Science.gov

Sample records for affinity chromatography purification

  1. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-04-01

    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.

  2. 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)

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

    PubMed

    Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

    2013-12-01

    The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.

  4. Purification of cytochrome c oxidase by lysine-affinity chromatography.

    PubMed

    Felsch, J; Kotake, S; Copeland, R A

    1992-02-01

    A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.

  5. Purification of baculovirus vectors using heparin affinity chromatography

    PubMed Central

    Nasimuzzaman, Md; Lynn, Danielle; van der Loo, Johannes CM; Malik, Punam

    2016-01-01

    Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses in vivo. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells. Based on the finding that heparan sulfate, which is structurally similar to heparin, is an attachment receptor for baculovirus, we developed a novel scalable baculovirus purification method using heparin-affinity chromatography. Baculovirus supernatants were loaded onto a POROS heparin column, washed to remove unbound materials, and eluted with 1.5 mol/l NaCl, which yielded a recovery of purified baculovirus of 85%. After ultracentrifugation, baculovirus titers increased from 200- to 700-fold with overall yields of 26–29%. We further show that baculovirus particles were infectious, normal in morphology and size, despite high-salt elution and shear forces used during purification and concentration. Our chromatography-based purification method is scalable and, together with ultracentrifugation and/or tangential flow filtration, will be suitable for large-scale manufacturing of baculovirus stocks for protein and vaccine production and in gene therapy applications. PMID:27933303

  6. 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.

  7. Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

    PubMed

    Fleminger, G; Neufeld, T; Star-Weinstock, M; Litvak, M; Solomon, B

    1992-04-24

    The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.

  8. 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.

  9. 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

    2016-04-08

    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.

  10. Polystyrene as an affinity chromatography matrix for the purification of antibodies.

    PubMed

    Staak, C; Salchow, F; Clausen, P H; Luge, E

    1996-08-14

    Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

  11. 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

    2011-02-04

    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.

  12. 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

  13. PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

    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.

  14. 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

  15. 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.

  16. Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

    PubMed

    Loukas, S; Mercouris, M; Panetsos, F; Zioudrou, C

    1994-05-10

    Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa.

  17. Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography

    PubMed Central

    Kuehne, Christian; Wedepohl, Stefanie; Dernedde, Jens

    2017-01-01

    l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin. PMID:28125045

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

    PubMed Central

    Chung, Jinhwa A.; Wollack, James W.; Hovlid, Marisa L.; Okesli, Ayse; Chen, Yan; Mueller, Joachim D.; Distefano, Mark D.; Taton, T. Andrew

    2009-01-01

    Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation. PMID:18834849

  3. Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

    PubMed Central

    Berman, Jonathan Dembitz; Young, Michael

    1971-01-01

    Affinity chromatography has been used to purify acetylcholinesterase both from the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic inhibitors of each protein were synthesized and joined covalently to an insoluble support resin. AchE is selectively retained by such inhibitor-resins when highly impure solutions are chromatographed upon them. After removal from the resin, both enzymes are electrophoretically homogeneous and they may be recovered in yields of 75% or more. Images PMID:5277092

  4. Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

    PubMed

    Baieli, María F; Urtasun, Nicolás; Martinez, María J; Hirsch, Daniela B; Pilosof, Ana M R; Miranda, María V; Cascone, Osvaldo; Wolman, Federico J

    2017-01-01

    Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

  5. [Prospects of application of the chitin-binding domains to isolation and purification of recombinant proteins by affinity chromatography: a review].

    PubMed

    Kurek, D V; Lopatin, S A; Varlamov, V P

    2009-01-01

    Properties of substrate-binding domains, some parameters of affinity sorbents, and a number of other special features that were necessary to take into account during creation of chromatographic system for isolation and purification of proteins with incorporated chitin-binding domain were discussed in this review. This method was shown to be successfully used along with metal-chelate affinity chromatography. The metal-chelate affinity chromatography with the use of polyhistidine peptides as affinity labels is successfully applied to isolation, purification, and investigation of recombinant proteins. However, this system had some disadvantages. At present, scientists attracted more and more attention to substrate-binding domains, including those chitin-binding, because they had a number of advantages being used as affinity label.

  6. Partial purification of the microsomal rat liver iodothyronine deiodinase. II. Affinity chromatography.

    PubMed

    Mol, J A; van den Berg, T P; Visser, T J

    1988-02-01

    Iodothyronine deiodinase has been solubilized and purified approximately 2400 times from liver microsomal fractions of male Wistar rats pretreated with thyroxine. The deiodinase was solubilized with 1% cholate, and stripped of adhering phospholipids by ammonium sulfate precipitation followed by solubilization with the non-ionic detergent Emulgen 911. The enzyme was further purified by successive ion-exchange chromatography on DEAE-Sephacel and Cellex-P and affinity chromatography on 3,3',5-triiodothyronine-Sepharose. Finally, the deiodinase was reacted with 6-propionyl-2-thiouracil-Sepharose, a derivative of the mechanism-based inhibitor 6-propyl-2-thiouracil. Covalent binding was observed only in the presence of substrate in agreement with the proposed mechanism of deiodination. The deiodinase was eluted from the affinity column by reduction of the enzyme-propylthiouracil mixed disulfide with 50 mM dithiothreitol. The enzyme was approximately 50% pure as judged by SDS-PAGE, exhibiting a subunit molecular weight of 25,000. This preparation was equally enriched in outer ring and inner ring deiodinase activities in keeping with the view that both are intrinsic to a single, type I deiodinase.

  7. 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

  8. Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.

    PubMed

    Zhuang, Ran; Zhang, Yuan; Zhang, Rui; Song, Chaojun; Yang, Kun; Yang, Angang; Jin, Boquan

    2008-05-01

    GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.

  9. Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

    PubMed

    Regnault, V; Rivat, C; Vallar, L; Geschier, C; Stolz, J F

    1992-12-11

    The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.

  10. Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

  11. Identification of the Cardiac Beta-Adrenergic Receptor Protein: Solubilization and Purification by Affinity Chromatography

    PubMed Central

    Lefkowitz, Robert J.; Haber, Edgar; O'Hara, Donald

    1972-01-01

    A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified β-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 × 105 liters/mol (as compared to two association constants, 107 and 106 liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for β-adrenergic drugs and antagonists. Images PMID:4507606

  12. Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nomega-homocysteinyl-aminohexyl-Agarose.

    PubMed

    Perła, Joanna; Undas, Anetta; Twardowski, Tomasz; Jakubowski, Hieronim

    2004-08-05

    Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.

  13. 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.

  14. Purification of a lectin from M. rubra leaves using immobilized metal ion affinity chromatography and its characterization.

    PubMed

    Sureshkumar, Thavamani; Priya, Sulochana

    2012-12-01

    Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52 kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7 %. Purified MRL was specific to three sugars, galactose, D-galactosamine and N-acetyl-D-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80 °C) and pH (4-11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe(2+), Ca(2+), Zn(2+), Ni(2+), Mn(2+), Na(+) and K(+), HA activity was reduced to 50 %, whereas the presence of trivalent metal ions (Fe3(+) and Al(3+)) and Cu(2+) did not affect the activity. In the presence of Mg(2+) and Hg(2+), only 25 % of HA activity remained.

  15. Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

    PubMed Central

    Gilbert, H J; Lowe, C R; Drabble, W T

    1979-01-01

    Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. PMID:44191

  16. Purification and characterization of a Cytisus-type Ulex europeus hemagglutinin II by affinity chromatography.

    PubMed

    Konami, Y; Tsuji, T; Matsumoto, I; Osawa, T

    1981-07-01

    Ulex europeus hemagglutinin II [Cytisus-type anti-H(O) hemagglutinin] inhibited most by di-N-acetylchitobiose has been purified by affinity chromatography on a column of chitobiose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. The purified hemagglutinin was homogeneous by ultracentrifugal analysis and gave a single band by electrophoresis on polyacrylamide gel, and had a molecular weight of 105 000 by sedimentation equilibrium and an isoelectric point of pH 6.66. This hemagglutinin was found to be composed of four, apparently identical, subunits of a molecular weight of 25 000 +/- 2 000 by dodecyl sulphate-polyacrylamide gel electrophoresis, and to contain 10.3% carbohydrate in which mannose (3.7%) was the predominant sugar, with smaller amounts of glucose, glucosamine, xylose, fucose and galactose. Amino acid analysis of the purified hemagglutinin II showed a large amount of aspartic acid and serine, but as little as 0.1 mol/100 mol of cystine or methionine could be detected.

  17. Purification of human immunoglobulin G autoantibodies to tumor necrosis factor using affinity chromatography and magnetic separation.

    PubMed

    Sennikov, S V; Golikova, E A; Kireev, F D; Lopatnikova, J A

    2013-04-30

    Autoantibodies to cytokines are important biological effector molecules that can regulate cytokine activities. The aim of the study was to develop a protocol to purify autoantibodies to tumor necrosis factor from human serum, for use as a calibration material to determine the absolute content of autoantibodies to tumor necrosis factor by enzyme-linked immunosorbent assay. The proposed protocol includes a set of affinity chromatography methods, namely, Bio-Gel P6DG sorbent to remove albumin from serum, Protein G Sepharose 4 Fast Flow to obtain a total immunoglobulin G fraction of serum immunoglobulins, and Affi-Gel 15 to obtain specifically antibodies to tumor necrosis factor. The addition of a magnetic separation procedure to the protocol eliminated contaminant tumor necrosis factor from the fraction of autoantibodies to tumor necrosis factor. The protocol generated a pure fraction of autoantibodies to tumor necrosis factor, and enabled us to determine the absolute concentrations of different subclasses of immunoglobulin G autoantibodies to tumor necrosis factor in apparently healthy donors.

  18. 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.

  19. Purification and characterization of two types of Cytisus multiflorus hemagglutinin by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Tsuji, T; Matsumoto, I; Osawa, T

    1983-10-01

    Two hemagglutinins were separated from extracts of Cytisus multiflorus seeds by successive affinity chromatographies on columns of galactose- and di- N-acetylchitobiose-Sepharose 4B. One was found to be inhibited by di- N-acetylchitobiose or tri- N-acetylchitotriose and shown to possess anti-H(O) activity [Cytisus-type anti-H(O) hemagglutinin designated as Cytisus multiflorus hemagglutinin I]. The other, which was not a blood group-specific hemagglutinin, was inhibited by galactose or lactose (hemagglutinin II). Hemagglutinins I and II were further purified by gel filtration on Sephacryl S-300. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular weights of the purified hemagglutinins I and II were found to be 86000 by sedimentation equilibrium analysis and 80000 by gel filtration. On disc gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, both hemagglutinins gave a single component of a molecular weight of 42000 +/- 2000, suggesting that these hemagglutinins are dimeric proteins of two identical subunits. Hemagglutinins I and II contain 2.7% and 1.5% carbohydrate, respectively, and only very small amounts of cystine and methionine were detected, but they are rich in aspartic acid and serine. Treatment of human O erythrocytes with a purified H-decomposing enzyme (alpha-L-fucosidase from Bacillus fulminans abolished the agglutinability of the cells with hemagglutinin I. This indicates that the L-fucosyl residue is important even for the H-specificity detected by this di-N-acetylchitobiose-specific hemagglutinin I.

  20. Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification.

    PubMed

    Arica, M Yakup; Yilmaz, Meltem; Yalçin, Emine; Bayramoğlu, Gülay

    2004-06-15

    Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.

  1. Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase.

    PubMed Central

    Srivastava, P N; Farooqui, A A

    1979-01-01

    Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C. Images Fig. 4. PMID:540029

  2. Purification of modified mycobacterial A60 antigen by affinity chromatography and its use for rapid diagnostic tuberculosis infection.

    PubMed

    Yari, Sh; Hadizadeh Tasbiti, A; Fateh, A; Karimi, A; Yari, F; Sakhai, F; Ghazanfari, M; Bahrmand, A

    2011-11-01

    Tuberculosis has been declared a global emergency. The mainstay for its control is the rapid and accurate identification of infected individual. Antibodies to A60, one of the macromolecular antigen complexes of mycobacteria were commonly used in the rapid detection of Mycobacterium tuberculosis. The aim of this study was to prepare specific antibodies against A60 for detection of tuberculosis infection. Specific polyclonal antibodies against A60, (A60-Ab) were prepared in rabbits using 2 boosted injections of the antigen (A60). The antibodies were purified and treated with normal oral flora to remove any non-specific and cross-reactive antibodies. These antibodies were conjugated to CNBr-activated Sepharose 4B and used to isolate subunits of A60 with more specificity for M. tuberculosis. A new affinity column was designed to prepare modified (purified) A60 antigen. Purified A60 antigen (PA60-Ag) was used to develop antibody production by Immunoaffinity chromatography. 113 patients with a confirmed diagnosis of pulmonary TB at Pasteur Institute were selected for the study. The specificity of the results was analyzed with TB-rapid test by using PA60-antibodies. TB-rapid test revealed that normal oral flora-absorbed antibodies could lead to more specific results than that of the non-absorbed antibodies. The developed, modified A60 antibodies, (PA60-Ab)-rapid test showed higher sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and overall efficiency (93.0%, 86.0%, 90.0%, 91.0%, and 90.0% respectively) for the detection of the Mycobacterium antigen. Moreover, PA60-Ag showed only two protein bands of molecular weight 45 and 66kDa in SDS-PAGE while untreated A60 showed multiple bands. Thus, our study helped in the purification of a novel and well characterized A60 antigen and good diagnostic potential for detecting tuberculosis infection.

  3. Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

    PubMed

    Chaga, G; Hopp, J; Nelson, P

    1999-02-01

    A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins.

  4. Chromatography on DEAE ion-exchange and Protein G affinity columns in tandem for the separation and purification of proteins.

    PubMed

    Qi, Y; Yan, Z; Huang, J

    2001-10-30

    A high-performance liquid-chromatographic method based on coupled DEAE anion-exchange and Protein G affinity columns has been developed for the simultaneous separation and purification of immunoglobulin G and albumin from mouse serum. The diluted mouse serum was injected directly into this system, and the proteins were eluted separately from the DEAE and Protein G columns, coupled in series, by the column-switching technique. The advantages of this method are that IgG and albumin can be separated and purified simultaneously, the expensive affinity column is protected from contamination by the impurities in the mouse serum, and it is fast, selective, robust, and reproducible.

  5. Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue.

    PubMed

    Wilson, J E

    1989-01-01

    The mitochondrial hexokinase from rat brain, selectively released from mitochondria by the action of glucose 6-phosphate, can be purified to greater than 90% homogeneity by a single affinity chromatography step on Affi-Gel Blue; the Cibacron Blue F3GA ligand bound to this matrix serves as an analog of ATP, the normal substrate for the enzyme, and selective elution is accomplished using glucose 6-phosphate which is a competitive ligand vs. ATP. With this and other modifications to the previously described procedure highly purified enzyme is readily obtained in good yield and with retention of the ability to rebind to mitochondria.

  6. Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins).

    PubMed Central

    Haddad, J G; Kowalski, M A; Sanger, J W

    1984-01-01

    The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) has been isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal-muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (approximately 58%) of purified binding protein was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialysed and analysed. These eluates contained the binding protein as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step also was effective in the isolation of the binding protein from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analysed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified binding protein which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein. Images Fig. 1. Fig. 3. Fig. 4. PMID:6547042

  7. 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

  8. Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

    PubMed

    Merten, O W; Reiter, S; Scheirer, W; Katinger, H

    1983-01-01

    The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

  9. Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography1

    PubMed Central

    Bonza, Cristina; Carnelli, Antonella; De Michelis, Maria Ida; Rasi-Caldogno, Franca

    1998-01-01

    The Ca2+-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca2+ concentrations, the Ca2+-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca2+-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca2+-ATPase by 125I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca2+-ATPase cross-reacted with an antiserum against a putative Ca2+-ATPase of the Arabidopsis thaliana chloroplast envelope. PMID:9490776

  10. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  11. Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.

