins in pancreatic cyst fluid using a high performance multiple lectin affinity chromatography platform

    PubMed Central

    Gbormittah, Francisca Owusu; Haab, Brian B.; Partyka, Katie; Garcia-Ott, Carolina; Hancapie, Marina; Hancock, William S.

    2014-01-01

    Currently, pancreatic cancer is the fourth cause of cancer death. In 2013, it is estimated that approximately 38,460 people will die of pancreatic cancer. Early detection of malignant cyst (pancreatic cancer precursor) is necessary to help prevent late diagnosis of the tumor. In this study, we characterized glycoproteins and non-glycoproteins on pooled mucinous (n=10) and non-mucinous (n=10) pancreatic cyst fluid to identify ‘proteins of interest’ to differentiate between mucinous cyst from non-mucinous cyst and investigate these proteins as potential biomarker targets. An automated multi-lectin affinity chromatography (M-LAC) platform was utilized for glycoprotein enrichment followed by nano-LC-MS/MS analysis. Spectral count quantitation allowed for the identification of proteins with significant differential levels in mucinous cysts from non-mucinous cysts of which one protein (periostin) was confirmed via immunoblotting. To exhaustively evaluate differentially expressed proteins, we used a number of proteomic tools including; gene ontology classification, pathway and network analysis, Novoseek data mining and chromosome gene mapping. Utilization of complementary proteomic tools, revealed that several of the proteins such as mucin 6 (MUC6), bile salt-activated lipase (CEL) and pyruvate kinase lysozyme M1/M2 with significant differential expression have strong association with pancreatic cancer. Further, chromosome gene mapping demonstrated co-expressions and co-localization of some proteins of interest including 14-3-3 protein epsilon (YWHAE), pigment epithelium derived factor (SERPINF1) and oncogene p53. PMID:24303806

  18. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  19. Analysis of Drug Interactions with Lipoproteins by High-Performance Affinity Chromatography

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2013-01-01

    Lipoproteins such as high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are known to interact with drugs and other solutes in blood. These interactions have been examined in the past by methods such as equilibrium dialysis and capillary electrophoresis. This chapter describes an alternative approach that has recently been developed for examining these interactions by using high-performance affinity chromatography. In this method, lipoproteins are covalently immobilized to a solid support and used within a column as a stationary phase for binding studies. This approach allows the same lipoprotein preparation to be used for a large number of binding studies, leading to precise estimates of binding parameters. This chapter will discuss how this technique can be applied to the identification of interaction models and be used to differentiate between systems that have interactions based on partitioning, adsorption or mixed-mode interactions. It is also shown how this approach can then be used for the measurement of binding parameters for HDL and LDL with drugs. Examples of these studies are provided, with particular attention being given to the use of frontal analysis to examine the interactions of R- and S-propranolol with HDL and LDL. The advantages and possible limitations of this method are described. The extension of this approach to other types of drug-lipoprotein interactions is also considered. PMID:25392741

  20. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S

    2014-08-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  1. The Lectin Frontier Database (LfDB), and data generation based on frontal affinity chromatography.

    PubMed

    Hirabayashi, Jun; Tateno, Hiroaki; Shikanai, Toshihide; Aoki-Kinoshita, Kiyoko F; Narimatsu, Hisashi

    2015-01-01

    Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms-from humans to microorganisms, including viruses-and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB)", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's). For Kd determination, an advanced system of frontal affinity chromatography (FAC) is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67) of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience. PMID:25580689

  2. Surface plasmon resonance spectroscopy-based high-throughput screening of ligands for use in affinity and displacement chromatography.

    PubMed

    Vutukuru, Srinavya; Kane, Ravi S

    2008-10-21

    We describe an approach that uses surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) for the high-throughput screening of ligands for use in displacement and affinity chromatographic processes. We identified a set of commercially available organic amines and allowed them to react with SAMs presenting interchain carboxylic anhydride groups; the resulting surfaces presented ligands of interest in a background of carboxylic acid groups. We used SPR spectroscopy to determine the extent of adsorption of two model proteinslysozyme and cytochrome conto these "multimodal" surfaces and to select promising "affinity" ligands for further characterization. The attachment of selected ligands to UltraLink Biosupport resulted in beads with a significantly greater affinity for lysozyme than for cytochrome c that would be suitable for use in affinity chromatographic processes. Furthermore, we also used the screens to design "affinity displacers"small molecules that selectively retain lysozyme on chromatographic resins, while displacing cytochrome c. The combination of SPR spectroscopy and SAMs represents a powerful technique for identifying novel ligands that enable the purification of complex protein mixtures.

  3. Preparative isolation and purification of iridoid glycosides from Fructus Corni by high-speed countercurrent chromatography

    PubMed Central

    Liang, Jinru; He, Jiao; Zhu, Sha; Zhao, Wenna; Zhang, Yongmin; Ito, Yoichiro; Sun, Wenji

    2012-01-01

    Using a two-phase solvent system composed of dichloromethane–methanol–n-butanol–water–acetic acid (5:5:3:4:0.1, v/v/v/v/v), high-speed countercurrent chromatography was successfully performed for isolation and purification of three iridoid glycosides from Fructus Corni for the first time. From 100 mg of a crude extract of Fructus Corni 7.9 mg of sweroside, 13.1 mg of morroniside, and 10.2 mg of loganin were obtained in less than 3 h with purities of 92.3, 96.3 and 94.2%, respectively. These target compounds were identified by ESI-MS, 1H NMR and 13C NMR. PMID:24899790

  4. Isolation and purification of glycoconjugates from complex biological sources by recycling high-performance liquid chromatography.

    PubMed

    Alley, William R; Mann, Benjamin F; Hruska, Vlastimil; Novotny, Milos V

    2013-11-01

    Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays.

  5. Aryl thioglycoside-based affinity purification of exo-acting cellulases.

    PubMed

    Piyachomkwan, K; Penner, M H

    1998-01-15

    The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters. PMID:9451508

  6. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    NASA Technical Reports Server (NTRS)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  7. Heterogeneous glycoform separation by process chromatography: I: Monomer purification and characterization.

    PubMed

    Li, Yi; Xu, Xuankuo; Shupe, Alan; Yang, Rong; Bai, Kevin; Das, Tapan; Borys, Michael C; Li, Zheng Jian

    2015-07-24

    Fc fusion proteins with high and low sialylation were purified and separated by preparative ion-exchange and hydrophobic interaction chromatography. Heterogeneity in sialylation and glycosylation led to variation in surface charge and hydrophobicity, and resulted in multiple distinct glycoform populations in response to various purification conditions. Monomer with high sialic acid content has higher surface charge and adsorbs stronger to ion-exchange resin, while the less sialylated monomer interacts more favorably with hydrophobic resin. Extensive biophysical characterization was carried out for purified monomers at different level of sialylation. In general, different monomeric glycoforms have different surface charge and hydrophobicity, different thermal stability, and different aggregation propensity. The surface charge corresponds well with sialic acid content, as evidenced by electrophoresis, N-link domain analysis, and zeta potential results. The sialylation also contributes to minor modification of protein size, molecular mass and tertiary structure. Notably, fluorescence emission spectra and thermal transition became less distinguishable when the monomers containing low and high sialic acid were prepared in high ionic strength solution. Such finding reiterates the fact that the electrostatic forces, which are largely dependent on sialic acid content of protein, plays a dominant role in many intra- and inter-molecular interactions. Overall, the characterization data agreed well with separation behaviors and provided valuable insight to control of glycoform profile in purification process.

  8. Purification of chymotrypsin by subunit exchange chromatography on the denatured protein

    SciTech Connect

    Carrea, G.; Antonini, E.; Pasta, P.

    1983-05-01

    The ..cap alpha..-chymotrypsin subunits immobilized under denaturing conditions (6M urea or 1% SDS) on CNBr-activated Sepharose 4B, were found to interact with soluble chymotrypsin subunits with the formation of oligomers higher than dimers. Subunits immobilized under nondenaturing conditions form only dimers. The effects of several parameters, such as organic solvents, cations, and anions of the lyotropic series, on the associating properties of the immobilized derivatives were examined. The interaction between immobilized and free enzyme was shown to be specific because extraneous proteins and compounds were not bound by the derivatives. Chymotrypsinogen, studied analogously, did not show appreciable self-associating capacity. Chymotrypsin subunits immobilized under denaturing conditions and packed in a column proved to be suitable for the purification of chymotrypsin from both bovine and porcine pancreatic extracts. The ''subunit exchange'' chromatography of such extracts, carried out between pH 2.5 and 4, gave an eightfold purification with a 93% recovery of chymotryptic activity. The specific activity was ca. 12,000 Schwert and Takenaka units/mg. Only 6% of the tryptic activity was bound by the column. The capacity of the matrix, 6 mg chymotrypsin/mL, dropped to about 70% of the original value.

  9. Purification of chymotrypsin by subunit exchange chromatography on the denatured protein.

    PubMed

    Carrea, G; Pasta, P; Antonini, E

    1983-05-01

    The alpha-chymotrypsin subunits immobilized under denaturing conditions (6 M urea or 1% SDS) on CNBr-activated Sepharose 4B, were found to interact with soluble chymotrypsin subunits with the formation of oligomers higher than dimers. Subunits immobilized under nondenaturing conditions form only dimers. The effects of several parameters, such as organic solvents, cations, and anions of the lyotropic series, on the associating properties of the immobilized derivatives were examined. The interaction between immobilized and free enzyme was shown to be specific because extraneous proteins and compounds were not bound by the derivatives. Chymotrypsinogen, studied analogously, did not show appreciable self-associating capacity. Chymotrypsin subunits immobilized under denaturing conditions and packed in a column proved to be suitable for the purification of chymotrypsin from both bovine and porcine pancreatic extracts. The "subunit exchange" chromatography of such extracts, carried out between pH 2.5 and 4, gave an eightfold purification with a 93% recovery of chymotryptic activity. The specific activity was ca. 12,000 Schwert and Takenaka units/mg. Only 6% of the tryptic activity was bound by the column. The capacity of the matrix, 6 mg chymotrypsin/mL, dropped to about 70% of the original value after.

  10. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography.

    PubMed

    Hilbrig, Frank; Freitag, Ruth

    2012-01-01

    Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.

  11. Heterogeneous glycoform separation by process chromatography: I: Monomer purification and characterization.

    PubMed

    Li, Yi; Xu, Xuankuo; Shupe, Alan; Yang, Rong; Bai, Kevin; Das, Tapan; Borys, Michael C; Li, Zheng Jian

    2015-07-24

    Fc fusion proteins with high and low sialylation were purified and separated by preparative ion-exchange and hydrophobic interaction chromatography. Heterogeneity in sialylation and glycosylation led to variation in surface charge and hydrophobicity, and resulted in multiple distinct glycoform populations in response to various purification conditions. Monomer with high sialic acid content has higher surface charge and adsorbs stronger to ion-exchange resin, while the less sialylated monomer interacts more favorably with hydrophobic resin. Extensive biophysical characterization was carried out for purified monomers at different level of sialylation. In general, different monomeric glycoforms have different surface charge and hydrophobicity, different thermal stability, and different aggregation propensity. The surface charge corresponds well with sialic acid content, as evidenced by electrophoresis, N-link domain analysis, and zeta potential results. The sialylation also contributes to minor modification of protein size, molecular mass and tertiary structure. Notably, fluorescence emission spectra and thermal transition became less distinguishable when the monomers containing low and high sialic acid were prepared in high ionic strength solution. Such finding reiterates the fact that the electrostatic forces, which are largely dependent on sialic acid content of protein, plays a dominant role in many intra- and inter-molecular interactions. Overall, the characterization data agreed well with separation behaviors and provided valuable insight to control of glycoform profile in purification process. PMID:26051082

  12. Purification of lipoteichoic acid by chromatography in water-organic solvent systems.

    PubMed Central

    Josephson, S L; Stinson, M W; Millar, S J; Cohen, R E

    1986-01-01

    Lipoteichoic acid (LTA), extracted from Streptococcus mutans 10449 by hot aqueous phenol, was partially purified by Sepharose 6B column chromatography in 0.01 M sodium acetate, pH 6.0, containing 0.25 M sodium chloride and 0.001 M EDTA. Nucleic acid and polysaccharide were precipitated from the LTA-containing column peak by the addition of 2 volumes of chloroform-methanol (1:5). The resulting single-phase chloroform-methanol-water (1:5:3) supernatant contained LTA and small amounts of several contaminating substances as indicated by reverse-phase high-pressure liquid chromatography and chemical analyses. LTA was purified further by DEAE-cellulose chromatography, using a concentration gradient of sodium chloride in chloroform-methanol-water (1:5:3). Two column peaks of LTA were found to contain phosphate, glycerol, glucose, and fatty acids at molar ratios of 1:1:0.11:0.10 and 1:1:0.09:0.04, respectively. The LTA polymers contained 18 and 22 repeating units of unsubstituted glycerophosphate and two glucose residues. The LTA in one column peak had two fatty acids per molecule, whereas that in the second peak contained only one. The yield of LTA was 1.68 mg per g of cell dry weight or 65 mg per g of phenol-water-extracted material. The specific activity of the LTA preparation was increased 128-fold by the purification scheme as determined by a erythrocyte-binding assay. Reverse-phase high-pressure liquid chromatography may be used for rapid separation of LTA molecules containing different numbers of acyl groups. PMID:3943892

  13. Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.

    PubMed

    Meininger, M; Stepath, M; Hennig, R; Cajic, S; Rapp, E; Rotering, H; Wolff, M W; Reichl, U

    2016-02-15

    Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.

  14. Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.

    PubMed

    Meininger, M; Stepath, M; Hennig, R; Cajic, S; Rapp, E; Rotering, H; Wolff, M W; Reichl, U

    2016-02-15

    Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO. PMID:26851523

  15. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  16. Affinity purification of antibodies using immobilized FB domain of protein A.

    PubMed

    Solomon, B; Raviv, O; Leibman, E; Fleminger, G

    1992-04-24

    A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A. PMID:1517325

  17. Affinity tag for protein purification and detection based on the disulfide-linked complex of InaD and NorpA.

    PubMed

    Kimple, Michelle E; Sondek, John

    2002-09-01

    Affinity tags are not only used for the expression and purification of recombinant proteins but also for the detection of protein-protein interactions. Common problems with many affinity tags are excessive length, which may interfere with the structure and function of tagged proteins, and low affinity and/or specificity for primary detection and purification agents. Preliminary results suggest that the C-terminalfive residues of the Drosophila protein NorpA, based on the short, covalent interaction they make with the N-terminal PDZ domain (PDZI) of InaD, are useful as a general affinity tag. First, a PDZI-alkaline phosphatase fusion protein specifically detects both its physiological ligand and a heterologous protein expressing the NorpA C-terminal five residues. The interaction of PDZI with a NorpA-tagged protein is reversible by a reducing agent, which allows nitrocellulose membranes to be stripped completely and reused. In addition, a NorpA-tagged protein can specifically bind to immobilized PDZI resin, while other cellular proteins are washed through. After washing, the NorpA-tagged protein is eluted by a reducing buffer. The NorpA tag's short length makes it the smallest affinity tag available, and its specific and high-affinity interaction with PDZI could yield a powerful system that improves on currently available technology.

  18. Purification of subunit B of Shiga toxin using a synthetic trisaccharide-based affinity matrix.

    PubMed

    Pozsgay, V; Trinh, L; Shiloach, J; Robbins, J B; Donohue-Rolfe, A; Calderwood, S B

    1996-01-01

    The blood group P1 antigenic trisaccharide (3), which is the receptor-binding ligand of Shiga-like toxins, is synthesized in a spacer-equipped form (32) from 2-(trimethylsilyl)ethyl glucoside 5 and the 1-thiogalactoside building blocks 10 and 22 in a stereocontrolled, stepwise fashion. Covalent attachment of 32 to hydrazine group-containing agarose gel by reductive amination provided the P1 trisaccharide-containing affinity sorbent which was used for preparative scale isolation of subunit B of Shiga toxin. PMID:8741990

  19. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

    PubMed

    Ulbrich, N; Lin, A; Wool, I G

    1979-09-10

    The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column. PMID:468846

  20. Isolation and purification of six iridoid glycosides from gardenia jasminoides fruit by medium-pressure liquid chromatography combined with macroporous resin chromatography.