  12. 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

  13. 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.

  14. Immobilized metal affinity chromatography in open-loop simulated moving bed technology: purification of a heat stable histidine tagged beta-glucosidase.

    PubMed

    Sahoo, Deepti; Andersson, Jonatan; Mattiasson, Bo

    2009-06-01

    Open-loop simulated moving bed (SMB) has been used for immobilized metal affinity chromatographic (IMAC) purification of his-tagged beta-glucosidase expressed in E. coli. A simplified approach based on an optimized single column protocol is used to design the open-loop SMB. A set of columns in the SMB represent one step in the chromatographic cycle i.e. there will be one set each of columns for load, wash, elution etc within the SMB. Only the wash and elution are operated with columns in sequence. The beta-glucosidase was purified to almost single band purity with a purification factor of 15 and a recovery of 91%. SMB-performance showed reduced buffer consumption, higher purification fold, a better yield and higher productivity.

  15. 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.

  16. Extension of the selection of protein chromatography and the rate model to affinity chromatography.

    PubMed

    Sandoval, G; Shene, C; Andrews, B A; Asenjo, J A

    2010-01-01

    The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.

  17. 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.

  18. 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)

  19. Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.

    PubMed

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

    2011-07-01

    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.

  20. Biomimetic affinity ligands for protein purification.

    PubMed

    Sousa, Isabel T; Taipa, M Angela

    2014-01-01

    The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

  1. Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis.

    PubMed

    Scopes, R K

    1984-02-01

    2-Keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) has been isolated from extracts of Zymomonas mobilis using differential dye-ligand chromatography and affinity elution with product/product analog. The one-step procedure gives an enzyme with specific activity 600 units mg-1. Only 1 out of 47 dyes, Procion Yellow MX-GR, bound the enzyme completely in 20 mM phosphate buffer, pH 6.5. A column of Navy HE-R adsorbent was used first to remove most of the potentially adsorbing proteins.

  2. [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.

  3. 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.

  4. Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library.

    PubMed

    Smith, Richard G; Missailidis, Sotiris; Price, Michael R

    2002-01-05

    A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.

  5. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2012-01-01

    The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.

  6. Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligand.

    PubMed

    Baieli, María Fernanda; Urtasun, Nicolás; Miranda, María Victoria; Cascone, Osvaldo; Wolman, Federico Javier

    2014-03-01

    The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.

  7. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    PubMed

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  8. Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA.

    PubMed

    Sousa, Fani; Prazeres, Duarte M F; Queiroz, João A

    2008-09-01

    The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids.

  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. Purification of polyclonal antibodies from Cohn fraction II + III, skim milk, and whey by affinity chromatography using a hexamer peptide ligand.

    PubMed

    Menegatti, Stefano; Naik, Amith D; Gurgel, Patrick V; Carbonell, Ruben G

    2012-11-01

    HWRGWV, a peptide that binds specifically to the Fc fragment of human immunoglobulin G (IgG), was used for the purification of IgG from Cohn fraction II + III of human plasma and from bovine skim milk and whey. The concentration of sodium chloride and sodium caprylate in the binding buffer as well as the pH of the elution buffer were optimized to achieve high IgG yield and purity. Under optimized conditions, IgG was recovered from plasma fractions with yield and purity up to 84% and 95%, respectively. IgG was also purified from skim milk with 74% yield and 92% purity and from whey with 85% yield and 93% purity. Purification experiments were also performed with Protein A resin and the results were found to be similar to those obtained with the peptide adsorbent.

  11. An inducible expression system of histidine-tagged proteins in Streptomyces lividans for one-step purification by Ni2+ affinity chromatography.

    PubMed

    Enguita, F J; de la Fuente, J L; Martín, J F; Liras, P

    1996-04-01

    An expression and purification cassette containing the aminoglycoside phosphotransferase gene (aph) as selective marker has been constructed in the Escherichia coli vector pULHis2. DNA fragments inserted in the cassette can be easily subcloned in pIJ699 to give vectors for overexpression of genes in Streptomyces and purification of proteins by a one-step procedure. The expression system uses the thiostrepton-inducible promoter tipA for expression and a six histidine coding nucleotide sequence that is fused in frame to the foreign gene inserted in the polylinker. The pULHis2-derived expression vector has been used satisfactorily to express and to purify the P7 and P8 proteins of Nocardia lactamdurans which carry out the methoxylation of cephalosporin C to 7-methoxycephalosporin C.

  12. Generation of an affinity column for antibody purification by intein-mediated protein ligation.

    PubMed

    Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2003-11-01

    Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

  13. 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.

  14. Solid support resins and affinity purification mass spectrometry.

    PubMed

    Havis, Spencer; Moree, Wilna J; Mali, Sujina; Bark, Steven J

    2017-02-28

    Co-affinity purification-mass spectrometry (CoAP-MS) is a primary technology for elucidating the protein-protein interactions that form the basis of all biological processes. A critical component of CoAP-MS is the affinity purification (AP) of the bait protein, usually by immobilization of an antibody to a solid-phase resin. This Minireview discusses common resins, reagents, tagging methods, and their consideration for successful AP of tagged proteins. We discuss our experiences with different solid supports, their impact in AP experiments, and propose areas where chemistry can advance this important technology.

  15. Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

    2014-09-07

    In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

  16. Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Alves, Nathan J; Bilgicer, Basar

    2013-05-21

    This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

  17. 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.

  18. A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart.

    PubMed

    Hermida, L; Rodríguez, R; Lazo, L; López, C; Márquez, G; Páez, R; Suárez, C; Espinosa, R; García, J; Guzmán, G; Guillén, G

    2002-03-28

    Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice. Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC). The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation. IMAC could be used as an alternative for high scale purification.

  19. 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.

  20. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu

    2014-09-12

    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.

  1. Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers.

    PubMed

    Hodson, B A; Pepper, D S; Dawes, J

    1991-04-19

    A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.

  2. Purification of matrix metalloproteinases by column chromatography.

    PubMed

    Imai, Kazushi; Okada, Yasunori

    2008-01-01

    Matrix metalloproteinases (MMPs) are zinc endopeptidases composed of 23 members in humans, which belong to a subfamily of the metzincin superfamily. They play important roles in many pathophysiological events including development, organogenesis, angiogenesis, tissue remodeling and destruction, and cancer cell proliferation and progression by degradation of extracellular matrix (ECM) and non-ECM proteins and interaction with various molecules. Here, we present standard protocols for purification of native proMMPs (proMMP-1, -2, -3, -7, -9 and -10) and recombinant MT1-MMP (MMP-14) using conventional column chromatography. Purification steps comprise the initial common step [diethylaminoethyl (DEAE)-cellulose, Green A Dyematrex gel and gelatin-Sepharose columns], the second step for removal of nontarget proMMPs by immunoaffinity columns (anti-MMP-1 and/or anti-MMP-3 IgG-Sepharose columns) and the final step for further purification (IgG-Sepharose, DEAE-cellulose, Zn2+-chelate-Sepharose and/or gel filtration columns). Purified proMMPs and MMP are functionally active and suitable for biochemical analyses. The basic protocol for the purification from culture media takes approximately 7-10 d.

  3. 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.

  4. 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.

  5. [Separation of osteoclasts by lectin affinity chromatography].

    PubMed

    Itokazu, M; Tan, A; Tanaka, S

    1991-09-01

    Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

  6. 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.

  7. 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).

  8. Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

    PubMed

    Qureshi, M S; Sheikh, Q I; Hill, R; Brown, P E; Dickman, M J; Tzokov, S B; Rice, D W; Gjerde, D T; Hornby, D P

    2013-08-01

    The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

  9. Characterization and affinity purification of juvenile hormone esterase from Bombyx mori.

    PubMed

    Shiotsuki, T; Bonning, B C; Hirai, M; Kikuchi, K; Hammock, B D

    2000-08-01

    Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.

  10. 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.

  11. Tandem affinity purification vectors for use in gram positive bacteria.

    PubMed

    Yang, Xiao; Doherty, Geoff P; Lewis, Peter J

    2008-01-01

    Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 degrees C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3xFLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product.

  12. False positive RNA binding activities after Ni-affinity purification from Escherichia coli.

    PubMed

    Milojevic, Tetyana; Sonnleitner, Elisabeth; Romeo, Alessandra; Djinović-Carugo, Kristina; Bläsi, Udo

    2013-06-01

    A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

  13. 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).

  14. Synthesis and characterisation of magnetised Dacron-heparin composite employed for antithrombin affinity purification.

    PubMed

    Mercês, Aurenice Arruda Dutra das; Silva, Ricardo de Souza; Silva, Karciano José Santos; Maciel, Jackeline da Costa; Oliveira, Givanildo Bezerra; Buitrago, Davian Martinez; de Aguiar, José Albino Oliveira; de Carvalho-Júnior, Luiz Bezerra

    2016-12-01

    Human antithrombin is a blood derivative widely used in the treatment of coagulation dysfunction. Affinity chromatography using heparin (HEP) derivatives is usually used for antithrombin purification. In this study, an affinity procedure based on a magnetic Dacron-HEP composite is proposed. Dacron was firstly converted to Dacron-hydrazide and magnetised by co-precipitation with of Fe(2+)/Fe(3+) (mDAC). HEP was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). EDX and infrared spectra analyses confirmed each synthesis step of mDAC-HEP. This composite exhibited superparamagnetism behaviour. Human plasma was incubated with mDAC-HEP (fresh and stored over a long period) and washed with phosphate buffer containing increasing concentrations of NaCl. Human plasma antithrombin activity was reduced by approximately 20% in the presence of the 1.0M NaCl fraction, and this eluate was able to prolong coagulation time (aPTT) using both preparations. Electrophoresis of the eluates revealed bands corresponding to the expected size of antithrombin (58kDa). The mDAC-HEP particles are reusable. This method presents the following advantages: easy, low-cost synthesis of the composite, magnet-based affinity purification steps, and reusability.

  15. DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

    PubMed

    Forier, Cynthia; Boschetti, Egisto; Ouhammouch, Mohamed; Cibiel, Agnès; Ducongé, Frédéric; Nogré, Michel; Tellier, Michel; Bataille, Damien; Bihoreau, Nicolas; Santambien, Patrick; Chtourou, Sami; Perret, Gérald

    2017-03-17

    Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.

  16. Highly efficient and low-cost purification of lysozyme: a novel tris(hydroxymethyl)aminomethane immobilized affinity column.

    PubMed

    Quan, Li; Cao, Qing; Li, Zhiyu; Li, Na; Li, Kean; Liu, Feng

    2009-03-01

    A highly efficient and low-cost affinity chromatography strategy for lysozyme (LZM) purification is reported. Using tris(hydroxymethyl)aminomethane (Tris) as ligand and macroporous silica spheres as matrix, a novel affinity column was prepared. The high specificity, stability and repeatability of this Tris immobilized affinity column were proved by LZM separations from protein mixture solutions for 20 circles and 6 months test. LZM purified from chicken egg white on the Tris affinity column had even higher purity than the commercial standard and well-maintained activity of 8287 U/mg (activity of commercial LZM was 8171 U/mg). The efficient affinity process avoiding expensive or fragile ligand would bring advantages to the routine production of LZM from chicken egg white.

  17. Cation exchange displacement batch chromatography of proteins guided by screening of protein purification parameters.

    PubMed

    Kotasińska, Marta; Richter, Verena; Thiemann, Joachim; Schlüter, Hartmut

    2012-11-01

    Displacement chromatography has been shown to be an effective alternative for protein purification. We investigated in this study sample displacement chromatography, which does not require a displacer molecule. Furthermore, we performed a screening for determination of parameters for an optimal sample displacement chromatography. We screened the affinities of cytochrome C, lysozyme, myoglobin, and ribonuclease A toward a cation exchange material as a function of different pH values and to presence of different concentrations of sodium chloride in the sample application buffer. Sample displacement chromatography in batch chromatography mode for the separation of the protein mixture was studied with a sample application buffer with a pH of 5 and 7. As predicted by the screening experiments, sample displacement chromatography was most effective at pH 7 since this pH guaranteed the largest differences of the affinities of the four proteins toward the stationary phase. In summary, we describe here sample displacement chromatography in the batch chromatography mode for the separation of proteins, which is a simple and fast alternative to conventional displacement chromatography. Systematic screening of chromatographic parameters prior to sample displacement chromatography promises a successful separation of a target protein.

  18. 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.

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

    PubMed

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

    2013-11-14

    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.

  20. 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.

  1. 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

    2005-10-05

    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).

  2. 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

  3. Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

    PubMed

    Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

    To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.

  4. Affinity purification of copper-chelating peptides from sunflower protein hydrolysates.

    PubMed

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

    2007-08-08

    Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. A new affinity approach to isolate Escherichia coli 6S RNA with histidine-chromatography.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2010-01-01

    6S RNA is an abundant non-coding RNA in Escherichia coli (E. coli), but its function has not been discovered until recently. The first advance on 6S RNA function was the demonstration of its ability to bind the σ(70)-holoenzyme form of RNA polymerase, inhibiting its activity and consequently the transcription process. The growing interest in the investigation of non-coding small RNAs (sRNA) calls for the development of new methods for isolation and purification of RNA. This work presents an optimized RNA extraction procedure and describes a new affinity chromatography method using a histidine support to specifically purify 6S RNA from other E. coli sRNA species. The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt). This improved method takes advantage of its simplicity and significant cost reduction, since some complex operations have been eliminated. A purification strategy was also developed to separate 6S RNA from an sRNA mixture. Pure RNA can be advantageously obtained using the histidine-affinity chromatography method, aiming at its application to structural or functional studies.

  10. Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

    PubMed

    Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

    2017-01-01

    In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10(5) U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

  11. DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin.

    PubMed

    Werner, P A; Galbraith, R M; Arnaud, P

    1983-10-01

    Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

  12. 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.

  13. Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure.

    PubMed

    Brazzolotto, Xavier; Wandhammer, Marielle; Ronco, Cyril; Trovaslet, Marie; Jean, Ludovic; Lockridge, Oksana; Renard, Pierre-Yves; Nachon, Florian

    2012-08-01

    Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ≈ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 Å. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.

  14. Generation of metastatic melanoma specific antibodies by affinity purification

    PubMed Central

    Schütz, Birgit; Koppensteiner, Anita; Schörghofer, David; Kinslechner, Katharina; Timelthaler, Gerald; Eferl, Robert; Hengstschläger, Markus; Missbichler, Albert; Hundsberger, Harald; Mikula, Mario

    2016-01-01

    Melanoma is the most aggressive type of skin cancer and one of the most frequent tumours in young adults. Identification of primary tumours prone to develop metastasis is of paramount importance for further patient stratification. However, till today, no markers exist that are routinely used to predict melanoma progression. To ameliorate this problem, we generated antiserum directed against metastatic melanoma tissue lysate and applied a novel approach to purify the obtained serum via consecutive affinity chromatography steps. The established antibody, termed MHA-3, showed high reactivity against metastatic melanoma cell lines both in vitro and in vivo. We also tested MHA-3 on 227 melanoma patient samples and compared staining with the melanoma marker S100b. Importantly, MHA-3 was able to differentiate between metastatic and non-metastatic melanoma samples. By proteome analysis we identified 18 distinct antigens bound by MHA-3. Combined expression profiling of all identified proteins revealed a significant survival difference in melanoma patients. In conclusion, we developed a polyclonal antibody, which is able to detect metastatic melanoma on paraffin embedded sections. Hence, we propose that this antibody will represent a valuable additional tool for precise melanoma diagnosis. PMID:27853253

  15. Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing.

    PubMed

    Hou, Ying; Brower, Mark; Pollard, David; Kanani, Dharmesh; Jacquemart, Renaud; Kachuik, Bradley; Stout, James

    2015-01-01

    Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput.

  16. 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.

  17. A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins.

    PubMed

    Huang, Yongdong; Zhang, Rongyue; Li, Juan; Li, Qiang; Su, Zhiguo; Ma, Guanghui

    2014-03-01

    Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100-500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.