    PubMed

    Wang, Yun; Liu, Hui; Shen, Lifeng; Yao, Lan; Ma, Yinlian; Yu, Dingrong; Chen, Jianhong; Li, Puling; Chen, Ying; Zhang, Cun

    2015-12-01

    Gardeniae fructus is one of the most frequently used herbs in traditional Chinese medicine. In the present study, a process for the enrichment of six iridoid glycosides from Gardeniae fructus was developed using medium-pressure liquid chromatography combined with macroporous resin and reversed-phase chromatography. The purities of different fractions from Gardeniae fructus were assessed using quantitative high-performance liquid chromatography. After fractionation using HPD-100 column chromatography, a 30% ethanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis to separate and purify. Based on the orientation analysis results, six compounds-deacetyl asperulosidic acid methyl ester, gardenoside, ixoroside, scandoside methyl ester, genipin-1-O-β-d-gentiobioside, and geniposide-were successfully isolated and purified in three to four combined steps from Gardeniae fructus. The purities of these compounds were found by high-performance liquid chromatography analysis to be 97.9, 98.1, 95.5, 96.3, 97.1, and 98.7%, respectively. Moreover, their structures were elucidated by NMR spectroscopy and liquid chromatography with tandem mass spectrometry. The separation process was highly efficient, rapid, and accurate, making it a potential approach for the large-scale production of iridoids in the laboratory and providing several marker compounds for quality control. This procedure may be meaningful for the purification of other natural products used in traditional Chinese medicine. PMID:26768549

  1. Isolation and purification of six iridoid glycosides from gardenia jasminoides fruit by medium-pressure liquid chromatography combined with macroporous resin chromatography.

    PubMed

    Wang, Yun; Liu, Hui; Shen, Lifeng; Yao, Lan; Ma, Yinlian; Yu, Dingrong; Chen, Jianhong; Li, Puling; Chen, Ying; Zhang, Cun

    2015-12-01

    Gardeniae fructus is one of the most frequently used herbs in traditional Chinese medicine. In the present study, a process for the enrichment of six iridoid glycosides from Gardeniae fructus was developed using medium-pressure liquid chromatography combined with macroporous resin and reversed-phase chromatography. The purities of different fractions from Gardeniae fructus were assessed using quantitative high-performance liquid chromatography. After fractionation using HPD-100 column chromatography, a 30% ethanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis to separate and purify. Based on the orientation analysis results, six compounds-deacetyl asperulosidic acid methyl ester, gardenoside, ixoroside, scandoside methyl ester, genipin-1-O-β-d-gentiobioside, and geniposide-were successfully isolated and purified in three to four combined steps from Gardeniae fructus. The purities of these compounds were found by high-performance liquid chromatography analysis to be 97.9, 98.1, 95.5, 96.3, 97.1, and 98.7%, respectively. Moreover, their structures were elucidated by NMR spectroscopy and liquid chromatography with tandem mass spectrometry. The separation process was highly efficient, rapid, and accurate, making it a potential approach for the large-scale production of iridoids in the laboratory and providing several marker compounds for quality control. This procedure may be meaningful for the purification of other natural products used in traditional Chinese medicine.

  2. Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

    PubMed Central

    Ruprecht, Benjamin; Koch, Heiner; Medard, Guillaume; Mundt, Max; Kuster, Bernhard; Lemeer, Simone

    2015-01-01

    Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems. PMID

  3. Optimization of human serum albumin monoliths for chiral separations and high-performance affinity chromatography

    PubMed Central

    Pfaunmiller, Erika L.; Hartmann, Mahli; Dupper, Courtney M.; Soman, Sony; Hage, David S.

    2012-01-01

    Various organic-based monoliths were prepared and optimized for immobilization of the protein human serum albumin (HSA) as a binding agent for chiral separations and high-performance affinity chromatography. These monoliths contained co-polymers based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or GMA and trimethylolpropane trimethacrylate (TRIM). A mixture of cyclohexanol and 1-dodecanol was used as the porogen, with the ratio of these solvents being varied along with the polymerization temperature to generate a library of monoliths. These monoliths were used with both the Schiff base and epoxy immobilization methods and measured for their final content of HSA. Monoliths showing the highest protein content were further evaluated in chromatographic studies using R/S-warfarin and d/l-tryptophan as model chiral solutes. A 2.6–2.7-fold increase in HSA content was obtained in the final monoliths when compared to similar HSA monoliths prepared according to the literature. The increased protein content made it possible for the new monoliths to provide higher retention and/or two-fold faster separations for the tested solutes when using 4.6 mm i.d. × 50 mm columns. These monoliths were also used to create 4.6 mm i.d. × 10 mm HSA microcolumns that could separate the same chiral solutes in only 1.5–6.0 min. The approaches used in this study could be extended to the separation of other chiral solutes and to the optimization of organic monoliths for use with additional proteins as binding agents. PMID:23010249

  4. Glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides

    PubMed Central

    Alvarez-Manilla, Gerardo; Warren, Nicole L.; Atwood, James; Orlando, Ron; Dalton, Stephen; Pierce, Michael

    2011-01-01

    Numerous studies have recently focused on the identification of specific glycan biomarkers; given the important roles that protein linked glycans play, for example, during development and disease progression. The identification of protein glycobiomarkers, which are part of a very complex proteome, has involved the use of fractionation techniques such as lectin affinity chromatography. In this study, the glycoproteomic characterization of pluripotent murine embryonic stem cells (ES) and from ES cells that were differentiated into embroid bodies (EB) was performed using immobilized Concanavalin A (ConA). This procedure allowed the isolation of glycopeptides that express biantennary and hybrid N-linked structures (ConA2 fraction) as well as high mannose glycans (ConA3 fraction), that were abundant in both ES and EB stages. A total of 293 unique N-linked glycopeptide sequences (from 180 glycoproteins) were identified in the combined data sets from ES and EB cells. Of these glycopeptides, a total of 119 sequences were identified exclusively in only one of the lectin bound fractions, (24 in the ES-ConA2, 15 in the ES-ConA3, 16 in the EB-ConA2 and 64 in the EB-ConA3). Results from this study allowed the identification of individual N-glycosylation sites of proteins that express specific glycan types. The absence of some of these lectin bound glycopeptides in a cell stage suggested that they were derived from proteins that were either expressed exclusively on a defined developmental stage, or were expressed in both cell stages but carried the lectin bound oligosaccharides in only one of them. Therefore, these lectin bound glycopeptides can be considered as stage specific glycobiomarkers. PMID:19545112

  5. Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants.

    PubMed

    Buyel, Johannes F; Fischer, Rainer

    2014-04-01

    Plants offer a valuable alternative to cultured mammalian cells for the production of recombinant biopharmaceutical proteins. However, the target protein typically represents only a minor fraction of the total protein in the initial plant extract, which means that the development of product-specific chromatography-based purification strategies is often laborious and expensive. To address this challenge, we designed a generic downstream process that is suitable for the purification of recombinant proteins with diverse properties from plant production platforms. This was achieved by focusing on the binding behavior of tobacco host cell proteins (HCPs) to a broad set of chromatography resins under different pH and conductivity conditions. Strong cation exchanger and salt-tolerant anion exchanger resins exhibited the best resolution of tobacco HCPs among the 13 tested resins, and their selectivity was easy to manipulate through the adjustment of pH and conductivity. The advantages, such as direct capture of a target protein from leaf extract, and limitations, such as low binding capacity, of various chromatography ligands and resins are discussed. We also address the most useful applications of the chromatography ligands, namely recovery of proteins with a certain pI, in a downstream process that aims to purify diverse plant-derived biopharmaceutical proteins. Based on these results, we describe generic purification schemes that are suitable for acidic, neutral, and basic target proteins, as a first step toward the development of industrial platform processes.

  6. Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants.