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

    PubMed

    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.

  19. [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.

  20. 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

  1. Affinity partitioning of restriction endonucleases. Application to the purification of EcoR I and EcoR V.

    PubMed

    Vlatakis, G; Bouriotis, V

    1991-02-01

    Partitioning of restriction endonucleases between two liquid aqueous phases can be strongly influenced by group-specific ligands included in the two-phase system. Three restriction endonucleases, namely EcoR I, EcoR V and BamH I, were partitioned within an aqueous dextran-polyethylene glycol (PEG) system. The enzymes could be extracted into the upper PEG phase by using either triazine dyes or herring DNA as affinity ligands. The influence of the endogenous bacterial nucleic acids, concentration of polymerbound dye and concentration of sodium chloride on the system were examined. A partial purification of EcoR I (up to 52-fold) and EcoR V (up to 37-fold) was achieved using a combination of affinity partitioning and ion-exchange chromatography, providing an extremely fast and economical method for the isolation of restriction endonucleases free from contaminating nuclease activities.

  2. 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.

  3. Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro.

    PubMed

    Hou, Shuai; Shi, Lei; Lei, Haixin

    2016-01-01

    RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.

  4. 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.

  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.

  6. 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.

  7. Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica.

    PubMed Central

    Luaces, A L; Barrett, A J

    1988-01-01

    We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion. Images Fig. 2. Fig. 4. PMID:2898937

  8. Fractionation of the genetic variants of human alpha 1-acid glycoprotein in the native form by chromatography on an immobilized copper(II) affinity adsorbent. Heterogeneity of the separate variants by isoelectrofocusing and by concanavalin A affinity chromatography.

    PubMed

    Hervé, F; Gomas, E; Duché, J C; Tillement, J P

    1993-05-19

    Fractionation of the three main genetic variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), in their native (sialylated) form, by chromatography on immobilized copper(II) affinity adsorbent was investigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had been previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different samples on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two protein peaks, irrespective of the origin of the native AAG sample used. The unbound peak 1 was found to consist of the F1, the S or both variants, depending on the phenotype of the AAG sample used in the chromatography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were found to be the same as those originally yielded by the desialylated protein. However, comparison of the interactions of native and desialylated AAG with immobilized copper(II) ions, using an affinity chromatographic method and a non-chromatographic equilibrium binding technique, respectively, showed that desialylation increased the non-specific interactions of the protein with immobilized copper(II) ions. The AAG variants were not fractionated when affinity chromatography was performed using immobilized zinc, nickel or cobalt(II) ions, instead of copper. After purification of each variant in the sialylated form (F1, S and A), their respective heterogeneity was studied by analytical isoelectrofocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants was investigated by affinity chromatography on immobilized concanavalin A.

  9. 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.

  10. An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase.

    PubMed Central

    Thomassin, H; Jacobson, M K; Guay, J; Verreault, A; Aboul-ela, N; Menard, L; Poirier, G G

    1990-01-01

    The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydroxyboronyl sepharose. An application is described for the purification of poly(ADP-ribose) glycohydrolase from calf thymus in which a single rapid affinity step was used to replace 3 column chromatographic steps yielding enzyme of greater than 90% purity with a 3 fold increase in yield. This matrix should also prove useful for other studies of ADP-ribose polymer metabolism and related clinical conditions. Images PMID:2395636

  11. Virus inactivation by protein denaturants used in affinity chromatography.

    PubMed

    Roberts, Peter L; Lloyd, David

    2007-10-01

    Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.

  12. 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.

  13. Use of immunoaffinity chromatography for purification of /sup 125/I-labeled human prolactin

    SciTech Connect

    Stuart, M.C.; Boscato, L.M.; Underwood, P.A.

    1983-02-01

    Researchers assessed a simple method for purifying /sup 125/I-labeled human prolactin, taking advantage of the abundant supplies of monoclonal antibodies available. /sup 125/I-labeled human prolactin purified by immunoaffinity chromatography is compared with that purified by gel filtration on Sephadex G-100. Researchers used monoclonal antibodies to prolactin to prepare the affinity chromatography columns. Prolactin was radiolabeled by the Chloramine T method, purified by each of the above procedures, and the binding and displacement characteristics were studied in radioimmunoassays in which either monoclonal antibodies or a rabbit anti-prolactin serum was the first antibody. A nonimmune fraction of /sup 125/I-labeled prolactin that co-eluted with the immunoreactive hormone from Sephadex G-100 was removed by affinity chromatography, which increased the antibody binding of /sup 125/I-labeled prolactin in the radioimmunoassay in the absence of unlabeled antigen (B/T0, in percent) twofold or more, increased the assay sensitivity, and increased the slope of antigen displacement measured by the 50% intercept. Several advantages make this the purification method of choice.

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

    PubMed

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

    2011-09-21

    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

  15. [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%.

  16. Preparation of multiprotein complexes from Arabidopsis chloroplasts using tandem affinity purification.

    PubMed

    Andrès, Charles; Agne, Birgit; Kessler, Felix

    2011-01-01

    Since its first description in 1998 (Rigaut et al., Nat Biotech 17:1030-1032, 1999), the TAP method, for Tandem Affinity Purification, has become one of the most popular methods for the purification of in vivo protein complexes and the identification of their composition by subsequent mass spectrometry analysis. The TAP method is based on the use of a tripartite tag fused to a target protein expressed in the organism of interest. A TAP tag has two independent binding regions separated by a protease cleavage site, and therefore allows two successive affinity purification steps. The most common TAP tag consists of two IgG binding repeats of Protein A from Staphylococcus aureus (ProtA) separated from a calmodulin-binding peptide by a Tobacco Etch Virus (TEV) protease cleavage site. Using the TAP method, native protein complexes can be purified efficiently with a reduced contaminant background when compared to single step purification methods. Initially developed in the yeast model system, the TAP method has been adapted to most common model organisms. The first report of the purification of protein complexes from plant tissue by the TAP method was published in 2004 by Rohila et al. (Plant J 38:172-181, 2004). The synthetic TAP tag gene described in this study has been optimized for use in plants, and since then, has been successfully used from single gene analyses to high-throughput studies of whole protein families (Rohila et al., PLoS ONE 4:e6685, 2009). Here, we describe a TAP tag purification method for the purification of protein complexes from total Arabidopsis extracts, that we employed successfully using a TAP-tagged chloroplast outer envelope protein.

  17. Two-step chromatography purification of IgGs possessing sialidase activity from human blood serum.

    PubMed

    Kit, Yury; Bilyy, Rostyslav; Korniy, Nataliya; Tomin, Andriy; Chop'yak, Valentyna; Tolstyak, Yaroslav; Antonyuk, Volodymyr; Stoika, Rostyslav

    2015-03-01

    Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti-inflammatory properties. Recently, we have found for the first time IgG-antibodies possessing sialidase-like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G-Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin-Sepharose). This matrix preferentially binds sialidase-like IgGs from a pool of sialidase-active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double-step chromatography purification of sialidase-like IgGs from human blood serum.

  18. New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes.

    PubMed

    Melissis, S C; Rigden, D J; Clonis, Y D

    2001-05-11

    Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg

  19. Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.

    PubMed

    Graumann, Johannes; Dunipace, Leslie A; Seol, Jae Hong; McDonald, W Hayes; Yates, John R; Wold, Barbara J; Deshaies, Raymond J

    2004-03-01

    A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT.

  20. Protein purification using chromatography: selection of type, modelling and optimization of operating conditions.

    PubMed

    Asenjo, J A; Andrews, B A

    2009-01-01

    To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (alpha-lactoalbumin, ovalbumin and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical

  1. 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.

  2. Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

    PubMed

    Abdelhamid, Mohamed A A; Ikeda, Takeshi; Motomura, Kei; Tanaka, Tatsuya; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

    2016-11-01

    We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification.

  3. Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes

    SciTech Connect

    Kanngiesser, U.; Nalik, P.; Pongs, O.

    1988-05-01

    Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca/sup 2 +/ channel) has been purified as a 340-kDa protein complex to approx.80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two step-chromatographic purification procedure. The equilibrium dissociation constant of (/sup 3/H) (+) -PN200-110 binding (K/sub d/; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approx.300 kDa. The 195-kDa subunit was affinity-labeled with (/sup 3/H)azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).

  4. 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.

  5. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.

    PubMed

    Mellacheruvu, Dattatreya; Wright, Zachary; Couzens, Amber L; Lambert, Jean-Philippe; St-Denis, Nicole A; 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

    2013-08-01

    Affinity purification coupled with mass spectrometry (AP-MS) is 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 (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. 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 its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

  6. Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

    PubMed

    Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

    2014-02-01

    Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis.

  7. A safe, effective, and facility compatible cleaning in place procedure for affinity resin in large-scale monoclonal antibody purification.

    PubMed

    Wang, Lu; Dembecki, Jill; Jaffe, Neil E; O'Mara, Brian W; Cai, Hui; Sparks, Colleen N; Zhang, Jian; Laino, Sarah G; Russell, Reb J; Wang, Michelle

    2013-09-20

    Cleaning-in-place (CIP) for column chromatography plays an important role in therapeutic protein production. A robust and efficient CIP procedure ensures product quality, improves column life time and reduces the cost of the purification processes, particularly for those using expensive affinity resins, such as MabSelect protein A resin. Cleaning efficiency, resin compatibility, and facility compatibility are the three major aspects to consider in CIP process design. Cleaning MabSelect resin with 50mM sodium hydroxide (NaOH) along with 1M sodium chloride is one of the most popular cleaning procedures used in biopharmaceutical industries. However, high concentration sodium chloride is a leading cause of corrosion in the stainless steel containers used in large scale manufacture. Corroded containers may potentially introduce metal contaminants into purified drug products. Therefore, it is challenging to apply this cleaning procedure into commercial manufacturing due to facility compatibility and drug safety concerns. This paper reports a safe, effective and environmental and facility-friendly cleaning procedure that is suitable for large scale affinity chromatography. An alternative salt (sodium sulfate) is used to prevent the stainless steel corrosion caused by sodium chloride. Sodium hydroxide and salt concentrations were optimized using a high throughput screening approach to achieve the best combination of facility compatibility, cleaning efficiency and resin stability. Additionally, benzyl alcohol is applied to achieve more effective microbial control. Based on the findings, the recommended optimum cleaning strategy is cleaning MabSelect resin with 25 mM NaOH, 0.25 M Na2SO4 and 1% benzyl alcohol solution every cycle, followed by a more stringent cleaning using 50 mM NaOH with 0.25 M Na2SO4 and 1% benzyl alcohol at the end of each manufacturing campaign. A resin life cycle study using the MabSelect affinity resin demonstrates that the new cleaning strategy

  8. 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.

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

    PubMed

    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----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----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 3200 pmol of [3H]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. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

  10. 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

  11. 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.

  12. A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.

    PubMed

    Honey, S; Schneider, B L; Schieltz, D M; Yates, J R; Futcher, B

    2001-02-15

    A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

  13. NADP-specific isocitrate dehydrogenase of Escherichia coli. IV. Purification by chromatography on Affi-Gel Blue.

    PubMed

    Vasquez, B; Reeves, H C

    1979-05-23

    Affinity chromatography on Affi-Gel Blue has been used to purify the NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) from Escherichia coli. The protocol permits rapid purification of the enzyme in milligram quantities with a yield of 50% and is carried out almost entirely at room temperature. The preparation was judged to be homogeneous by non-denaturing electrophoresis at pH 7.5 and denaturing electrophoresis in the presence of sodium dodecyl sulfate. The subunit molecular weight of 53 000, determined by sodium dodecyl sulfate gel electrophoresis, is in reasonable agreement with the value of 46 900 estimated from the amino acid composition data.

  14. Development of an affinity cryogel for one step purification of lysozyme from chicken egg white.

    PubMed

    Mól, Paula Chequer Gouveia; Veríssimo, Lizzy Ayra Alcântara; Eller, Monique Renon; Minim, Valéria Paula Rodrigues; Minim, Luis Antonio

    2017-02-15

    In this study, a supermacroporous polyacrylamide cryogel was produced by cryo-polymerization and activated with Tris(hydroxymethyl)aminomethane (Tris-cryogel) to be applied as an affinity ligand for a one step purification of lysozyme (LYZ), directly from chicken egg white (EW). The Tris-cryogel presented interconnected pores with size varying in the range of 20-80μm and swelling capacity of 19.6±0.9g/g. The axial dispersion of the Tris-cryogel was analyzed at different flow velocities and mobile phase viscosities. It was verified that higher viscosity resulted in a higher degree of dispersion, causing the HETP values to increase from 0.04cm to 0.8cm. Adsorption isotherms were measured at 15°C and 35°C at pH 7.5. A Langmuir model was fitted to the equilibrium data, with a maximum adsorptive capacity of 285mg/g at 15°C and 363mg/g at 35°C. Thermodynamic analysis based on the Van't Hoff relationship showed that the process was spontaneous and enthalpically driven. Lysozyme was purified directly from egg white in a one step purification process at different pH values (7.5, 8.5 and 9.5). Independent of the pH, the specificity of Tris-cryogel for lysozyme adsorption was confirmed. At pH 7.5, yield and purification fold were higher (30% and 45). In addition, the effect of the dilution rate on egg white and flow velocity were also analyzed and it was shown that flow velocity did not affected purification and column efficiency, and that diluting the egg white increased yield to 70% with a purification fold of 23. Results show Tris-cryogel is a promising matrix for use in high throughput purification of lysozyme from egg white.

  15. 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)

  16. Efficient wheat germ agglutinin purification with a chitosan-based affinity chromatographic matrix.

    PubMed

    Baieli, María F; Urtasun, Nicolás; Miranda, María V; Cascone, Osvaldo; Wolman, Federico J

    2012-01-01

    An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini-spheres cross-linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA - calculated from the corresponding isotherms - was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini-spheres cross-linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP-HPLC was developed.

  17. 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

  18. Observations on different resin strategies for affinity purification mass spectrometry of a tagged protein.

    PubMed

    Mali, Sujina; Moree, Wilna J; Mitchell, Morgan; Widger, William; Bark, Steven J

    2016-12-15

    Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS.

  19. Purification of Capping Protein Using the Capping Protein Binding Site of CARMIL as an Affinity Matrix

    PubMed Central

    Remmert, Kirsten; Uruno, Takehito; Hammer, John A.

    2009-01-01

    Capping Protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function and regulation. PMID:19427903

  20. Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix.

    PubMed

    Remmert, Kirsten; Uruno, Takehito; Hammer, John A

    2009-10-01

    Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Protein purification-free method of binding affinity determination by microscale thermophoresis.

    PubMed

    Khavrutskii, Lyuba; Yeh, Joanna; Timofeeva, Olga; Tarasov, Sergey G; Pritt, Samuel; Stefanisko, Karen; Tarasova, Nadya

    2013-08-15

    Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

  6. A benzoboroxole-based affinity ligand for glycoprotein purification at physiological pH.

    PubMed

    Rowe, Laura; El Khoury, Graziella; Lowe, Christopher R

    2016-05-01

    Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre-enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio-pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one-pot, multi-component synthesis reaction (Ugi synthesis) and an amine-functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis-diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff-type boronic acid. The solid-phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate-dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid-functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid-based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold.

  7. 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.

  8. 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

  9. Copper(II)-based metal affinity chromatography for the isolation of the anticancer agent bleomycin from Streptomyces verticillus culture.

    PubMed

    Gu, Jiesi; Codd, Rachel

    2012-10-01

    The glycopeptide-based bleomycins are structurally complex natural products produced by Streptomyces verticillus used in combination therapy against testicular and other cancers. Bleomycin has a high affinity towards a range of transition metal ions with the 1:1 Fe(II) complex relevant to its mechanism of action in vivo and the 1:1 Cu(II) complex relevant to its production from culture. The affinity between Cu(II) and bleomycin was the underlying principle for using Cu(II)-based metal affinity chromatography in this work to selectively capture bleomycin from crude S. verticillus culture. A solution of standard bleomycin was retained at a binding capacity of 300 nmol mL(-1) on a 1-mL bed volume of Cu(II)-loaded iminodiacetate (IDA) resin at pH 9 via the formation of the heteroleptic immobilized complex [Cu(IDA)(bleomycin)]. Bleomycin was eluted from the resin at pH 5 as the metal-free ligand under conditions where pK(a) (IDA)purification of bleomycin from complex culture supernatant under aqueous conditions in a single step demonstrates the potential of Cu(II)-based metal affinity chromatography as a green chemistry platform for streamlined access to this high-value therapeutic agent.

  10. 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.

  11. Protein purification with polymeric affinity membranes containing functionalized poly(acid) brushes.

    PubMed

    Jain, Parul; Vyas, Mukesh Kumar; Geiger, James H; Baker, Gregory L; Bruening, Merlin L

    2010-04-12

    Porous nylon membranes modified with poly(acid) brushes and their derivatives can rapidly purify proteins via ion-exchange and metal-ion affinity interactions. Membranes containing poly(2-(methacryloyloxy)ethyl succinate) (poly(MES)) brushes bind 118 +/- 8 mg of lysozyme per cm(3) of membrane and facilitate purification of lysozyme from chicken egg white. Moreover, functionalization of the poly(MES) brushes with nitrilotriacetate (NTA)-Ni(2+) complexes yields membranes that bind poly(histidine)-tagged (His-tagged) ubiquitin with a capacity of 85 +/- 2 mg of protein per cm(3) of membrane. Most importantly, the membranes modified with poly(MES)-NTA-Ni(2+) allow isolation of His-tagged cellular retinaldehyde-binding protein directly from a cell extract in <10 min, and the protein purity is comparable to that achieved with commercial affinity columns. Therefore, porous nylon membranes containing functionalized poly(MES) brushes are attractive candidates for rapid, high-capacity purification of His-tagged proteins from cell extracts.

  12. Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry.

    PubMed

    Eyckerman, Sven; Impens, Francis; Van Quickelberghe, Emmy; Samyn, Noortje; Vandemoortele, Giel; De Sutter, Delphine; Tavernier, Jan; Gevaert, Kris

    2016-10-07

    Protein complexes are essential in all organizational and functional aspects of the cell. Different strategies currently exist for analyzing such protein complexes by mass spectrometry, including affinity purification (AP-MS) and proximal labeling-based strategies. However, the high sensitivity of current mass spectrometers typically results in extensive protein lists mainly consisting of nonspecifically copurified proteins. Finding the true positive interactors in these lists remains highly challenging. Here, we report a powerful design based on differential labeling with stable isotopes combined with nonequal mixing of control and experimental samples to discover bona fide interaction partners in AP-MS experiments. We apply this intelligent mixing of proteomes (iMixPro) concept to overexpression experiments for RAF1, RNF41, and TANK and also to engineered cell lines expressing epitope-tagged endogenous PTPN14, JIP3, and IQGAP1. For all baits, we confirmed known interactions and found a number of novel interactions. The results for RNF41 and TANK were compared to a classical affinity purification experiment, which demonstrated the efficiency and specificity of the iMixPro approach.

  13. Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin

    PubMed Central

    Nguyen, Hung Huy; Varadi, Mihaly; Tompa, Peter

    2017-01-01

    Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies. PMID:28319173

  14. 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.

  15. Urinary 19-norandrosterone purification by immunoaffinity chromatography: application to gas chromatography/combustion/isotope ratio mass spectrometric analysis.

    PubMed

    Desroches, M C; Mathurin, J C; Richard, Y; Delahaut, P; de Ceaurriz, J

    2002-01-01

    The detection of exogenous 19-norandrosterone (19-NA) in urines was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). 19-NA is, for the first time to our knowledge, isolated from urinary matrix by specific immunoaffinity chromatography (IAC) before analysis. The sample preparation consisted of a preliminary purification of urine by solid-phase extraction after hydrolysis by beta-glucuronidase. Unconjugated 19-NA was thus isolated by IAC and directly analysed by GC/C/IRMS. Optimisation of IAC purification was achieved and the reliability of the technique for anti-doping control is discussed.

  16. 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.

  17. 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.

  18. Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry.

    PubMed

    Hesketh, Geoffrey G; Youn, Ji-Young; Samavarchi-Tehrani, Payman; Raught, Brian; Gingras, Anne-Claude

    2017-01-01

    Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost. A more recently developed method called BioID employs the expression of bait proteins fused to a nonspecific biotin ligase, BirA*, that induces in vivo biotinylation of proximal proteins. Coupling this method to biotin affinity enrichment and mass spectrometry negates many of the solubility and interaction strength issues inherent in traditional AP-MS methods, and provides unparalleled spatial context for protein interactions. Here we describe the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.

  19. 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.

  20. Mullerian inhibiting substance fractionation by dye affinity chromatography.

    PubMed

    Budzik, G P; Powell, S M; Kamagata, S; Donahoe, P K

    1983-08-01

    Mullerian inhibiting substance (MIS), a large glycoprotein secreted by the fetal and neonatal testis, is responsible for regression of the Mullerian ducts in the male embryo. This fetal growth regulator has been purified more than 2000-fold from crude testicular incubation medium following fractionation on a triazinyl dye affinity support. A high yield of 60% recovered activity was achieved in the absence of exogenous carrier protein by stabilizing MIS with 2-mercaptoethanol, EDTA, and Nonidet-P40 and eliminating losses in the handling and concentration of MIS fractions. Although affinity elution with nucleotides has proved successful in other systems, MIS could not be eluted with ATP, GTP, or AMP, with or without divalent metal ions. Nucleotide elution, however, does remove contaminating proteins prior to MIS recovery with high ionic strength. The 2000-fold-purified MIS fraction, although not homogeneous, shows a reduction-sensitive band after SDS-gel electrophoresis that has been proposed to be the MIS dimer.

  1. 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.

  2. 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.

  3. Evaluation of flash supercritical fluid chromatography and alternate sample loading techniques for pharmaceutical medicinal chemistry purifications.

    PubMed

    Miller, Larry; Mahoney, Max

    2012-08-10

    Flash chromatography is the preferred approach for small molecule purification in pharmaceutical discovery. This paper will discuss the potential for flash supercritical fluid chromatography (SFC) as an alternative technology for these purifications. It was shown that the high sample loadings seen with flash LC could also be achieved using flash SFC. The dry load injection technique greatly increases the amount of sample that can be applied to a flash SFC column while still achieving separation. Flash SFC has much lower solvent usage and higher purification productivities relative to flash LC. Product concentrations post purification were higher for flash SFC vs. flash LC, reducing the time required to isolate dry product. There still exist a number of technical details to be worked out with flash SFC, mainly around the equipment and column/cartridge technology.

  4. Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald; Dardick, Chris; Canlas, Patrick; Xu, Xia; Gribskov, Michael; Kanrar, Siddhartha; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2006-04-01

    Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

  5. 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.

  6. 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

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

    PubMed

    Huang, He; Alvarez, Sophie; Bindbeutel, Rebecca; Shen, Zhouxin; Naldrett, Michael J; Evans, Bradley S; Briggs, Steven P; Hicks, Leslie M; Kay, Steve A; Nusinow, Dmitri 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

  8. Synthesis of medronic acid monoesters and their purification by high-performance countercurrent chromatography or by hydroxyapatite

    PubMed Central

    Vepsäläinen, Jouko; Turhanen, Petri A

    2016-01-01

    Summary We achieved the synthesis of important medronic acid monoalkyl esters via the dealkylation of mixed trimethyl monoalkyl esters of medronic acid. Two methods were developed for the purification of medronic acid monoesters: 1) small scale (10–20 mg) purification by using hydroxyapatite and 2) large scale (tested up to 140 mg) purification by high-performance countercurrent chromatography (HPCCC). PMID:27829921

  9. 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.

  10. Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants.

    PubMed

    Rohila, Jai S; Chen, Mei; Cerny, Ronald; Fromm, Michael E

    2004-04-01

    A synthetic gene encoding the tandem affinity purification (TAP) tag has been constructed, and the TAP tag assayed for its effects on expression levels and subcellular localization by fusion to green fluorescent protein (GFP) as well as for its effects on steroid-dependent translocation to the nucleus and transcription when fused to a hybrid glucocorticoid receptor. A nuclear localization signal (NLS) was detected in the calmodulin-binding protein (CBP) domain and removed by mutation to improve the usefulness of the TAP tag. Additionally, purification improvements were made, including inhibition of a co-purifying protease, and adding a protein cross-linking step to increase the recovery of interacting proteins. The improved synthetic TAP tag gene and methods were used to isolate proteins interacting with the hybrid glucocorticoid receptor and to identify them by mass spectrometry. The two proteins identified, HSP70 and HSP90, are known to interact with the glucocorticoid receptor in vivo in mammalian cells and in vitro in plants.

  11. 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

  12. A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix

    PubMed Central

    Wu, Sau-Ching; Wang, Chris; Hansen, Dave; Wong, Sui-Lam

    2017-01-01

    SAVSBPM18 is an engineered streptavidin for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags. To develop a user-friendly approach for the preparation of the SAVSBPM18-based affinity matrices, a designer fusion protein containing SAVSBPM18 and a galactose binding domain was engineered. The galactose binding domain derived from the earthworm lectin EW29 was genetically modified to eliminate a proteolytic cleavage site located at the beginning of the domain. This domain was fused to the C-terminal end of SAVSBPM18. It allows the SAVSBPM18 fusions to bind reversibly to agarose and can serve as an affinity handle for purification of the fusion. Fluorescently labeled SAVSBPM18 fusions were found to be stably immobilized on Sepharose 6B-CL. The enhanced immobilization capability of the fusion to the agarose beads results from the avidity effect mediated by the tetrameric nature of SAVSBPM18. This approach allows the consolidation of purification and immobilization of SAVSBPM18 fusions to Sepharose 6B-CL in one step for affinity matrix preparation. The resulting affinity matrix has been successfully applied to purify both SBP tagged β-lactamase and biotinylated proteins. No significant reduction in binding capacity of the column was observed for at least six months. PMID:28220817

  13. Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

    PubMed

    London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

    2015-04-01

    Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis.

  14. 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...

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

    PubMed Central

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

    2015-01-01

    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. PMID:26715090

  16. Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis.

    PubMed

    Toriello, Nicholas M; Liu, Chung N; Blazej, Robert G; Thaitrong, Numrin; Mathies, Richard A

    2007-11-15

    A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.

  17. 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

    2015-12-29

    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.

  18. Cross-linked leucaena seed gum matrix: an affinity chromatography tool for galactose-specific lectins.

    PubMed

    Seshagirirao, Kottapalli; Leelavathi, Chaganti; Sasidhar, Vemula

    2005-05-31

    A cross-linked leucaena (Leucaena leucocephala) seed gum (CLLSG) matrix was prepared for the isolation of galactose-specific lectins by affinity chromatography. The matrix was evaluated for affinity with a known galactose-specific lectin from the seeds of snake gourd (Trichosanthes anguina). The matrix preparation was simple and inexpensive when compared to commercial galactose-specific matrices (i.e. about 1.5 US dollars/100 ml of matrix). The current method is also useful for the demonstration of the affinity chromatography technique in laboratories. Since leucaena seeds are abundant and inexpensive, and the matrix preparation is easy, CLLSG appears to be a promising tool for the separation of galactose-specific lectins.

  19. Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

    PubMed Central

    Li, Shangyong; Wang, Linna; Xu, Ximing; Lin, Shengxiang; Wang, Yuejun; Hao, Jianhua; Sun, Mi

    2016-01-01

    Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA) as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects. PMID:28036010

  20. Identification of inositol 1,4,5-trisphosphate-binding proteins by heparin-agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa.

    PubMed

    Nie, Yanli; Huang, Feifei; Dong, Shujun; Li, Lin; Gao, Ping; Zhao, Heping; Wang, Yingdian; Han, Shengcheng

    2014-10-01

    Inositol 1,4,5-trisphosohate (IP3 ) and its receptors play a pivotal role in calcium signal transduction in mammals. However, no homologs of mammalian IP3 receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP3 -binding proteins by heparin-agarose affinity purification. The IP3 -binding activities of these protein fractions were determined by [(3) H] IP3 -binding assay. SDS-PAGE and MS analysis were then performed to characterize these proteins. We have identified 297 proteins from the eluates of heparin-agarose column chromatography, which will provide insight into the IP3 signaling pathways in plants. All MS data have been deposited in the ProteomeXchange with identifier PXD000763 (http://proteomecentral.proteomexchange.org/dataset/PXD000763).

  1. Performance of protein-A-based affinity membranes for antibody purification.

    PubMed

    Uzun, Lokman; Türkmen, Deniz; Karakoç, Veyis; Yavuz, Handan; Denizli, Adil

    2011-01-01

    The preparation of affinity membranes for application in antibody purification studies is described here. Protein-A-attached poly(hydroxyethyl methacrylate-N-methacryloyl-L-alanine) (PHEMAAL) membranes were produced by a photopolymerization technique and then characterized by swelling tests, surface area measurements, contact angle and scanning electron microscopy (SEM) studies. The water swelling ratio of the PHEMAAL membrane was 133.2%. PHEMAAL membranes have large pores with a size in the range of 5-10 μm. Protein A was covalently attached onto the PHEMAAL membranes via cyanogen bromide (CNBr) activation. Maximum protein A loading was 4.7 mg/g. There was a very low non-specific IgG adsorption onto the PHEMAAL membranes, about 0.38 mg/g. The maximum IgG adsorption on the PHEMAAL-protein A membrane was found to be 9.8 mg/g at pH 7.4 from aqueous solutions. Higher adsorption amount was observed from human plasma (up to 37.3 mg/g). Adsorbed IgG was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 93%. PHEMAAL-protein A membrane was used for repetitive adsorption/elution of IgG without noticeable loss in IgG adsorption amount after 10 cycles. The PHEMAAL-protein A membrane showed several advantages, such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption-elution cycles.

  2. 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

  3. Purification of antibodies by precipitating impurities using Polyethylene Glycol to enable a two chromatography step process.

    PubMed

    Giese, Glen; Myrold, Adam; Gorrell, Jeffrey; Persson, Josefine

    2013-11-01

    The purification of antibodies by precipitating impurities using Polyethylene Glycol (PEG) was assessed with the objective of developing a two chromatography column purification process. A PEG precipitation method was evaluated for use in the industrial purification of recombinant monoclonal antibodies (MAbs). Effective and robust precipitation conditions including PEG concentration, pH, temperature, time, and protein concentration were identified for several different MAbs. A recovery process using two chromatography steps in combination with PEG precipitation gave acceptable yield and purity levels for IgG1 and IgG4 antibodies with a broad range of isoelectric points (pI). PEG precipitation removed host cell proteins (HCPs), high molecular weight species (HMWS), leached Protein A ligand, and host cell DNA to acceptable levels when run under appropriate conditions, and some endogenous virus removal was achieved.

  4. 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.

  5. New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.

    PubMed

    Lauer, I; Bonnewitz, B; Meunier, A; Beverini, M

    2000-01-14

    Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also

  6. 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.

  7. Affinity chromatography of Band 3, the anion transport protein of erythrocyte membranes.

    PubMed

    Pimplikar, S W; Reithmeier, R A

    1986-07-25

    Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.

  8. Single-Step Affinity Purification of ERK Signaling Complexes Using the Streptavidin-Binding Peptide (SBP) Tag.

    PubMed

    Yang, Liu; Veraksa, Alexey

    2017-01-01

    Elucidation of biological functions of signaling proteins is facilitated by studying their protein-protein interaction networks. Affinity purification combined with mass spectrometry (AP-MS) has become a favorite method to study protein complexes. Here we describe a procedure for single-step purification of ERK (Rolled) and associated proteins from Drosophila cultured cells. The use of the streptavidin-binding peptide (SBP) tag allows for a highly efficient isolation of native ERK signaling complexes, which are suitable for subsequent analysis by mass spectrometry. Our analysis of the ERK interactome has identified both known and novel signaling components. This method can be easily adapted for SBP-based purification of protein complexes in any expression system.