    PubMed

    Buyel, Johannes F; Fischer, Rainer

    2014-04-01

    Plants offer a valuable alternative to cultured mammalian cells for the production of recombinant biopharmaceutical proteins. However, the target protein typically represents only a minor fraction of the total protein in the initial plant extract, which means that the development of product-specific chromatography-based purification strategies is often laborious and expensive. To address this challenge, we designed a generic downstream process that is suitable for the purification of recombinant proteins with diverse properties from plant production platforms. This was achieved by focusing on the binding behavior of tobacco host cell proteins (HCPs) to a broad set of chromatography resins under different pH and conductivity conditions. Strong cation exchanger and salt-tolerant anion exchanger resins exhibited the best resolution of tobacco HCPs among the 13 tested resins, and their selectivity was easy to manipulate through the adjustment of pH and conductivity. The advantages, such as direct capture of a target protein from leaf extract, and limitations, such as low binding capacity, of various chromatography ligands and resins are discussed. We also address the most useful applications of the chromatography ligands, namely recovery of proteins with a certain pI, in a downstream process that aims to purify diverse plant-derived biopharmaceutical proteins. Based on these results, we describe generic purification schemes that are suitable for acidic, neutral, and basic target proteins, as a first step toward the development of industrial platform processes. PMID:24478119

  7. Affinity purification of proteins binding to kinase inhibitors immobilized on self-assembling monolayers.

    PubMed

    Bantscheff, Marcus; Hobson, Scott; Kuster, Bernhard

    2012-01-01

    Kinase inhibitors represent a relatively new class of drugs that offer novel therapies targeting specific -malfunctioning kinase-mediated signaling pathways in oncology and potentially inflammation. As the ATP binding sites of the ∼500 human kinases are structurally conserved and because most current drugs target the ATP binding site, there is a need to profile all the kinases that a drug may bind and/or inhibit. We have developed a chemical proteomics method that affinity purifies kinases from cell or tissue lysates using kinase inhibitors immobilized on self-assembling monolayers. The method can be applied to assess the selectivity of a given kinase inhibitor and thus to guide its preclinical or clinical development.

  8. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  9. A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei.

    PubMed

    Nassa, Giovanni; Tarallo, Roberta; Ambrosino, Concetta; Bamundo, Angela; Ferraro, Lorenzo; Paris, Ornella; Ravo, Maria; Guzzi, Pietro H; Cannataro, Mario; Baumann, Marc; Nyman, Tuula A; Nola, Ernesto; Weisz, Alessandro

    2011-01-01

    Estrogen receptors α (ER-α) and β (ER-β) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-β exerting a modulatory activity on ER-α-mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER-β fused to a tandem affinity purification-tag in estrogen-responsive MCF-7 cells and applied tandem affinity purification and nanoLC-MS/MS to identify the ER-β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells. PMID:21182203

  10. From pathways to networks: connecting dots by establishing protein-protein interaction networks in signaling pathways using affinity purification and mass spectrometry

    PubMed Central

    Li, Xu; Wang, Wenqi; Chen, Junjie

    2015-01-01

    Signal transductions are the basis of biological activities in all living organisms. Studying the signaling pathways, especially under physiological conditions, has become one of the most important facets of modern biological research. During the last decade, mass spectrometry has been used extensively in biological research and is proven to be effective in addressing important biological questions. Here, we review the current progress in the understanding of signaling networks using mass spectrometry approaches. We will focus on studies of protein-protein interactions that use affinity purification followed by mass spectrometry approach. We discuss obstacles to affinity purification, data processing, functional validation, and identification of transient interactions and provide potential solutions for pathway-specific proteomics analysis, which we hope one day will lead to a comprehensive understanding of signaling networks in humans. PMID:25137225

  11. From pathways to networks: connecting dots by establishing protein-protein interaction networks in signaling pathways using affinity purification and mass spectrometry.

    PubMed

    Li, Xu; Wang, Wenqi; Chen, Junjie

    2015-01-01

    Signal transductions are the basis of biological activities in all living organisms. Studying the signaling pathways, especially under physiological conditions, has become one of the most important facets of modern biological research. During the last decade, MS has been used extensively in biological research and is proven to be effective in addressing important biological questions. Here, we review the current progress in the understanding of signaling networks using MS approaches. We will focus on studies of protein-protein interactions that use affinity purification followed by MS approach. We discuss obstacles to affinity purification, data processing, functional validation, and identification of transient interactions and provide potential solutions for pathway-specific proteomics analysis, which we hope one day will lead to a comprehensive understanding of signaling networks in humans. PMID:25137225

  12. Identification of BZR1-interacting Proteins as Potential Components of the Brassinosteroid Signaling Pathway in Arabidopsis Through Tandem Affinity Purification*

    PubMed Central

    Wang, Chunming; Shang, Jian-Xiu; Chen, Qi-Xiu; Oses-Prieto, Juan A.; Bai, Ming-Yi; Yang, Yihong; Yuan, Min; Zhang, Yu-Lan; Mu, Cong-Cong; Deng, Zhiping; Wei, Chuang-Qi; Burlingame, Alma L.; Wang, Zhi-Yong; Sun, Ying

    2013-01-01

    Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14–3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. PMID:24019147

  13. PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

    PubMed Central

    Schildbach, Stefan; Blumert, Conny; Horn, Friedemann; von Bergen, Martin; Labudde, Dirk

    2016-01-01

    The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6. PMID:26966684

  14. Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification.

    PubMed

    Wang, Chunming; Shang, Jian-Xiu; Chen, Qi-Xiu; Oses-Prieto, Juan A; Bai, Ming-Yi; Yang, Yihong; Yuan, Min; Zhang, Yu-Lan; Mu, Cong-Cong; Deng, Zhiping; Wei, Chuang-Qi; Burlingame, Alma L; Wang, Zhi-Yong; Sun, Ying

    2013-12-01

    Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. PMID:24019147

  15. Using ProHits to store, annotate and analyze affinity purification - mass spectrometry (AP-MS) data

    PubMed Central

    Liu, Guomin; Zhang, Jianping; Choi, Hyungwon; Lambert, Jean-Philippe; Srikumar, Tharan; Larsen, Brett; Nesvizhskii, Alexey I.; Raught, Brian; Tyers, Mike; Gingras, Anne-Claude

    2012-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is a robust technique used to identify protein-protein interactions. With recent improvements in sample preparation, and dramatic advances in MS instrumentation speed and sensitivity, this technique is becoming more widely used throughout the scientific community. To meet the needs of research groups both large and small, we have developed software solutions for tracking, scoring and analyzing AP-MS data. Here, we provide details for the installation and utilization of ProHits, a Laboratory Information Management System designed specifically for AP-MS interaction proteomics. This protocol explains: (i) how to install the complete ProHits system, including modules for the management of mass spectrometry files and the analysis of interaction data, and (ii) alternative options for the use of pre-existing search results in simpler versions of ProHits, including a virtual machine implementation of our ProHits Lite software. We also describe how to use the main features of the software to analyze AP-MS data. PMID:22948730

  16. Purification of C70 using charcoal as a stationary phase in a flash chromatography column. Technical report

    SciTech Connect

    Scrivens, W.A.; Cassell, A.M.; Kinsey, K.E.; Tour, J.M.

    1995-06-07

    Described is a method for the purification of C60 and C70 using a flash chromatography column that contains charcoal as the stationary phase. A number of functionalized aromatic solvents are studied and their efficacy for extraction, NMR spectral acquisition, and chromatographic purification of fullerenes is discussed. Ortho-dichlorobenzene was chosen as the best solvent for these applications and examples of its use in the extraction of higher fullerenes (>C84) and in the rapid acquisition of (13)C NMR spectra are given. Finally, single column purification of both C60 and C70 is discussed. Starting with a typical arc-derived mixture of soluble fullerenes, 5.97 g of C60 at >99.9% purity and 1.58 g of C70 at >97% purity were produced in a single column pass.

  17. Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps.

    PubMed

    Roberts, Peter L

    2014-01-01

    The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG-1 & -3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion-exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non-enveloped viruses over the life-time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion-exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X-100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process.

  18. Gel compression considerations for chromatography scale-up for protein C purification.

    PubMed

    He, W; Bruley, D F; Drohan, W N

    1998-01-01

    This work is to establish theoretical and experimental relationships for the scale-up of Immobilized Metal Affinity Chromatography (IMAC) and Immuno Affinity Chromatography for the low cost production of large quantities of Protein C. The external customer requirements for this project have been established for Protein C deficient people with the goal of providing prophylactic patient treatment. Deep vein thrombosis is the major symptom for protein C deficiency creating the potential problem of embolism transport to important organs, such as, lung and brain. Gel matrices for protein C separation are being analyzed to determine the relationship between the material properties of the gel and the column collapse characteristics. The fluid flow rate and pressure drop is being examined to see how they influence column stability. Gel packing analysis includes two considerations; one is bulk compression due to flow rate, and the second is gel particle deformation due to fluid flow and pressure drop. Based on the assumption of creeping flow, Darcy's law is being applied to characterize the flow through the gel particles. Biot's mathematical description of three-dimensional consolidation in porous media is being used to develop a set of system equations. Finite difference methods are being utilized to obtain the equation solutions. In addition, special programs such as finite element approaches, ABAQUS, will be studied to determine their application to this particular problem. Experimental studies are being performed to determine flow rate and pressure drop correlation for the chromatographic columns with appropriate gels. Void fraction is being measured using pulse testing to allow Reynolds number calculations. Experimental yield stress is being measured to compare with the theoretical calculations. Total Quality Management (TQM) tools have been utilized to optimize this work. For instance, the "Scatter Diagram" has been used to evaluate and select the appropriate gels and

  19. Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography.