  9. 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.

  10. Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli.

    PubMed

    Babu, Mohan; Butland, Gareth; Pogoutse, Oxana; Li, Joyce; Greenblatt, Jack F; Emili, Andrew

    2009-01-01

    Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).

  11. 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.

  12. 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

  13. Purification of papain by metal affinity partitioning in aqueous two-phase polyethylene glycol/sodium sulfate systems.

    PubMed

    Jiang, Zhi-Guo; Zhang, Hai-De; Wang, Wei-Tao

    2015-05-01

    A simple and inexpensive aqueous two-phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu(2+) was attached to the polyethylene glycol-iminodiacetic acid. The chelated Cu(2+) content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase-forming components (polyethylene glycol 12-20% w/w and sodium sulfate 12-20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol-iminodiacetic acid-Cu(2+) and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6-fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.

  14. Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies

    PubMed Central

    Wyler, Emanuel; Zimmermann, Mirjam; Widmann, Barbara; Gstaiger, Matthias; Pfannstiel, Jens; Kutay, Ulrike; Zemp, Ivo

    2011-01-01

    Tandem affinity purification (TAP) is an efficient method for the purification and characterization of large macromolecular complexes. To elucidate the role of specific components of such complexes, it is important to address the question of how loss of a specific factor affects complex composition. Here, we introduce a method that combines TAP of large macromolecular assemblies with inducible shRNA-mediated protein depletion in human somatic cells. As a proof of principle, we have applied this method to the purification of human pre-ribosomal particles. Using inducible expression of ribosome assembly factors as bait proteins, different pre-40S particles could be isolated and characterized, revealing high conservation of the ribosome biogenesis pathway from yeast to human cells. Besides known ribosome maturation factors, C21orf70 was identified as a novel pre-40S component. By combining TAP of pre-40S particles with shRNA-mediated depletion of the pre-40S-associated protein kinase Rio2, we observed that increased levels of the nuclear HEAT-repeat protein Rrp12 are associated with 40S precursors in absence of Rio2. Further analyses revealed that Rrp12 is partially mislocalized to the cytoplasm and trapped on late 40S precursors upon loss of Rio2, and therefore fails to efficiently recycle to the nucleus. Thus, the combination of tandem affinity purification and shRNA induction provided further insights into late cytoplasmic 40S maturation steps, demonstrating the high potential of this method. PMID:21097556

  15. Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies.

    PubMed

    Wyler, Emanuel; Zimmermann, Mirjam; Widmann, Barbara; Gstaiger, Matthias; Pfannstiel, Jens; Kutay, Ulrike; Zemp, Ivo

    2011-01-01

    Tandem affinity purification (TAP) is an efficient method for the purification and characterization of large macromolecular complexes. To elucidate the role of specific components of such complexes, it is important to address the question of how loss of a specific factor affects complex composition. Here, we introduce a method that combines TAP of large macromolecular assemblies with inducible shRNA-mediated protein depletion in human somatic cells. As a proof of principle, we have applied this method to the purification of human pre-ribosomal particles. Using inducible expression of ribosome assembly factors as bait proteins, different pre-40S particles could be isolated and characterized, revealing high conservation of the ribosome biogenesis pathway from yeast to human cells. Besides known ribosome maturation factors, C21orf70 was identified as a novel pre-40S component. By combining TAP of pre-40S particles with shRNA-mediated depletion of the pre-40S-associated protein kinase Rio2, we observed that increased levels of the nuclear HEAT-repeat protein Rrp12 are associated with 40S precursors in absence of Rio2. Further analyses revealed that Rrp12 is partially mislocalized to the cytoplasm and trapped on late 40S precursors upon loss of Rio2, and therefore fails to efficiently recycle to the nucleus. Thus, the combination of tandem affinity purification and shRNA induction provided further insights into late cytoplasmic 40S maturation steps, demonstrating the high potential of this method.

  16. Affinity chromatography of porcine pepsin A using quinolin-8-ol as ligand.

    PubMed

    Novotná, Lenka; Hrubý, Martin; Benes, Milan J; Kucerová, Zdenka

    2005-08-19

    Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.

  17. Identification of potential cellular targets of aloisine A by affinity chromatography.

    PubMed

    Corbel, Caroline; Haddoub, Rose; Guiffant, Damien; Lozach, Olivier; Gueyrard, David; Lemoine, Jérôme; Ratin, Morgane; Meijer, Laurent; Bach, Stéphane; Goekjian, Peter

    2009-08-01

    Affinity chromatography was used to identify potential cellular targets of aloisine A (7-n-butyl-6-(4'-hydroxyphenyl)-5H-pyrrolo[2,3b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine.

  18. Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.

    PubMed

    Franco-Medrano, Diana Ivonne; Guerrero-Germán, Patricia; Montesinos-Cisneros, Rosa María; Ortega-López, Jaime; Tejeda-Mansir, Armando

    2017-03-01

    The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

  19. Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

    PubMed

    Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

    2016-11-11

    A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml(-1) and 0.48mgml(-1) for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10(6)M(-1) affinity constants and Qmax values of 19.11±2.60ugg(-1) and 79.39ugg(-1) for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.

  20. Caprylic acid precipitation method for impurity reduction: an alternative to conventional chromatography for monoclonal antibody purification.

    PubMed

    Brodsky, Yan; Zhang, Cheng; Yigzaw, Yinges; Vedantham, Ganesh

    2012-10-01

    We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes.

  1. Preparation of adsorbents for affinity chromatography using TSKgel Tresyl-Toyopearl 650M.

    PubMed

    Nakamura, K; Toyoda, K; Kato, Y; Shimura, K; Kasai, K

    1989-09-08

    The optimum conditions for the coupling of proteins were investigated using TSKgel Tresyl-Toyopearl 650M. They were dependent on the proteins coupled. For example, when soybean trypsin inhibitor was coupled at pH 8 the coupling was completed within 1 h and the subsequent adsorption capacity for trypsin was maximal. Longer coupling times decreased the adsorption capacity due to multi-point attachment. The adsorbents obtained were successfully used for affinity chromatography in a short time.

  2. Optimising the design and operation of semi-continuous affinity chromatography for clinical and commercial manufacture.

    PubMed

    Pollock, James; Bolton, Glen; Coffman, Jon; Ho, Sa V; Bracewell, Daniel G; Farid, Suzanne S

    2013-04-05

    This paper presents an integrated experimental and modelling approach to evaluate the potential of semi-continuous chromatography for the capture of monoclonal antibodies (mAb) in clinical and commercial manufacture. Small-scale single-column experimental breakthrough studies were used to derive design equations for the semi-continuous affinity chromatography system. Verification runs with the semi-continuous 3-column and 4-column periodic counter current (PCC) chromatography system indicated the robustness of the design approach. The product quality profiles and step yields (after wash step optimisation) achieved were comparable to the standard batch process. The experimentally-derived design equations were incorporated into a decisional tool comprising dynamic simulation, process economics and sizing optimisation. The decisional tool was used to evaluate the economic and operational feasibility of whole mAb bioprocesses employing PCC affinity capture chromatography versus standard batch chromatography across a product's lifecycle from clinical to commercial manufacture. The tool predicted that PCC capture chromatography would offer more significant savings in direct costs for early-stage clinical manufacture (proof-of-concept) (∼30%) than for late-stage clinical (∼10-15%) or commercial (∼5%) manufacture. The evaluation also highlighted the potential facility fit issues that could arise with a capture resin (MabSelect) that experiences losses in binding capacity when operated in continuous mode over lengthy commercial campaigns. Consequently, the analysis explored the scenario of adopting the PCC system for clinical manufacture and switching to the standard batch process following product launch. The tool determined the PCC system design required to operate at commercial scale without facility fit issues and with similar costs to the standard batch process whilst pursuing a process change application. A retrofitting analysis established that the direct cost

  3. Monoclonal antibodies to human interferon-gamma: production, affinity purification and radioimmunoassay.

    PubMed Central

    Novick, D; Eshhar, Z; Fischer, D G; Friedlander, J; Rubinstein, M

    1983-01-01

    Human interferon-gamma (IFN-gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN-gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN-gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN-gamma. Moreover, IFN-gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN-gamma subtypes are immunologically cross-reactive. Immune precipitation of partially purified 125I-labelled IFN-gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN-gamma gave a 50-fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid-phase radioimmunoassay, allowing the detection of IFN-gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h. Images Fig. 1. Fig. 2. PMID:11892806

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

    PubMed Central

    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.

    2016-01-01

    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

  5. 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.

  6. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

    PubMed

    Brgles, Marija; Kurtović, Tihana; Kovačič, Lidija; Križaj, Igor; Barut, Miloš; Lang Balija, Maja; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

  7. 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.

  8. 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.

  9. 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.

  10. Affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells.

    PubMed

    Chase, Geoffrey P; Schwemmle, Martin

    2012-06-03

    Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized. Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by

  11. Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

    PubMed

    Ngo, That T; Narinesingh, Dyer

    2008-01-01

    The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.

  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. Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH.

    PubMed

    Li, Xiaobao; Pennington, Justin; Stobaugh, John F; Schöneich, Christian

    2008-01-15

    Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.

  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

    2011-09-09

    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. Orthogonal ion pairing reversed phase liquid chromatography purification of oligonucleotides with bulky fluorophores.

    PubMed

    Anacleto, Concordio; Ouye, Randall; Schoenbrunner, Nancy

    2014-02-14

    A dual labeled oligonucleotide used as TaqMan® or 5' nuclease probe for in vitro diagnostic has been purified through orthogonal ion-pairing reversed phase chromatography, using polymeric semi-preparative and preparative PRP-1 column. We studied the mechanism of separation of oligonucleotides using ion-pairing reversed phase chromatography. We found that elution profiles of dye labeled oligonucleotides can be controlled by use of specific ion-pairing reagents. Here, we report a method for purification of an oligonucleotide containing an internally positioned rhodamine dye using two orthogonal chromatographic steps, in which the primary step resolves mostly by differences in hydrophobicity by using a weak ion-pairing reagent, and a secondary step uses a strong ion-pairing reagent for separation of length variants. Purification is demonstrated for both 1 and 15μmol scale syntheses, and amenable to further scale up for commercial lot production.

  16. Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography.

    PubMed Central

    Vockley, J; Harris, H

    1984-01-01

    We have used the technique of monoclonal antibody immunoaffinity chromatography to purify adult and foetal intestinal alkaline phosphatases. Pure adult intestinal enzyme was obtained from a crude tissue extract with a single immunoaffinity chromatographic step in yields exceeding 95%. An additional ion-exchange chromatographic step was necessary for purification of the foetal enzyme, but yields still exceeded 70%. Experiments to optimize the efficiency of the monoclonal antibody immunoaffinity chromatography procedure suggest that the relative strength of binding of an antibody to its antigen is the most important factor to consider when constructing such columns. A column made from an antibody of too low an avidity will not retain the enzyme, while one of too high an avidity will make elution of enzyme in the active state difficult. A scheme is suggested for the application of this technique to a general approach to enzyme purification. Images Fig. 2. PMID:6365087

  17. Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography.

    PubMed

    Guerrero-Germán, Patricia; Montesinos-Cisneros, Rosa Ma; Prazeres, D Miguel F; Tejeda-Mansir, Armando

    2011-01-01

    A novel downstream bioprocess was developed to obtain purified plasmid DNA (pDNA) from Escherichia coli ferments. The intermediate recovery and purification of the pDNA in cell lysate was conducted using hollow-fiber tangential filtration and frontal anion-exchange membrane and elution hydrophobic chromatographies. The purity of the solutions of pDNA obtained during each process stage was investigated. The results show that the pDNA solution purity increased 30-fold and more than 99% of RNA in the lysate was removed during the process operations. The combination of membrane operations and hydrophobic interaction chromatography resulted in an efficient way to recover pDNA from cell lysates. A better understanding of membrane-based technology for the purification of pDNA from clarified E. coli lysate was developed in this research.

  18. 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.

  19. 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.

  20. 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.

  1. Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences.

    PubMed

    Carvalho, Rimenys Junior; Cabrera-Crespo, Joaquin; Tanizaki, Martha Massako; Gonçalves, Viviane Maimoni

    2012-05-01

    Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

  2. Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level.

    PubMed

    Bartlow, Patrick; Uechi, Guy T; Cardamone, John J; Sultana, Tamanna; Fruchtl, McKinzie; Beitle, Robert R; Ataai, Mohammad M

    2011-08-01

    Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.

  3. Phosphoprotein affinity purification identifies proteins involved in S-adenosyl-L-methionine-induced enhancement of antibiotic production in Streptomyces coelicolor.

    PubMed

    Meng, Lingzhu; Yang, Seung Hwan; Palaniyandi, Sasikumar Arunachalam; Lee, Sung-Kwon; Lee, In-Ae; Kim, Tae-Jong; Suh, Joo-Won

    2011-01-01

    Streptomycetes are the major natural source of clinical antibiotics. The enhanced secondary metabolite production of many streptomycetes by S-adenosylmethionine (SAM) in previous studies suggested the existence of a common SAM regulatory effect. We screened nine proteins using the phosphoprotein purification column from Streptomyces coelicolor. Among them, genes (SCO5477, SCO5113, SCO4647, SCO4885 and SCO1793) for five proteins were disrupted by insertion mutation. The undecylprodigiosin and actinorhodin productions were changed in all mutations. The SAM-induced enhancement of actinorhodin production was abolished by all mutations except SCO4885 mutation, which reduced the production of actinorhodin and undecylprodigiosin with SAM treatment. This study demonstrates that phosphoprotein affinity purification can be used as a screening method to identify the proteins involved SAM signaling.

  4. 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

  5. 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.

  6. 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.

  7. Discovery of protein complexes with core-attachment structures from Tandem Affinity Purification (TAP) data.

    PubMed

    Wu, Min; Li, Xiao-Li; Kwoh, Chee-Keong; Ng, See-Kiong; Wong, Limsoon

    2012-09-01

    Many cellular functions involve protein complexes that are formed by multiple interacting proteins. Tandem Affinity Purification (TAP) is a popular experimental method for detecting such multi-protein interactions. However, current computational methods that predict protein complexes from TAP data require converting the co-complex relationships in TAP data into binary interactions. The resulting pairwise protein-protein interaction (PPI) network is then mined for densely connected regions that are identified as putative protein complexes. Converting the TAP data into PPI data not only introduces errors but also loses useful information about the underlying multi-protein relationships that can be exploited to detect the internal organization (i.e., core-attachment structures) of protein complexes. In this article, we propose a method called CACHET that detects protein complexes with Core-AttaCHment structures directly from bipartitETAP data. CACHET models the TAP data as a bipartite graph in which the two vertex sets are the baits and the preys, respectively. The edges between the two vertex sets represent bait-prey relationships. CACHET first focuses on detecting high-quality protein-complex cores from the bipartite graph. To minimize the effects of false positive interactions, the bait-prey relationships are indexed with reliability scores. Only non-redundant, reliable bicliques computed from the TAP bipartite graph are regarded as protein-complex cores. CACHET constructs protein complexes by including attachment proteins into the cores. We applied CACHET on large-scale TAP datasets and found that CACHET outperformed existing methods in terms of prediction accuracy (i.e., F-measure and functional homogeneity of predicted complexes). In addition, the protein complexes predicted by CACHET are equipped with core-attachment structures that provide useful biological insights into the inherent functional organization of protein complexes. Our supplementary material can

  8. 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.

  9. 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.

  10. Preparation of affinity membranes using thermally induced phase separation for one-step purification of recombinant proteins.

    PubMed

    Honjo, Takafumi; Hoe, Kazuki; Tabayashi, Shunsuke; Tanaka, Tsutomu; Shimada, Josui; Goto, Masahiro; Matsuyama, Hideto; Maruyama, Tatsuo

    2013-03-15

    We synthesized several surfactant-like ligands and prepared affinity membranes by introducing them into porous polymeric membranes using the thermally induced phase separation method. The ligands (nitrilotriacetate, iminodiacetate, and glutathione) were successfully displayed on the surfaces of cellulose diacetate membranes. Membranes functionalized with nitrilotriacetate and glutathione captured and released hexahistidine-tagged enhanced green fluorescent protein (His-tag GFP) and glutathione S-transferase (GST) selectively under appropriate conditions. The affinity membranes also enabled highly selective purification of target proteins (GFP and GST) from cell lysates. The protein-binding capacity was 15 μg/cm(2) for His-tag GFP and 13 μg/cm(2) for GST. The application-specific membranes described in this work will aid high-throughput screening and high-throughput analysis of recombinant proteins.

  11. Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography.

    PubMed

    Cao, Xue-Li; Xu, Ya-Tao; Zhang, Guang-Ming; Xie, Sheng-Meng; Dong, Ying-Mao; Ito, Yoichiro

    2006-09-15

    High-speed counter-current chromatography (HSCCC) is applied to the purification of coenzyme Q(10) (CoQ(10)) for the first time. CoQ(10) was obtained from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile-dichloromethane (12:7:3.5, v/v/v) was selected by analytical HSCCC and used for purification of CoQ(10) from 500 mg of the crude extract. The separation yielded 130 mg of CoQ(10) at an HPLC purity of over 99%. The overall results of the present studies show the advantages of HSCCC over an alternative of silica gel chromatography followed by recrystallization. These advantages extend to higher purity (97.8% versus 93.3%), recovery (88% versus 74.3%) and yield (26.4% versus 23.4%). An effort to avoid the toxic, expensive solvent CH(2)Cl(2) was unsuccessful, but at least its percentage is low in the solvent system.

  12. [Separation and purification of lysozyme from egg white by high performance cation-exchange chromatography].

    PubMed

    Li, Rong; Chen, Guo-liang

    2002-05-01

    A new method used to separate and purify lysozyme from egg white by high performance cation-exchange chromatography has been established. The process conditions for purifying lysozyme were also discussed in detail. The procedure involved that homogenization of the egg white sample, preliminary purification with sodium chloride, and chromatographic separation by the weak cation exchange column (XIDACE-WCX). The experimental results showed that the purified lysozyme and other impurity proteins were completely separated. By using bioactivity assay, the recovery of lysozyme was 107%, and the specific activity was 15,467 U/mg through the column. Its purity was raised 5.6-fold. The collected fraction with activity was detected by size-exclusion chromatography (SEC). The purified lysozyme was homogeneous. Compared with the traditional soft-based low pressure ion-exchange chromatography, the developed method is rapid and effective.

  13. 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

  14. 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.

  15. Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein.

    PubMed

    Uhlik, Ondrej; Kamlar, Marek; Kohout, Ladislav; Jezek, Rudolf; Harmatha, Juraj; Macek, Tomas

    2008-12-22

    The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.

  16. Application of Frontal Affinity Chromatography to Study the Biomolecular Interactions with Trypsin.

    PubMed

    Hu, YuanYuan; Qian, Junqing; Guo, Hui; Jiang, ShengLan; Zhang, Zheng

    2015-07-01

    Trypsin is a serine protease that has been proposed as a potential therapeutic target for metabolic disorders and malignancy diseases, thus the identification of biomolecular interactions of compounds to trypsin could be of great therapeutic importance. In this study, trypsin was immobilized on a monolithic silica capillary column via sol-gel. The binding properties of four small molecules (daidzin, genistin, matrine and oxymatrine) to trypsin were examined using the trypsin affinity columns by frontal analysis. The results indicate that the matrine (dissociation constant, Kd = 7.904 μM) has stronger interaction with trypsin than the oxymatrine (Kd = 8.204 μM), whereas daidzin and genistin were nearly have no affinity with trypsin. The results demonstrated that the frontal affinity chromatography can be used for the direct determination of protein-protease inhibitor binding interactions and have several significant advantages, including easy fabricating, reproducible, minimal technological requirements and potential to become a reliable alternative for quantitative studies of biomolecular interactions.

  17. 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

  18. A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification.

    PubMed

    Fernandes, Cláudia S M; Pina, Ana Sofia; Dias, Ana M G C; Branco, Ricardo J F; Roque, Ana Cecília Afonso

    2014-09-30

    The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions.

  19. Experimental and theoretical investigation of effect of spacer arm and support matrix of synthetic affinity chromatographic materials for the purification of monoclonal antibodies.

    PubMed

    Zamolo, Laura; Salvalaglio, Matteo; Cavallotti, Carlo; Galarza, Benedict; Sadler, Chris; Williams, Sharon; Hofer, Stefan; Horak, Jeannie; Lindner, Wolfgang

    2010-07-29

    The aim of this study was to elucidate the influence of each material component-the support, the spacer, and the surface chemistry-on the overall material performance of an affinity type purification media for the capture of immunoglobulin G (IgG). Material properties were investigated in terms of an experimental evaluation using affinity chromatography as well as computer modeling. The biomimetic triazine-based A2P affinity ligand was chosen as a fixed point, while spacer and support were varied. The investigated spacers were 1-2-diaminoethane (2LP), 1,3-propanedithiol (SS3), 3,6-dioxo-1,8-octanedithiol (DES), and a 1,4-substituted [1,2,3]-triazole spacer (TRZ). The support media considered were the agarose (AG) resins, PuraBead, the polyvinylether, Fractoprep, the polymethacrylate, Fractogel, and the porous silica, Fractosil. All materials were tested with pure IgG standard solution, with a mock feed solution as well as real cell culture supernatant. The interaction between IgG and A2P linked through the investigated spacers to AG was studied using molecular dynamics. The effect of a modification of the support chemical structure or of the protein-ligand binding site on the material performances was studied through target oriented simulations. Dynamic binding experiments (DBC) revealed that the performances of materials containing 2LP spacers were significantly decreased in the presence of Pluronic F68. The simulations indicated that this is probably determined by the establishment of intermolecular interactions between the 2LP charged amino group and the ether oxygen of Pluronic F68. The spacer giving the highest IgG dynamic binding capacity when Pluronic F68 was present in the feed was TRZ. The simulations showed that, among the investigated spacers, TRZ is the only one that prevents the adsorption of A2P on the support surface, thus suggesting that the mobility and lack of interaction of the ligand with the support is an important property for an affinity

  20. Application of a New Dual Localization-Affinity Purification Tag Reveals Novel Aspects of Protein Kinase Biology in Aspergillus nidulans

    PubMed Central

    De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Osmani, Stephen A.

    2014-01-01

    Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients

  1. 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.

  2. Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography.

    PubMed

    Peram, Thanmaya; McDonald, Paul; Carter-Franklin, Jayme; Fahrner, Robert

    2010-01-01

    The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.

  3. 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.

  4. 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.

  5. 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

    2012-11-12

    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

  6. [Purification of immunoglobulin and serum albumin from serum via strong anion exchange chromatography coupled with molecular exclusion chromatography].

    PubMed

    Qin, Zonghua; Chen, Ting; Li, Renqiang

    2012-08-01

    The isoelectric points of immunoglobulin (Ig) and serum albumin (SA) in animal serum are about 7.8 and 4.8, respectively. Based on their larger difference of isoelectric points, Q Sepharose-XL strong anion exchange chromatography coupled with molecular exclusion chromatography was used to purify Ig and SA simultaneously from the high immune rabbit serum. After the Q Sepharose-XL strong anion exchange column was equilibrated with 0.02 mol/L Tris-HCl buffer of pH 8.0, a 10-fold dilution sample of rabbit serum was loaded onto the column and the pH gradient elution was performed. With the low flow rate of elution of 0.3 mL/min, the high-purity Ig was obtained when the elution pH was at 6.0. Continuously eluted at pH 4.0 with the same flow rate of elution, the SA was obtained and its purity was greater than 95% after molecular exclusion chromatography through Sephadex G-75. The purified Ig and SA were demonstrated to maintain normal activities by activity analysis. The results of protein content showed that the purification recoveries of Ig and SA were over 95% and 90%, respectively. The method has the advantages of simple operation and rapidity, and the Ig and SA purified simultaneously from the animal serum could maintain normal activities.

  7. 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.

  8. 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.

  9. Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

    PubMed

    Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

    2017-01-01

    Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

  10. Purification of Mouse Monoclonal Immunoglobulin M by Ion-Exchange Liquid Chromatography

    DTIC Science & Technology

    1989-01-01

    Retention times of the IgM peaks were 27 min for each. The IgM concentration of the pooled peak fractions was determined by ELISA with mouse myeloma IgM as...PURIFICATION OF MOUSE MONOCLONAL IMMUNOGLOBULIN M BY ION-EXCHANGE LIQUID CHROMATOGRAPHY SARA W. ROTHMAN1 , MARY KAY GENTRY 1, ROGER D. GAWNE 1...antibodies have been difficult to purify. By eluting with a gradient of increasing salt and decreasing pH, we succeeded in highly purifying mouse

  11. A low-cost affinity purification system using β-1,3-glucan recognition protein and curdlan beads.

    PubMed

    Horiuchi, Masataka; Takahasi, Kiyohiro; Kobashigawa, Yoshihiro; Ochiai, Masanori; Inagaki, Fuyuhiko

    2012-08-01

    Silkworm β-1,3-glucan recognition protein (βGRP) tightly and specifically associates with β-1,3-glucan. We report here an affinity purification system named the 'GRP system', which uses the association between the β-1,3-glucan recognition domain of βGRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble β-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4-6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.

  12. 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.

  13. Contamination of ribosome inactivating proteins with ribonucleases, separated by affinity chromatography on red sepharose.

    PubMed

    Wang, H X; Ng, T B; Cheng, C H K; Fong, W P

    2003-05-01

    Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

  14. Reinforcement of frontal affinity chromatography for effective analysis of lectin-oligosaccharide interactions.

    PubMed

    Hirabayashi, J; Arata, Y; Kasai, K

    2000-08-25

    Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 microm diameter), and packed into a miniature column (e.g., 10 x 4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 microg/ml for protein) analyses of lectin-carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.

  15. Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography.

    PubMed

    Wiley, J P; Hughes, K A; Kaiser, R J; Kesicki, E A; Lund, K P; Stolowitz, M L

    2001-01-01

    Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

  16. Partial Purification and Properties of Bovine and Ovine Spinal Cord Acetylcholinesterases,

    DTIC Science & Technology

    1985-08-01

    The yellow bands which developed were preserved by photography within 1 hr due to subsequent fading of the colour . Affinity Chromatography The...McCormick. Flavin affinity chromatography: General methods for purification of proteins that bind riboflavin . Anal. Biochem., 89, 87-102 (1978). 10

  17. Using affinity chromatography to engineer and characterize pH-dependent protein switches.

    PubMed

    Sagermann, Martin; Chapleau, Richard R; DeLorimier, Elaine; Lei, Margarida

    2009-01-01

    Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione-S-transferase to convert this protein into a pH-dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH-dependent GSH-binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector-specific allostery into a protein structure.

  18. Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

    PubMed Central

    Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

    2016-01-01

    Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

  19. 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.

  20. 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

  1. Alternative separation steps for monoclonal antibody purification: combination of centrifugal partitioning chromatography and precipitation.

    PubMed

    Oelmeier, Stefan A; Ladd-Effio, Christopher; Hubbuch, Jürgen

    2013-12-06

    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.

  2. A flow-through chromatography process for influenza A and B virus purification.

    PubMed

    Weigel, Thomas; Solomaier, Thomas; Peuker, Alessa; Pathapati, Trinath; Wolff, Michael W; Reichl, Udo

    2014-10-01

    Vaccination is still the most efficient measure to protect against influenza virus infections. Besides the seasonal wave of influenza, pandemic outbreaks of bird or swine flu represent a high threat to human population. With the establishment of cell culture-based processes, there is a growing demand for robust, economic and efficient downstream processes for influenza virus purification. This study focused on the development of an economic flow-through chromatographic process avoiding virus strain sensitive capture steps. Therefore, a three-step process consisting of anion exchange chromatography (AEC), Benzonase(®) treatment, and size exclusion chromatography with a ligand-activated core (LCC) was established, and tested for purification of two influenza A virus strains and one influenza B virus strain. The process resulted in high virus yields (≥68%) with protein contamination levels fulfilling requirements of the European Pharmacopeia for production of influenza vaccines for human use. DNA was depleted by ≥98.7% for all strains. The measured DNA concentrations per dose were close to the required limits of 10ng DNA per dose set by the European Pharmacopeia. In addition, the added Benzonase(®) could be successfully removed from the product fraction. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for production of cell culture-derived influenza vaccines.

  3. Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.

    PubMed Central

    Dean, N; Berk, A J

    1987-01-01

    Recently, it has been shown that mammalian transcription factor IIIC (TFIIIC) activity can be separated by anion exchange FPLC chromatography into two functional components (1), both of which are required for transcription of tRNA and the adenovirus VA RNA genes. Here we show that these two functional components, designated TFIIIC1 and TFIIIC2, can also be separated by sequence specific DNA affinity chromatography. These results confirm the observation that TFIIIC can be fractionated into two components, which are both required for transcription of VA I and tRNA genes in vitro. Thus in the mammalian reconstituted system, a minimum of three proteins, in addition to RNA polymerase III, are required for the transcription of the VA and tRNA genes in vitro. The DNA binding component, TFIIIC2, binds specifically to the 3' segment of the internal promoter (the B block), demonstrated by its ability to protect this region from digestion by DNase I. TFIIIC2 is the limiting, titratable component in the phosphocellulose C fraction required for the formation of a stable pre-initiation complex on the VAI RNA gene in vitro, as demonstrated with a template competition and rescue assay. Images PMID:3697084

  4. Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification.

    PubMed

    Rashid, Zahra; Ghahremanzadeh, Ramin; Nejadmoghaddam, Mohammad-Reza; Nazari, Mahboobeh; Shokri, Mohammad-Reza; Naeimi, Hossein; Zarnani, Amir-Hassan

    2017-03-24

    In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc)2.4H2O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe2O4@SiO2@Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg(-1) (protein/MnFe2O4@SiO2@Ni-Salen complex). Collectively, purification process with MnFe2O4@SiO2@Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems.

  5. Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag

    PubMed Central

    Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G.; Jalah, Rashmi; Bear, Jenifer; Rosati, Margherita; Pavlakis, George N.; Felber, Barbara K.; Zaharoff, David A.; Kumar, Thallapuranam Krishnaswamy Suresh

    2014-01-01

    Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous.Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ~98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichrosim, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31 ± 0.05 mg/ mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app) = 69 ± 1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics. PMID:25123642

  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. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    PubMed

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

  8. An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments.

    PubMed

    Knappik, A; Plückthun, A

    1994-10-01

    The commercially available monoclonal antibodies M1 and M2 were raised against and bind the FLAG sequence DYKDDDDK with high specificity. Using the calcium-dependent M1 antibody and the FLAG tag attached to the N terminus of various fragments of the antibody McPC603 expressed in Escherichia coli, we found that the M1 antibody binds with almost the same affinity to a much shorter version of this sequence (DYKD). Since most antibody light chains start with an aspartate, the addition of only three additional amino acids to the N terminus is sufficient to detect and quantify the expressed antibody fragments using standard immunological methods. Similarly, the heavy chain can be detected specifically with the sequence DYKD, which requires four additional amino acids since most heavy chains do not start with Asp. The signal sequence of both chains that is necessary for the transport of the chains to the periplasm of E. coli is processed correctly. Furthermore, we investigated the influence of the amino acid at the fifth position of the FLAG sequence on the binding affinity of the M1 antibody and found that a glutamate at this position increased the sensitivity in Western blots sixfold over the original long FLAG sequence containing an aspartate residue at this position. Together, the improved FLAG is a versatile tool for both sensitive detection and one-step purification of recombinant proteins.