    PubMed

    Du, Xiping; Dong, Congcong; Wang, Kai; Jiang, Zedong; Chen, Yanhong; Yang, Yuanfan; Chen, Feng; Ni, Hui

    2016-09-01

    An effective high-speed counter-current chromatography (HSCCC) method was established for the preparative isolation and purification of astaxanthin from Phaffia rhodozyma. With a two-phase solvent system composed of n-hexane-acetone-ethanol-water (1:1:1:1, v/v/v/v), 100mg crude extract of P. rhodozyma was separated to yield 20.6mg of astaxanthin at 92.0% purity. By further one step silica gel column chromatography, the purity reached 99.0%. The chemical structure of astaxanthin was confirmed by thin layer chromatography (TLC), UV spectroscopy scanning, high performance liquid chromatography with a ZORBAX SB-C18 column and a Waters Nova-pak C18 column, and ESI/MS/MS. PMID:27433984

  20. Affinity selection-based two-dimensional chromatography coupled with high-performance liquid chromatography-mass spectrometry for discovering xanthine oxidase inhibitors from Radix Salviae Miltiorrhizae.

    PubMed

    Fu, Yu; Mo, Hua-Yan; Gao, Wen; Hong, Jia-Ying; Lu, Jun; Li, Ping; Chen, Jun

    2014-08-01

    Xanthine oxidase (XOD) is a key oxidative enzyme to the pathogenesis of hyperuricemia and certain diseases induced by excessive reactive oxygen species. XOD inhibitors could provide an important therapeutic approach to treat such diseases. A new method using affinity selection-based two-dimensional chromatography coupled with liquid chromatography-mass spectrometry was developed for the online screening of potential XOD inhibitors from Radix Salviae Miltiorrhizae. Based on our previous study, the two-dimensional, turbulent-flow chromatography (TFC) was changed to a mixed-mode anion-exchange/reversed-phase column and one reversed-phase column. The developed method was validated to be selective and sensitive for screening XOD-binding compounds, especially weak acidic ones, in the extracts. Three salvianolic acids were screened from the Radix Salviae Miltiorrhizae extract via the developed method. The XOD inhibitory activities of salvianolic acid C and salvianolic acid A were confirmed, and their inhibitory modes were measured. Salvianolic acid C exhibited potent XOD inhibitory activity with an IC(50) of 9.07 μM. This work demonstrated that the developed online, two-dimensional TFC/LC-MS method was effective in discovering the binding affinity of new compounds from natural extracts for target proteins, even at low concentrations.

  1. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS).

    PubMed

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A

    2016-09-01

    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1).

  2. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS).

    PubMed

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A

    2016-09-01

    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1). PMID:27274533

  3. Affinity column for purification of the human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor

    SciTech Connect

    Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

    1987-05-01

    The TXA/sub 2//PGH/sub 2/ receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific /sup 3/H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA/sub 2//PGH/sub 2/ receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor.

  4. A Versatile and Inexpensive Enzyme Purification Experiment for Undergraduate Biochemistry Labs.

    ERIC Educational Resources Information Center

    Farrell, Shawn O.; Choo, Darryl

    1989-01-01

    Develops an experiment that could be done in two- to three-hour blocks and does not rely on cold room procedures for most of the purification. Describes the materials, methods, and results of the purification of bovine heart lactate dehydrogenase using ammonium sulfate fractionation, dialysis, and separation using affinity chromatography and…

  5. Identification of Novel Surface-Exposed Proteins of Rickettsia rickettsii by Affinity Purification and Proteomics

    PubMed Central

    Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2014-01-01

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens. PMID:24950252

  6. Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

    PubMed Central

    Bieberich, Erhard

    2011-01-01

    The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane

  7. Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography.

    PubMed

    Zaveckas, Mindaugas; Snipaitis, Simas; Pesliakas, Henrikas; Nainys, Juozas; Gedvilaite, Alma

    2015-06-01

    Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impact on swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccine candidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizes were examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. Q Sepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some host cell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6B and CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPs after chromatography on Heparin Sepharose CL-6B was only 4-7% and the recovery of VLPs was 5-7%. Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purity of about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 monolith was 15-18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electron microscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate was developed using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith.

  8. Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography*

    PubMed Central

    Hoehenwarter, Wolfgang; Thomas, Martin; Nukarinen, Ella; Egelhofer, Volker; Röhrig, Horst; Weckwerth, Wolfram; Conrath, Uwe; Beckers, Gerold J. M.

    2013-01-01

    Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment. PMID:23172892

  9. Screening of Potential Xanthine Oxidase Inhibitors in Gnaphalium hypoleucum DC. by Immobilized Metal Affinity Chromatography and Ultrafiltration-Ultra Performance Liquid Chromatography-Mass Spectrometry.

    PubMed

    Zhang, Hong-Jian; Hu, Yi-Juan; Xu, Pan; Liang, Wei-Qing; Zhou, Jie; Liu, Pei-Gang; Cheng, Lin; Pu, Jin-Bao

    2016-01-01

    In this study, a new method based on immobilized metal affinity chromatography (IMAC) combined with ultrafiltration-ultra performance liquid chromatography-mass spectrometry (UF-UPLC-MS) was developed for discovering ligands for xanthine oxidase (XO) in Gnaphalium hypoleucum DC., a folk medicine used in China for the treatment of gout. By IMAC, the high flavonoid content of G. hypoleucum could be determined rapidly and efficiently. UF-UPLC-MS was used to select the bound xanthine oxidase ligands in the mixture and identify them. Finally, two flavonoids, luteolin-4'-O-glucoside and luteolin, were successfully screened and identified as the candidate XO inhibitors of G. hypoleucum. They were evaluated in vitro for XO inhibitory activity and their interaction mechanism was studied coupled with molecular simulations. The results were in favor of the hypothesis that the flavonoids of G. hypoleucum might be the active content for gout treatment by inhibiting XO. PMID:27649136

  10. Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

    PubMed

    Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana

    2011-08-01

    Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.

  11. Use of a Phosphatidylinositol Phosphate Affinity Chromatography (PIP Chromatography) for the Isolation of Proteins Involved in Protein Quality Control and Proteostasis Mechanisms in Plants.

    PubMed

    Farmaki, T

    2016-01-01

    Protein functionality depends directly on its accurately defined three-dimensional organization, correct and efficient posttranslational modification, and transport. However, proteins are continuously under a hostile environment threatening with folding aberrations, aggregation, and mistargeting. Therefore, proteins must be constantly "followed up" by a tightly regulated homeostatic mechanism specifically known as proteostasis. To this end other proteins ensure this close surveillance including chaperones as well as structural and functional members of the proteolytic mechanisms, mainly the autophagy and the proteasome related. They accomplish their action via interactions not only with other proteins but also with lipids as well as cytoskeletal components. We describe a protocol based on an affinity chromatographic approach aiming at the isolation of phosphatidyl inositol phosphate binding proteins, a procedure which results into the enrichment and purification of several members of the proteostasis mechanism, e.g. autophagy and proteasome, among other components of the cell signaling pathways. PMID:27424758

  12. Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography.

    PubMed

    Batas, B; Schiraldi, C; Chaudhuri, J B

    1999-02-19

    The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.

  13. Cored anion-exchange chromatography media for antibody flow-through purification.

    PubMed

    Wang, Chen; Soice, Neil P; Ramaswamy, Senthilkumar; Gagnon, Brian A; Umana, Joaquin; Cotoni, Kristen A; Bian, Nanying; Cheng, Kwok-Shun C

    2007-06-29

    Agarose-based anion-exchangers (e.g. quaternary amine, Q) have been widely used in monoclonal antibody flow-through purification to remove trace levels of impurities. Such media are often packed in a large column and the operation is usually robust but with limited throughput due to the compressibility of agarose and consequentially low bed permeability. In order to address this limitation, cored Q beads consisting of a rigid core and a thin agarose gel coating were developed and evaluated for protein flow-through chromatography. Using laboratory-scale columns it was found that, the cored beads indeed provide significantly enhanced rigidity and flow permeability relative to conventional homogeneous agarose resins. Depending on the structure and size of the cored beads, the permeability was 2-4-fold higher than that of a commonly used commercial agarose resin. Good virus and host cell protein clearance was achieved with the cored Q beads even at increased flow velocities. In addition, the impermeable core allows for more efficient use of buffers without loss of useful capacity in polishing applications. Process analyses based upon the experimental data demonstrated that the enhanced permeability achieved with the cored beads can significantly improve process throughput and economics.