  9. 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.

  10. Adsorption of human serum proteins onto TREN-agarose: purification of human IgG by negative chromatography.

    PubMed

    Bresolin, Igor Tadeu Lazzarotto; Borsoi-Ribeiro, Mariana; Caro, Juliana Rodrigues; dos Santos, Francine Petit; de Castro, Marina Polesi; Bueno, Sonia Maria Alves

    2009-01-01

    Tris(2-aminoethyl)amine (TREN) - a chelating agent used in IMAC - immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90-95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.

  11. Purification of tracer for somatomedin C radioimmunoassay by hydrophobic interaction chromatography

    SciTech Connect

    Baxter, R.C.; Brown, A.S.

    1982-03-01

    A tracer for use in the somatomedin C radiommunoassay by hydrophobic interaction chromatography was purified. 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 NH/sub 4/HCO/sub 3/, 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.

  12. A carboxy-terminal affinity tag for the purification and mass spectrometric characterization of integral membrane proteins.

    PubMed

    Wong, Julie P; Reboul, Emmanuelle; Molday, Robert S; Kast, Juergen

    2009-05-01

    G-protein-coupled receptors (GPCRs) and other structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tag-specific, high-affinity monoclonal antibody. When two GPCRs as well as two related ATP binding cassette (ABC) transporters are expressed in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 as compared against other entries in an annotated spectrum library. Furthermore, specific enzyme combinations were necessary to produce suitable peptides for deducing N-glycosylation sites on CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.

  13. 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.

  14. 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.

  15. Affinity purification of HC-Pro of potyviruses with Ni2+-NTA resin.

    PubMed

    Kadouri, D; Peng, Y; Wang, Y; Singer, S; Huet, H; Raccah, B; Gal-On, A

    1998-12-01

    The HC-Pro of zucchini yellow mosiac virus (ZYMV) was found to bind to Ni2+-NTA resin with or without His-tagging. The binding stringency was similar to that observed in proteins with a zinc finger motif like the HC-Pro. Using this characteristic we developed an efficient and rapid method (2-3 h) for purification of the HC-Pro of several potyviruses. A dominant protein of about 150 kDa was extracted and identified as the HC-Pro of ZYMV by means of immunoblotting. About 150 microg of HC-Pro were partially purified from the soluble fraction of 1 g of leaves. High titers of HC-Pro protein were obtained from plants infected with four potyviruses [ZYMV, watermelon mosaic virus II (WMVII), papaya ringspot virus (PRSV) and turnip mosaic virus (TuMV)]. The HC-Pros of potato virus Y (PVY) and tobacco vein mottling virus (TVMV) did not bind to the Ni2+-NTA resin. The ZYMV-HC-Pro purified by the Ni2+-NTA resin could bind in vitro to ZYMV virions blotted onto a membrane. All the HC-Pros which had been successfully purified by the Ni2+-NTA resin were bound in vitro to membrane-blotted ZYMV coat protein. However, only the HC-Pros of ZYMV and WMVII were able to mediate aphid transmission of purified ZYMV virions. The purification procedure described herein is efficient and convenient, and enables HC-Pro for a number of potyviruses to be obtained in larger amounts and at higher purity than possible by means of most existing methods, based on ultracentrifugation.

  16. Anion-exchange purification of recombinant factor IX from cell culture supernatant using different chromatography supports.

    PubMed

    Ribeiro, Daniel A; Passos, Douglas F; Ferraz, Helen C; Castilho, Leda R

    2013-11-01

    Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose(®) FF and Fractogel(®) TMAE, the monolith CIM(®) QA and the membrane adsorber Sartobind(®) Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca(2+) ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.

  17. Modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin.

    PubMed

    Moraes, Caroline Costa; Mazutti, Marcio A; Maugeri, Francisco; Kalil, Susana Juliano

    2013-03-15

    This work is focused on the experimental evaluation and mathematical modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin from crude fermentative broth containing Spirulina platensis cells. Experiments were carried out in different expansion degree to evaluate the process performance. The experimental breakthrough curves were used to estimate the mass transfer and kinetics parameters of the proposed model, using the Particle Swarm Optimization algorithm (PSO). The proposed model satisfactorily fitted the experimental data. The results from the model application pointed out that the increase in the initial bed height does not influence the process efficiency, however enables the operation of expanded-bed column at high volumetric flow rates, improving the productivity. It was also shown that the use of mathematical modeling was a good and promising tool for the optimization of chromatographic processes.

  18. 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

    2013-11-05

    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.

  19. [Isolation and purification of puerarin from puerarin extractive by chelate complex chromatography].

    PubMed

    Pan, Jian; Yuan, Chuanxun; Dai, Yuqing

    2006-09-01

    Puerarin, an isoflavone compound, is an important ingredient in traditional Chinese medicine, which has the good medical effects on hypertension and angina. A new type of separation and purification method for isolating puerarin was developed using chelate complex chromatography. The stationary phase was prepared with silica gel containing 7% of Cu(OAc)2 in the chloroform. After the chloroform was evaporated, it was used as chelate complex chromatographic stationary phase to purify puerarin extractive. Pure puerarin was obtained when a mixture of chloroform and methanol (10:1, v/v) was used as eluent. In comparison with a common silica gel column, the chelate complex chromatographic column was more convenient and efficient. The purity and recovery of puerarin, and column capacity were improved 11%, 12% and 200%, respectively. Under the optimized experimental conditions, the purity of puerarin could be as high as with a recovery of 92%.

  20. Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

    PubMed

    Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

    2014-06-01

    The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification.

  1. 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.

  2. Purification of the Caenorhabditis elegans transposase Tc1A refolded during gel filtration chromatography.

    PubMed

    García-Sáez, I; Plasterk, R H

    2000-08-01

    Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12-16 mg/liter E. coli culture, in a form suitable for crystallization studies.

  3. 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.

  4. 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

  5. NMR screening of new carbocyanine dyes as ligands for affinity chromatography.

    PubMed

    Cruz, Carla; Boto, Renato E F; Drzazga, Anna K; Almeida, Paulo; Queiroz, João A

    2014-04-01

    Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N-carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α-chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un-substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α-chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α-chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD-NMR technique was successfully used to screen cyanine-protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography.

  6. 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

  7. Characterization of minor site probes for human serum albumin by high-performance affinity chromatography.

    PubMed

    Sengupta, A; Hage, D S

    1999-09-01

    This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents.

  8. 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%.

  9. Centrifugal partition chromatography: A preparative tool for isolation and purification of xylindein from Chlorociboria aeruginosa.

    PubMed

    Boonloed, Anukul; Weber, Genevieve L; Ramzy, Kelly M; Dias, Veronica R; Remcho, Vincent T

    2016-12-23

    A centrifugal partition chromatography (CPC) method was developed for the preparative-scale isolation and purification of xylindein from the wood-staining fungi, Chlorociboria aeruginosa. Xylindein, a blue-green pigment naturally secreted from the hyphae and fruiting bodies of the fungus, has great value in the decorative wood industry and textile coloration. Xylindein has great potential for use as a fluorescent labeling agent as well as in organic semiconductor applications. However, a primary limitation of xylindein is its poor solubility in most common HPLC solvents. Consequently, it is arduous to purify using preparative liquid chromatography or solid-phase extraction (SPE). Support-free, liquid-liquid chromatographic methods, including CPC, where solutes are separated based on their different distribution coefficients (KD) between two immiscible solvent systems, are promising alternatives for the purification of the compound on a preparative scale. In this work, a new biphasic solvent system suitable for CPC separation of xylindein was developed. Various groups of solvents were assessed for their suitability as xylindein extractants. A new solvent system suitable for CPC separation of xylindein, composed of heptane/THF/MEK/acetonitrile/acetic acid/water, was developed. This solvent system yielded a KD value for xylindein of 1.54±0.04, as determined by HPLC (n=3). The compositions of the upper phase and lower phase of the solvent system were determined by Heteronuclear Single Quantum Correlation (HSQC) NMR and proton NMR. A CPC system, equipped with a fraction collector, was used for the isolation of xylindein from crude extracts. The xylindein fractions isolated by the CPC were then analyzed using HPLC and presented as a fractogram. Based on the CPC fractogram, the purified xylindein fractions were achieved after 30min CPC separation time, yielding 71% extraction efficiency. The developed CPC method allowed for isolation of this naturally sourced xylindein

  10. Receptor affinity purification of a lipid-binding adhesin from Helicobacter pylori.

    PubMed Central

    Lingwood, C A; Wasfy, G; Han, H; Huesca, M

    1993-01-01

    Our previous work has shown that Helicobacter pylori specifically recognizes gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro. This binding specificity is shared by exoenzyme S from Pseudomonas aeruginosa, and monoclonal antibodies against this adhesin prevent the attachment of H. pylori to its lipid receptors. We now report the use of a novel, versatile affinity matrix to purify a 63-kDa exoenzyme S-like adhesin from H. pylori which is responsible for the lipid-binding specificity of this organism. Images PMID:8500882

  11. 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.

  12. PEG precipitation coupled with chromatography is a new and sufficient method for the purification of botulinum neurotoxin type B [corrected].

    PubMed

    Zhao, Yao; Kang, Lin; Gao, Shan; Gao, Xing; Xin, Wenwen; Wang, Jinglin

    2012-01-01

    Clostridium botulinum neurotoxins are used to treat a variety of neuro-muscular disorders, as well as in cosmetology. The increased demand requires efficient methods for the production and purification of these toxins. In this study, a new purification process was developed for purifying type B neurotoxin. The kinetics of C.botulinum strain growth and neurotoxin production were determined for maximum yield of toxin. The neurotoxin was purified by polyethylene glycol (PEG) precipitation and chromatography. Based on design of full factorial experiment, 20% (w/v) PEG-6000, 4 °C, pH 5.0 and 0.3 M NaCl were optimal conditions to obtain a high recovery rate of 87% for the type B neurotoxin complex, as indicated by a purification factor of 61.5 fold. Furthermore, residual bacterial cells, impurity proteins and some nucleic acids were removed by PEG precipitation. The following purification of neurotoxin was accomplished by two chromatography techniques using Sephacryl™ S-100 and phenyl HP columns. The neurotoxin was recovered with an overall yield of 21.5% and the purification factor increased to 216.7 fold. In addition, a mouse bioassay determined the purified neurotoxin complex possessed a specific toxicity (LD(50)) of 4.095 ng/kg.

  13. Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

    PubMed

    Roy, Ipsita; Jain, Sulakshana; Teotia, Sunita; Gupta, Munishwar Nath

    2004-01-01

    Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.

  14. Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification.

    PubMed

    Perçin, Işık; Sağlar, Emel; Yavuz, Handan; Aksöz, Erol; Denizli, Adil

    2011-05-01

    The aim of this study is to prepare supermacroporous pseudospecific cryogel which can be used for the purification of plasmid DNA (pDNA) from bacterial lysate. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the pseudospecific ligand and/or comonomer. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Compared with the PHEMA cryogel (50 μg/g polymer), the pDNA adsorption capacity of the PHEMAH cryogel (13,350 μg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The amount of pDNA bound onto the PHEMAH cryogel disks first increased and then reached a saturation value (i.e., 13,350μg/g) at around 300 μg/ml pDNA concentration. pDNA adsorption amount decreased from 1137 μg/g to 160 μg/g with the increasing NaCl concentration. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 90%. The PHEMAH cryogel could be used 3 times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH cryogel disks promise high selectivity for pDNA.

  15. 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.

  16. Purification of semiconducting single-walled carbon nanotubes by spiral counter-current chromatography.

    PubMed

    Knight, Martha; Lazo-Portugal, Rodrigo; Ahn, Saeyoung Nate; Stefansson, Steingrimur

    2017-02-03

    Over the last decade man-made carbon nanostructures have shown great promise in electronic applications, but they are produced as very heterogeneous mixtures with different properties so the achievement of a significant commercial application has been elusive. The dimensions of single-wall carbon nanotubes are generally a nanometer wide, up to hundreds of microns long and the carbon nanotubes have anisotropic structures. They are processed to have shorter lengths but they need to be sorted by diameter and chirality. Thus counter-current chromatography methods developed for large molecules are applied to separate these compounds. A modified mixer-settler spiral CCC rotor made with 3 D printed disks was used with a polyethylene glycol-dextran 2-phase solvent system and a surfactant gradient to purify the major species in a commercial preparation. We isolated the semi-conducting single walled carbon nanotube chiral species identified by UV spectral analysis. The further development of spiral counter-current chromatography instrumentation and methods will enable the scalable purification of carbon nanotubes useful for the next generation electronics.

  17. 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.

  18. 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.

  19. Purification of d-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) by a temperature-modulated silica gel column chromatography: use of Taguchi method to optimize purification conditions.

    PubMed

    Chang, Yinzi; Cao, Yucheng; Zhang, Jin; Wen, Yangyi; Ren, Qilong

    2011-12-05

    The demand for high purity d-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) is increasing with the exploitation of TPGS-related products. Previously, we synthesized a TPGS mixture by esterifying vitamin E succinate with polyethyleneglycol 1000. In this study, a temperature-modulated silica gel chromatographic column was used to purify the synthesized TPGS. Taguchi method was used to optimize purification conditions associated with column temperature, loading amount, feedstock concentration and flow rate of mobile phases. Purification efficacy under the Taguchi optimized conditions was predicted theoretically and the predicted results were verified experimentally. High-performance liquid chromatography was used to quantify the unpurified and purified TPGS. The Taguchi-based analysis separately produced an optimum combination of purification conditions for TPGS purity and recovery. Under the optimized conditions, both the theoretical prediction and the confirmatory experiment yielded TPGS purity and recovery approximating to 98% each. Impressively, the study also found that column temperature had a considerable effect on purification efficacy, in particular on TPGS purity, although it was a less influential factor compared to loading amount and feedstock concentration.

  20. Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina.

    PubMed Central

    Piccinini, M; Merighi, A; Bruno, R; Cascio, P; Curto, M; Mioletti, S; Ceruti, C; Rinaudo, M T

    1996-01-01

    Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity. PMID:8809066

  1. Hemoglobin Ypsilanti: a high-oxygen-affinity hemoglobin demonstrated by two automated high-pressure liquid chromatography systems.

    PubMed

    Mais, Daniel D; Boxer, Laurence A; Gulbranson, Ronald D; Keren, David F

    2007-11-01

    Hemoglobin (Hb) Ypsilanti is a rare high-oxygen-affinity hemoglobin. Like other high-oxygen-affinity hemoglobins, Hb Ypsilanti manifests as erythrocytosis. Because the migration of many high-oxygen-affinity variants on alkaline and acid gels does not differ from that of HbA, oxygen-hemoglobin dissociation studies are often used to document their presence. Hb Ypsilanti is a notable exception because its electrophoresis pattern on alkaline gel is highly characteristic, exemplifying the phenomenon of hybrid formation in variant hemoglobins. In the past few years, several laboratories have begun to use high-pressure liquid chromatography (HPLC) as a screen for hemoglobinopathies. We demonstrate the elution profile of Hb Ypsilanti on the 2 most widely used HPLC methods.

  2. Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing.

    PubMed

    Motejadded, Hassan; Altenbuchner, Josef

    2009-04-01

    An E. coli vector system was constructed which allows the expression of fusion genes via a L: -rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.

  3. Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments

    PubMed Central

    Nesvizhskii, Alexey I.