  14. [Isolation and purification of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5 using chromatography].

    PubMed

    Ma, Lina; Wu, Dan; Bian, Liujiao

    2012-08-01

    The Kringle 5 domain of plasminogen is one of the most potent angiogenesis inhibitors known to date, which can inhibit cell proliferation and migration efficiently. In the study, on the foundation of successful clone and expression of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5, a two-step chromatographic method, including the use of SP Sepharose Fast Flow cation exchanger and Sephacryl S-100 HR size exclusion chromatography in sequence, was established to separate and purify angiogenesis inhibitor Kringle 5. On the SP Sepharose Fast Flow column, the buffer A consisted of 50.0 mmol/L acetic acid-sodium acetate (pH 5.2), and the buffer B consisted of buffer A with the addition of 0.5 mol/L sodium chloride (pH 5.2); on Sephacryl S-100 HR column, the elution buffer was 5.0 mmol/L phosphate solution (pH 7.0). Through the two-step chromatographic purification process, the purity of the obtained Kringle 5 was more than 98%. In addition, it was found that the obtained Kringle 5 could inhibit the blood vessel growth of chick embryo chorioallantoic membrane effectively. Finally it is concluded that this method can effectively separate active recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5.

  15. Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody.

    PubMed

    Yu, Deqiang; Chen, Xiaonong; Pelton, Robert; Ghosh, Raja

    2008-04-15

    This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.

  16. Preparative purification of plasmin activity stimulating phenolic derivatives from Gastrodia elata using centrifugal partition chromatography.

    PubMed

    Jeon, Je-Seung; Kim, Jeeyoung; Park, Soyoung; Ryou, Chongsuk; Kim, Chul Young

    2016-06-01

    Gastrodia rhizome, a dried and steamed tuber of Gastrodia elata Blume (Orchidaceae), has been traditionally used in Korea, China and Japan for the treatment of neurological and nervous disorders such as headaches, dizziness, vertigo and convulsive illnesses. The ethyl acetate and water extracts of G. elata stimulated plasmin activity. The active ethyl acetate fraction was subjected to centrifugal partition chromatography (CPC) with a two-phase solvent system, composed of n-hexane-ethyl acetate-methanol-water (3:7:4:6, v/v) followed by semi-preparative HPLC purification to separate active compounds and the water fraction was purified by Diaion HP-20 resin and semi-preparative HPLC. In ethyl acetate extract, 4-hydroxybenzyl alcohol (1), 4-hydroxybenzoic acid (2), 4-hydroxybenzaldehyde (3), 4-ethoxymethylphenol (4), 4,4'-oxybis(methylene)diphenol (5) and 4,4'-methylenediphenol (6) were obtained with high purities. Parishin (7) and parishin B (8) were isolated from water extract. Among isolated compounds, 4-hydroxybenzyl alcohol (1), 4-hydroxybenzaldehyde (3) and 4-ethoxymethylphenol (4) significantly stimulated plasmin activity. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

    PubMed

    Dutta, Amit K; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A; Zhang, Ada W; Tustian, Andrew D; Zydney, Andrew L; Shinkazh, Oleg

    2015-11-10

    Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products.

  18. Engineered protein A ligands, derived from a histidine-scanning library, facilitate the affinity purification of IgG under mild acidic conditions

    PubMed Central

    2014-01-01

    Background In antibody purification processes, the acidic buffer commonly used to elute the bound antibodies during conventional affinity chromatograph, can damage the antibody. Herein we describe the development of several types of affinity ligands which enable the purification of antibodies under much milder conditions. Results Staphylococcal protein A variants were engineered by using both structure-based design and combinatorial screening methods. The frequency of amino acid residue substitutions was statistically analyzed using the sequences isolated from a histidine-scanning library screening. The positions where the frequency of occurrence of a histidine residue was more than 70% were thought to be effective histidine-mutation sites. Consequently, we identified PAB variants with a D36H mutation whose binding of IgG was highly sensitive to pH change. Conclusion The affinity column elution chromatograms demonstrated that antibodies could be eluted at a higher pH (∆pH**≧2.0) than ever reported (∆pH = 1.4) when the Staphylococcal protein A variants developed in this study were used as affinity ligands. The interactions between Staphylococcal protein A and IgG-Fab were shown to be important for the behavior of IgG bound on a SpA affinity column, and alterations in the affinity of the ligands for IgG-Fab clearly affected the conditions for eluting the bound IgG. Thus, a histidine-scanning library combined with a structure-based design was shown to be effective in engineering novel pH-sensitive proteins. PMID:25057290

  19. Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography.

    PubMed

    Sun, Bo; Yu, XiangHui; Yin, Yuhe; Liu, Xintao; Wu, Yongge; Chen, Yan; Zhang, Xizhen; Jiang, Chunlai; Kong, Wei

    2013-09-01

    The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 μg/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 μg/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA.

  20. Preparative separation and purification of gingerols from ginger (Zingiber officinale Roscoe) by high-speed counter-current chromatography.

    PubMed

    Zhan, Kunyou; Xu, Kun; Yin, Hongzong

    2011-06-15

    A novel method for purifying gingerols from ginger was developed using a high-speed counter-current chromatography (HSCCC). The two-phase solvent system such as light petroleum (bp 60-90°C)-ethyl acetate-methanol-water (5:5:6.5:3.5, v/v/v/v) was applied to the separation and purification of 6-, 8- and 10-gingerol from a crude extract of ginger. The experiment yielded 30.2mg of 6-gingerol, 40.5mg of 8-gingerol, 50.5mg of 10-gingerol from 200mg of crude extract in one-step separation. And the purity of these compounds was 99.9%, 99.9% and 99.2%, respectively, as determined by high-performance liquid chromatography (HPLC). Their structures were identified by gas chromatography-mass spectrometry (GC/MS) and (1)H, (13)C nuclear magnetic resonance (NMR). PMID:25213983

  1. Myogenic Growth Factor Present in Skeletal Muscle is Purified by Heparin-Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Kardami, Elissavet; Spector, Dennis; Strohman, Richard C.

    1985-12-01

    A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

  2. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    PubMed

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. PMID:26616099

  3. Proteome-wide identification of novel binding partners to the oncogenic fusion gene protein, NPM-ALK, using tandem affinity purification and mass spectrometry.

    PubMed

    Wu, Fang; Wang, Peng; Young, Leah C; Lai, Raymond; Li, Liang

    2009-02-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

  4. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    PubMed

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.

  5. Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

    PubMed

    Kollmar, Martin

    2006-08-15

    The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. PMID:16516959

  6. Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

    SciTech Connect

    Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.; Belchik, Sara M.; Shi, Liang; Monroe, Matthew E.; Smith, Richard D.; Lipton, Mary S.

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  7. Detection and identification of heme c-modified peptides by histidine affinity chromatography, high-performance liquid chromatography-mass spectrometry, and database searching.

    PubMed

    Merkley, Eric D; Anderson, Brian J; Park, Jea; Belchik, Sara M; Shi, Liang; Monroe, Matthew E; Smith, Richard D; Lipton, Mary S

    2012-12-01

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, two bacterial decaheme cytochromes, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide-spectrum matches to heme c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for 4 of the 10 expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 1×10(-4) was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c. PMID:23082897

  8. Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

    PubMed Central

    Brinkworth, R I; Masters, C J; Winzor, D J

    1975-01-01

    Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase. PMID:175784

  9. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

    PubMed Central

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

    2011-01-01

    In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

  10. Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Brock, Jonathan W.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John; Smith, Richard D.; Metz, Thomas O.

    2007-06-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.

  11. Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography.

    PubMed

    Grein, Tanja A; Michalsky, Ronald; Vega López, Maria; Czermak, Peter

    2012-08-01

    Significant progress in the application of viral vectors for gene delivery into mammalian cells and the use of viruses as biopesticides requires downstream processing that can satisfy application-specific demands on performance. In the present work the stability and ion exchange membrane chromatography of a recombinant of Autographa californica M nucleopolyhedrovirus is studied. To adjust the degree of purification the effect of ionic conductivity or pH on the viral infectivity was assessed (0.77-78.00mS/cm, pH 3-8). Infectivity decreased rapidly by several orders of magnitude at below 5mS/cm (i.e., 0.49MPa osmotic pressure change) or at below pH 5.5 (rationalized with particle aggregation). The virus was concentrated and purified via adsorption (0.2-1.1×10(16)pfu/m(3) chromatographic bed volume, 0.6-1.1×10(12)pfu/m(2) membrane area facing the incident fluid flow) and elution at pH 6.1 and 6.35mS/cm from three strong anion exchange membranes. Virus recovery and concentration in accord with the volume reduction were obtained using a polyether sulfone-based membrane with quaternary ammonium ligands. The level of host cell protein (down to below the detection limit) and suspended DNA (below 93pg DNA per 10(6)pfu) are reported for each membrane employed, for the purpose of comparability, under equal adsorption or elution conditions respectively.

  12. Purification of six lignans from the stems of Schisandra chinensis by using high-speed counter-current chromatography combined with preparative high-performance liquid chromatography.

    PubMed

    Zhu, Lijie; Li, Bin; Liu, Xiuying; Huang, Guohui; Meng, Xianjun

    2015-11-01

    A method for the preparative purification of lignans from Schisandra chinensis was established using a combination of high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). The crude extracts obtained from S. chinensis by using 70% ethanol were separated on a macroporous resin column and then eluted with a graded ethanol series. A two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) was used for HSCCC, and a mobile phase of acetonitrile-water (50:50, v/v) was used for preparative HPLC. The results obtained using HSCCC were compared with those obtained using preparative HPLC, and their advantages were further integrated to improve the separation efficiency. Six known lignans were identified by electrospray ionisation mass spectrometry and (1)H nuclear magnetic resonance (NMR) and (13)C NMR analyses; the purities of all the compounds were more than 91%.

  13. Preparative high-performance liquid chromatography for the purification of natural acylated anthocyanins from red radish (Raphanus sativus L.).

    PubMed

    Liu, Yonghong; Murakami, Nobutoshi; Wang, Lishu; Zhang, Si

    2008-09-01

    Preparative high-performance liquid chromatography (HPLC) is applied to the purification of anthocyanins from raw extracts of red radish (Raphanus sativus L). For each separation, the chromatographic conditions are optimized to achieve an efficient purification in the shortest time. In addition, UV-vis characterization is carried out on all purified anthocyanins. The current work shows that analytical chromatographic experiments alone are useful for the prediction of scale-up conditions of preparative HPLC separations. Ten known acylated anthocyanins (See the Appendix for compounds 1-10) are isolated from the red radish by isocratic HPLC. The structures are established on the basis of nuclear magnetic resonance and mass spectrometric analysis. The acylated anthocyanins are all based on pelargonidin 3-sophoroside-5-glucoside, acylated with caffeoyl, feruloyl, or p-coumaroyl moieties.

  14. Fundamental and practical studies on high-performance liquid affinity chromatography of biopolymers with novel stationary phases

    SciTech Connect

    Bacolod, M.D.

    1992-01-01

    Rigid microparticulate stationary phases having surface-bound metal chelating functions were developed and evaluated in high performance metal chelate affinity chromatography of proteins. Silica- and polystyrene-divinylbenzene-based metal chelate sorbents were produced in wide pore and in non-porous type of column packings. A major effort has been placed on development of non-porous highly crosslinked polystyrene-divinylbenzene (PSDVB). These PSDVB microparticles were produced by a two-step swelling polymerization, and exhibited excellent mechanical strength over a wide range of flow-rates and composition used in high performance liquid chromatography (HPLC). Simple and reproducible hydrophilic coatings were developed for the surface modification of hydrophobic PSDVB supports. A tetradentate metal chelating ligand, ethylenediamine-N, N[prime]-diacetic acid (EDDA), was covalently bound to the surface of the various supports. Sorbents having iminodiacetic acid (IDA) metal chelating functions were also evaluated. The hydrophilic character and surface coverage of various stationary phases were assessed chromatographically. Studies concerning the effects of eluent pH as well as the nature and concentration of salts on retention and selectivity with different metal chelate stationary phases having various immobilized metal ions were carried out. Elution schemes were developed for rapid separation of proteins in metal chelate affinity chromatography. EDDA stationary phases in metal forms can be viewed as complementary to IDA stationary phases since they afforded different selectivity and retentivity toward proteins. Hydrophilic PSDVB could be functionalized with IDA or EDDA metal chelating ligands or lectins. The non-porous metal chelate stationary phases afforded rapid separation of proteins by the development of multiple gradient systems, which permitted higher column peak capacity, enabling the separation of a greater number of proteins in a single chromatographic run.

  15. Hydrophilic interaction liquid chromatography for the separation, purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz.

    PubMed

    Liang, Tu; Fu, Qing; Li, Fangbing; Zhou, Wei; Xin, Huaxia; Wang, Hui; Jin, Yu; Liang, Xinmiao

    2015-08-01

    A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid-phase extraction followed by hydrophilic interaction liquid chromatography at semi-preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible. PMID:26011699

  16. Affinity purification of antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  17. Integrative refolding and purification of histidine-tagged protein by like-charge facilitated refolding and metal-chelate affinity adsorption.

    PubMed

    Liu, Hu; Du, Wen-Jie; Dong, Xiao-Yan; Sun, Yan

    2014-05-30

    This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.

  18. Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.

    PubMed

    Barbato, O; Sousa, N M; Klisch, K; Clerget, E; Debenedetti, A; Barile, V L; Malfatti, A; Beckers, J F

    2008-12-01

    The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

  19. sup 32 P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase

    SciTech Connect

    Reddy, M.V.; Bleicher, W.T.; Blackburn, G.R. )

    1991-04-01

    Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive {sup 32}P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO{sub 4}). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO{sub 4}-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO{sub 4} selectively forms cis-Tg adducts. With OsO{sub 4}-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO{sub 4}-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.

  20. Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography.

    PubMed

    Zhang, Qiang; Chen, Li-Juan; Ye, Hao-Yu; Gao, Lei; Hou, Wenli; Tang, Minghai; Yang, Guangli; Zhong, Zhenhua; Yuan, Yuan; Peng, Aihua

    2007-08-01

    A high-speed counter-current chromatography method was developed for the separation and purification of bioactive flavonol glycosides from a crude ethanol extract of Ginkgo biloba leaves. The separation was performed with a two-phase solvent system composed of n-hexane-butanol-ethyl acetate-methanol-0.5% acetic acid (1:0.5:3.5:1:4, v/v) and three pure compounds were eluted in high purities in a one-step separation. Their purities were determined by HPLC and identified by MS,(1)H-NMR, and(13)C-NMR.

  1. Isolation and purification of oridonin from the whole plant of Isodon rubescens by high-speed counter-current chromatography.

    PubMed

    He, Fa; Bai, Yuhua; Wang, Jing; Wei, Jing; Yu, Chunyue; Li, Sen; Yang, Weili; Han, Chenghua

    2011-09-14

    Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of oridonin from Isodon rubescens by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (2.8:5:2.8:5, v/v/v/v). The targeted compound isolated, collected and purified by HSCCC was analyzed by high performance liquid chromatography (HPLC). A total of 40.6 mg of oridonin with the purity of 73.5% was obtained in less than 100 min from 100 mg of crude Isodon rubescens extract. The chemical structure of the compound was identified by IR, 1H-NMR and 13C-NMR.

  2. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    PubMed Central

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-01-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  3. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate.

  4. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. PMID:26363185

  5. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography.

    PubMed

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-05-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  6. Multi-Parameter Cell Affinity Chromatography: Separation and Analysis in a Single Microfluidic Channel

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2012-01-01

    The ability to sort and capture more than one cell type from a complex sample will enable a wide variety of studies of cell proliferation, death, and the analysis of disease states. In this work, we integrated a pneumatic actuated control layer to an affinity separation layer to create different antibody coating regions on the same fluidic channel. The comparison of different antibody capture capabilities to the same cell line was demonstrated by flowing Ramos cells through anti-CD19 and anti-CD71 coated regions in the same channel, respectively. It was determined that cell capture density on anti-CD19 region was 2.44±0.13 times higher than on anti-CD71 coated region. This approach can be used to test different affinity molecules for selectivity and capture efficiency using a single cell line in one separation. Selective capture of Ramos and HuT 78 cells from a mixture was also demonstrated using two antibody regions in the same channel. Greater than 90% purity was obtained on both capture areas in both continuous flow and stop flow separation modes. A four-region antibody coated device was then fabricated to study the simultaneous, serial capture of three different cell lines. In this case the device showed effective capture of cells in a single separation channel, opening up the possibility of multiple cell sorting. Multi-parameter sequential blood sample analysis was also demonstrated with high capture specificity (>97% for both CD19+ and CD4+ leukocytes). The chip can also be used to selectively treat cells after affinity separation. PMID:22958145

  7. [Renaturation with simultaneous purification of the recombinant human Flt3 ligand from inclusion bodies by high performance hydrophobic interaction chromatography].

    PubMed

    Jia, Jia; Wang, Lili; Gao, Dong; Geng, Xindu

    2010-06-01

    Flt3 ligand (FL) is a class of cytokines with the functions of promoting early hematopoiesis. It has important clinical value in promoting growth and development of hematopoietic cells and hematopoietic mobilization. In order to obtain large quantities of recombinant human FL (rhFL) by genetic engineering methods for clinic and research, in this work, rhFL was expressed in E. coli as inclusion bodies. The inclusion bodies were recovered, cleaned and solubilized in 8 mol/L urea, the solubilized rhFL was renatured by high performance hydrophobic interaction chromatography (HPHIC) with simultaneous purification, the retention feature and renaturation regularity were studied. The results showed that when the denatured protein concentration was 8.51 g/L, and the end group of stationary phase was PEG800, under the conditions of mobile phase of pH 7.0 and with the addition of 4 mol/L urea, 1.8 mmol/L glutathione (GSH) and 0.3 mmol/L oxidative glutathione (GSSG), a mass recovery of 36.9% and a purity of 94.5% were obtained after refolding with simultaneous purification. The obtained rhFL was successfully renatured with simultaneous purification in only one step of HPHIC, and it provided a foundation for the manufacturing of high quality rhFL.

  8. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  9. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  10. Ribonucleic acid purification.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2014-08-15

    Research on RNA has led to many important biological discoveries and improvement of therapeutic technologies. From basic to applied research, many procedures employ pure and intact RNA molecules; however their isolation and purification are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality. The current techniques to isolate and purify RNA molecules still have several limitations and the requirement for new methods able to improve RNA quality to meet regulatory demands is growing. In fact, as basic research improves the understanding of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the purification of biological compounds, requiring high purity degree. In addition, its application in biopharmaceutical manufacturing is well established. This paper discusses the importance and the progress of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In particular and in view of the high specificity, affinity chromatography has been recently applied to RNA purification processes. Accordingly, recent chromatographic investigations based on biorecognition phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential contribution of these methods to overcome the challenges of RNA purification. PMID:24951289

  11. Aqueous two-phase extraction combined with chromatography: new strategies for preparative separation and purification of capsaicin from capsicum oleoresin.

    PubMed

    Zhao, Pei-Pei; Lu, Yan-Min; Tan, Cong-Ping; Liang, Yan; Cui, Bo

    2015-01-01

    Capsaicin was preparatively separated and purified from capsicum oleoresin with a new method combined with aqueous two-phase extraction (ATPE) and chromatography. Screening experiments of ATPE systems containing salts and hydrophilic alcohols showed that potassium carbonate/ethanol system was the most suitable system for capsaicin recovery among the systems considered. Response surface methodology was used to determine an optimized aqueous two-phase system for the extraction of capsaicin from capsaicin oleoresin. In a 20 % (w/w) ethanol/22.3 % (w/w) potassium carbonate system, 85.4 % of the capsaicin was recovered in the top ethanol-rich phase while most oil and capsanthin ester were removed in the interphase. The capsaicinoid extract was then subjected to two chromatographic steps using D101 macroporous resin and inexpensive SKP-10-4300 reverse-phase resin first applied for the purification of capsaicin. After simple optimization of loading/elution conditions for D101 macroporous resin chromatography and SKP-10-4300 reverse-phase resin chromatography, the purities of capsaicin were improved from 7 to 85 %. In the two chromatography processes, the recoveries of capsaicin were 93 and 80 % respectively; the productivities of capsaicin were 1.86 and 4.2 (g capsaicin/L resin) per day respectively. It is worth mentioning that a by-product of capsaicin production was also obtained with a high purity (90 %). PMID:25355002

  12. Aqueous two-phase extraction combined with chromatography: new strategies for preparative separation and purification of capsaicin from capsicum oleoresin.

    PubMed

    Zhao, Pei-Pei; Lu, Yan-Min; Tan, Cong-Ping; Liang, Yan; Cui, Bo

    2015-01-01

    Capsaicin was preparatively separated and purified from capsicum oleoresin with a new method combined with aqueous two-phase extraction (ATPE) and chromatography. Screening experiments of ATPE systems containing salts and hydrophilic alcohols showed that potassium carbonate/ethanol system was the most suitable system for capsaicin recovery among the systems considered. Response surface methodology was used to determine an optimized aqueous two-phase system for the extraction of capsaicin from capsaicin oleoresin. In a 20 % (w/w) ethanol/22.3 % (w/w) potassium carbonate system, 85.4 % of the capsaicin was recovered in the top ethanol-rich phase while most oil and capsanthin ester were removed in the interphase. The capsaicinoid extract was then subjected to two chromatographic steps using D101 macroporous resin and inexpensive SKP-10-4300 reverse-phase resin first applied for the purification of capsaicin. After simple optimization of loading/elution conditions for D101 macroporous resin chromatography and SKP-10-4300 reverse-phase resin chromatography, the purities of capsaicin were improved from 7 to 85 %. In the two chromatography processes, the recoveries of capsaicin were 93 and 80 % respectively; the productivities of capsaicin were 1.86 and 4.2 (g capsaicin/L resin) per day respectively. It is worth mentioning that a by-product of capsaicin production was also obtained with a high purity (90 %).

  13. Preparative purification of five bioactive components from Agrimonia pilosa Ledeb by high-speed counter-current chromatography.

    PubMed

    Wang, Yan; Liu, Mozhen; Zheng, Lingli; Yin, Lianhong; Xu, Lina; Qi, Yan; Ma, Xiaochi; Liu, Kexin; Peng, Jinyong

    2012-08-01

    High-speed counter-current chromatography (HSCCC) coupled with ultraviolet (UV) detection or evaporative light-scattering detection was successfully applied for preparative separation of five bioactive compounds from Agrimonia pilosa Ledeb. In preliminary process, D101 macroporous resin was used to separate the crude extract of the plant and four fractions (20, 40, 50, and 60% aqueous ethanol elutions) were produced. Then, these fractions were directly subjected to HSCCC purification. Five chemicals including taxifolin-3-glucoside (6.4 mg), quercetin-3-rhamnoside (13.0 mg), tiliroside (14.7 mg), agrimonolide (21.4 mg), and tormentic acid (29.8 mg) with the purities of 94.24, 95.37, 97.42, 95.29, and 96.34% were separated from each 200 mg prepared fraction. The purities were analyzed by high-performance liquid chromatography, and the chemical structures of the products were identified by UV detection, mass spectrometry, nuclear magnetic resonance, and the standards. This paper used a simple method to separate five bioactive compounds from A. pilosa Ledeb, and it could provide a new idea for the purification of bioactive compounds from other medicinal plants.

  14. NON-COMPETITIVE PEAK DECAY ANALYSIS OF DRUG-PROTEIN DISSOCIATION BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Jianzhong; Schiel, John E.; Hage, David S.

    2009-01-01

    The peak decay method is an affinity chromatographic technique that has been used to examine the dissociation of solutes from immobilized ligands in the presence of excess displacing agent. However, it can be difficult to find a displacing agent that does not interfere with detection of the eluting analyte. In this study, a non-competitive peak decay method was developed in which no displacing agent was required for analyte elution. This method was evaluated for the study of drug-protein interactions by using it along with high-performance affinity chromatography to measure the dissociation rate constants for R- and S-warfarin from columns containing immobilized human serum albumin (HSA). Several factors were considered in the optimization of this method, including the amount of applied analyte, the column size, and the flow rate. The dissociation rate constants for R- and S-warfarin from HSA were measured at several temperatures by this approach, giving values of 0.56 (± 0.01) and 0.66 (± 0.01) s−1 at pH 7.4 and 37°C. These results were in good agreement with previous values obtained by other methods. This approach is not limited to warfarin and HSA but could be employed in studying additional drug-protein interactions or other systems with weak-to-moderate binding. PMID:19472288