    2013-01-01

    Analysis of protein interaction networks and protein complexes using affinity purification and mass spectrometry (AP/MS) is among most commonly used and successful applications of proteomics technologies. One of the foremost challenges of AP/MS data is a large number of false positive protein interactions present in unfiltered datasets. Here we review computational and informatics strategies for detecting specific protein interaction partners in AP/MS experiments, with a focus on incomplete (as opposite to genome-wide) interactome mapping studies. These strategies range from standard statistical approaches, to empirical scoring schemes optimized for a particular type of data, to advanced computational frameworks. The common denominator among these methods is the use of label-free quantitative information such as spectral counts or integrated peptide intensities that can be extracted from AP/MS data. We also discuss related issues such as combining multiple biological or technical replicates, and dealing with data generated using different tagging strategies. Computational approaches for benchmarking of scoring methods are discussed, and the need for generation of reference AP/MS datasets is highlighted. Finally, we discuss the possibility of more extended modeling of experimental AP/MS data, including integration with external information such as protein interaction predictions based on functional genomics data. PMID:22611043

  4. 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

  5. Using ProHits to store, annotate, and analyze affinity purification-mass spectrometry (AP-MS) data.

    PubMed

    Liu, Guomin; Zhang, Jianping; Choi, Hyungwon; Lambert, Jean-Philippe; Srikumar, Tharan; Larsen, Brett; Nesvizhskii, Alexey I; Raught, Brian; Tyers, Mike; Gingras, Anne-Claude

    2012-09-01

    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.

  6. Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins

    PubMed Central

    Qin, Guochen; Ma, Jun; Chen, Xiaomei; Chu, Zhaoqing; She, Yi-Min

    2017-01-01

    Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using 14N/15N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins. PMID:28224978

  7. 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

  8. Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins.

    PubMed

    Qin, Guochen; Ma, Jun; Chen, Xiaomei; Chu, Zhaoqing; She, Yi-Min

    2017-02-22

    Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using (14)N/(15)N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins.

  9. AraUTP-Affi-Gel 10: a novel affinity absorbent for the specific purification of DNA polymerase alpha-primase.

    PubMed

    Izuta, S; Saneyoshi, M

    1988-10-01

    For the specific purification of eukaryotic DNA-dependent DNA polymerase alpha, we prepared two novel affinity resins bearing 5-(E)-(4-aminostyryl) araUTP as a ligand. One of them was araUTP-Sepharose 4B which was coupled directly with the ligand and the other was araUTP-Affi-Gel 10 which was coupled with the ligand through a spacer. No DNA polymerase alpha-primase activity from cherry salmon (Oncorhynchus masou) testes was bound on the araUTP-Sepharose 4B in all cases examined. On the other hand, the araUTP-Affi-Gel 10 retains this enzyme activity when poly(dA) or poly(dA)-oligo(dT)12-18 is present. The retained enzyme activity was sharply eluted around 100-mM KCl concentrations as a single peak, and this fraction showed a specific activity of about 170,000 units/mg as alpha-polymerase activity. The highly purified DNA polymerase alpha-primase isolated using the araUTP-Affi-Gel 10 contained only three polypeptides, which showed Mr values of 120,000, 62,000, and 58,000, respectively, as judged using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  10. Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein.

    PubMed

    Sommer, Benjamin; Friehs, Karl; Flaschel, Erwin; Reck, Michael; Stahl, Frank; Scheper, Thomas

    2009-03-25

    Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to alpha-1,4-glucans.

  11. Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of antibodies for early diagnosis of multiple sclerosis patients.

    PubMed

    Horak, Daniel; Hlidkova, Helena; Kit, Yurii; Stoika, Rostyslav; Antonyuk, Volodymyr; Myronovsky, Severyn

    2017-03-28

    The aim of this work is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ca. 4 µm in diameter and containing ~ 1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and [(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier-transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS-PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

  12. Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.

    PubMed

    Zacharis, Constantinos K; Kalaitzantonakis, Eftichios A; Podgornik, Ales; Theodoridis, Georgios

    2007-03-09

    In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).

  13. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high performance affinity chromatography.

    PubMed

    Li, Haiyan; Ge, Jingwen; Guo, Tao; Yang, Shuo; He, Zhonggui; York, Peter; Sun, Lixin; Xu, Xu; Zhang, Jiwen

    2013-08-30

    It is challenging and extremely difficult to measure the kinetics of supramolecular systems with extensive, weak binding (Ka<10(5)M(-1)), and fast dissociation, such as those composed of cyclodextrins and drugs. In this study, a modified peak profiling method based on high performance affinity chromatography (HPAC) was established to determine the dissociation rate constant of cyclodextrin supramolecular systems. The interactions of β-cyclodextrin with acetaminophen and sertraline were used to exemplify the method. The retention times, variances and the plate heights of the peaks for acetaminophen or sertraline, conventional non-retained substance (H2O) on the β-cyclodextrin bonded column and a control column were determined at four flow rates under linear elution conditions. Then, plate heights for the theoretical non-retained substance were estimated by the modified HPAC method, in consideration of the diffusion and stagnant mobile phase mass transfer. As a result, apparent dissociation rate constants of 1.82 (±0.01)s(-1) and 3.55 (±0.37)s(-1) were estimated for acetaminophen and sertraline respectively at pH 6.8 and 25°C with multiple flow rates. Following subtraction of the non-specific binding with the support, dissociation rate constants were estimated as 1.78 (±0.00) and 1.91 (±0.02)s(-1) for acetaminophen and sertraline, respectively. These results for acetaminophen and sertraline were in good agreement with the magnitude of the rate constants for other drugs determined by capillary electrophoresis reported in the literature and the peak fitting method we performed. The method described in this work is thought to be suitable for other supramolecules, with relatively weak, fast and extensive interactions.

  14. Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography.

    PubMed

    Guo, Xiujie; Zhang, Xiuli; Feng, Jiatao; Guo, Zhimou; Xiao, Yuansheng; Liang, Xinmiao

    2013-04-01

    Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C18 columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C18 column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C18 and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C18 column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.

  15. Process for purification of monoclonal antibody expressed in transgenic Lemna plant extract using dextran-coated charcoal and hexamer peptide affinity resin.

    PubMed

    Naik, Amith D; Menegatti, Stefano; Reese, Hannah R; Gurgel, Patrick V; Carbonell, Ruben G

    2012-10-19

    The production of therapeutic proteins using transgenic plants offers several advantages, including low production cost, absence of human pathogens, presence of glycosylation mechanisms, and the ability to fold complex therapeutic proteins into their proper conformation. However, impurities such as phenolic compounds and pigments encountered during purification are quite different from those faced during purification from mammalian cell culture supernatants. This paper deals with the development of a pretreatment and affinity separation process for the purification of a monoclonal antibody from transgenic Lemna plant extract. A pretreatment step is described using dextran-coated charcoal for the removal of pigments and phenolic compounds without reducing the antibody concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial polymethacrylate resin is used for the capture and purification of MAb from the pretreated plant extract. The final yield and purity of the MAb obtained were 90% and 96% respectively. The performance of the hexamer peptide resin after the pretreatment step was found to be similar to that obtained with a commercial Protein A resin.

  16. 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…

  17. High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.

    PubMed

    Madadkar, Pedram; Nino, Sergio Luna; Ghosh, Raja

    2016-11-01

    We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution.

  18. Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry.

    PubMed

    Nwokeoji, Alison O; Kung, An-Wen; Kilby, Peter M; Portwood, David E; Dickman, Mark J

    2017-02-10

    RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry.

  19. 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.

  20. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    PubMed

    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.

  1. Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

    PubMed Central

    Murphy, Patrick J. M.

    2014-01-01

    Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

  2. A comprehensive classification of solvent systems used for natural product purifications in countercurrent and centrifugal partition chromatography.

    PubMed

    Skalicka-Woźniak, Krystyna; Garrard, Ian

    2015-11-01

    Using both library paper copies and modern electronic copies, every known, published, English-language journal paper that employs either countercurrent or centrifugal partition chromatography solvent systems for natural product purifications has been studied and the solvent systems classified in a comprehensive database. Papers were studied from the earliest found examples containing natural product separations in 1984 until the end of 2014. In total, 2594 solvent systems have been classified, of which 272 are gradient systems. To observe any trends or patterns in the data, the natural product solutes were divided into 21 classes and the solvent systems into 7 different types. The complete database, sorted according to natural product class, is available for download to assist separation scientists in future liquid-liquid chromatography purifications.

  3. 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.

  4. Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form.

    PubMed

    Imamura, Morikazu; Kato, Nobuko; Iwamaru, Yoshifumi; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

    2017-01-02

    We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrP(Sc)) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrP(C) source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.

  5. Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

    PubMed Central

    Fogen, Dawson; Wu, Sau-Ching; Ng, Kenneth Kai-Sing; Wong, Sui-Lam

    2015-01-01

    To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability. PMID:26406477

  6. Large-scale purification of IgM from human sera. Comparison of three optimized procedures utilizing protein A chromatography.

    PubMed

    Mauch, H; Kümel, G; Hammer, H J

    1980-01-01

    for the preparation of gram amounts of IgM from human sera sedimentation at 100,000 g or treatment with ZnSO4 of the redissolved "euglobulin"-precipitate was compared to direct precipitation from the clarified serum by boric acid. Three alternative large scale purification procedures were developed, leading to an IgM-sample characterized as pure by various criteria. Inclusion of protein A chromatography proved to enhance the yield very considerably.

  7. Industrial case study on alkaloids purification by pH-zone refining centrifugal partition chromatography.

    PubMed

    Kotland, Alexis; Chollet, Sébastien; Diard, Catherine; Autret, Jean-Marie; Meucci, Jeremy; Renault, Jean-Hugues; Marchal, Luc

    2016-11-25

    The industrial potential of pH-zone refining centrifugal partition chromatography has been evaluated by studying the purification of pharmaceutical ingredients at the pilot scale. For the first time, a scale up methodology based on both column capacity and mass transfer efficiency as invariants was developed. The purification of catharanthine and vindoline from an industrial crude extract of aerial parts of Catharanthus roseus, was used as a case of study. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system, triethylamine as retainer in the organic stationary phase and sulphuric acid as displacer in the aqueous mobile phase. The separation intensification was performed on a 36mL CPC column equipped with 832 partition twin-cells. The combined effects of four parameters (displacer and retainer concentrations for intensive parameters, flow rate and rotational speed for extensive parameters) were studied by design of experiment in order to maximize both recoveries and productivities. Then, scale change was done on two larger columns (305mL and 1950mL of capacity) equipped with only 231 and 238 partition cells. For this step, it has been shown that the global mass transfer coefficient k0a (the efficiency of a column design) and the stationary phase retention (the capacity of the column) were relevant and useful scale up invariants. A CPC model based on acid-base equilibriums and interfacial mass transfer in continuously stirred tank reactors in series was used to predict fully separations on larger CPC column at the optimized operating conditions and to guide the CPC user in its scale-up strategy. The experimental validation on pilot CPC column, by injecting up to 150g of Catharanthus roseus crude extract on the 1950mL column highlighted the preservation of the separation quality, the non-linear character of the scale up in centrifugal partition chromatography and that a productivity of about 4kg of processed crude extract per day can be reached

  8. 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.

  9. Purification of a catalase from Thermus thermophilus via IMAC chromatography: effect of the support.

    PubMed

    Hidalgo, Aurelio; Betancor, Lorena; Mateo, Cesar; Lopez-Gallego, Fernando; Moreno, Renata; Berenguer, Jose; Guisán, José M; Fernández-Lafuente, Roberto

    2004-01-01

    A hexameric Mn-catalase was purified from crude extracts of Thermus thermophilus using ammonium sulfate precipitation and ion metal-chelate affinity chromatography (IMAC). Eupergit 250 and Sepabeads FP-EP3 epoxy supports derivatized with iminodiacetic acid (IDA) and copper were used, at similar micromole/packed milliliter of support. Although Eupergit 250-IDA-Cu support adsorbed 80% of the total proteins in the extract, it exhibited a minimum affinity for the catalase. On the other hand, Sepabeads FP-EP3-IDA-Cu allowed the full adsorption of the catalase activity, which could be desorbed in fractions of different purity. This was attributed to a different geometrical congruence of the support surfaces with the enzyme surface, resulting in a different ability to form multipoint interactions with the proteins. Thus, by a cleanup step, followed by a negative chromatographic step using Eupergit 250-IDA-Cu2+ and by the adsorption of the catalase on Sepabeads-IDA-Cu2+ support, a pure enzyme fraction was obtained and its N-terminal end was sequenced.

  10. Fully automated protein purification

    PubMed Central

    Camper, DeMarco V.; Viola, Ronald E.

    2009-01-01

    Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that will allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion-exchange, metal affinity, hydrophobic interaction and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol. PMID:19595984

  11. 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.

  12. G-quadruplex on oligo affinity support (G4-OAS): an easy affinity chromatography-based assay for the screening of G-quadruplex ligands.

    PubMed

    Musumeci, Domenica; Amato, Jussara; Randazzo, Antonio; Novellino, Ettore; Giancola, Concetta; Montesarchio, Daniela; Pagano, Bruno

    2014-05-06

    A simple, cheap, and highly reproducible affinity chromatography-based method has been developed for the screening of G-quadruplex binders. The tested compounds were flowed through a polystyrene resin functionalized with an oligonucleotide able to form, in proper conditions, a G-quadruplex structure. Upon cation-induced control of the folding/unfolding processes of the immobilized G-quadruplex-forming sequence, small molecules specifically interacting with the oligonucleotide structure were first captured and then released depending on the used working solution. This protocol, first optimized for different kinds of known G-quadruplex ligands and then applied to a set of putative ligands, has allowed one to fully reuse the same functionalized resin batch, recycled for several tens of experiments without loss in efficiency and reproducibility.

  13. [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.

  14. Sample injection strategy to increase throughput in counter-current chromatography: Case study of Honokiol purification.

    PubMed

    Peng, Aihua; Hewitson, Peter; Ye, Haoyu; Zu, Liansuo; Garrard, Ian; Sutherland, Ian; Chen, Lijuan; Ignatova, Svetlana

    2016-12-09

    Counter-current chromatography (CCC) has been widely used as a preparative separation method to purify natural products from plant extracts and fermentation broths. Traditionally, throughput optimization in CCC has focused on sample concentration and sample volume. In this paper sample injection was considered as consisting of three variables: injection flow rate, post-injection flow rate and sample solvent. The effects of these parameters were studied using a honokiol purification from a Magnolia officinalis bark extract as a case study aiming to achieve the highest throughput/yield ratio for greater than 99% purity of this potential anti-cancer drug obtained for submission to the Chinese FDA. An injection method was established that increased the throughput of honokiol by 46.5% (from 3.05g/h to 4.47g/h), and decreased the solvent consumption of mobile phase and stationary phase per gram of honokiol by 40.0% (from 0.68L/g to 0.41L/g) and 48.4% (from 0.40L/g to 0.21L/g) respectively. These results show the importance of understanding the whole injection process when optimizing a given CCC separation.

  15. Purification of human seminal plasma no. 7 antigen by immunoaffinity chromatography on bound monoclonal antibody.

    PubMed Central

    Isojima, S; Koyama, K; Fujiwara, N

    1982-01-01

    Human seminal plasma (HSP) No. 7 antigen was purified by immunoaffinity chromatography on bound 1C4 monoclonal antibody (Moab) (Shigeta et al., 1980b). The pooled HSP protein was applied to a CNBr-activated Sepharose 4B column of bound 1C4 Moab gamma globulin and the antibody bound fraction (fr) eluted was further purified by rechromatography in the same way. The purified antigen in the antibody bound fr obtained by rechromatography gave a single band on SDS-PAGE in a position corresponding to a molecular weight of 15,000 daltons. This preparation was 196.2 times more effective than the original HSP protein in neutralizing the sperm immobilizing activity of 1C4 Moab. The purified HSP No. 7 antigen contained iron, but was different from lactoferrin and transferrin. It did not show any enzymatic activities, such as those of acid phosphatase, LDH or trypsin inhibitor, and shared antigenicity with human milk protein. It was present in seminal plasma as a molecule with a higher molecular weight but seemed to be cleaved to a monomer of 15,000 daltons during purification procedures. This antigen is present on spermatozoa as sperm-coating antigen and the corresponding antibody can immobilize spermatozoa with complement. Images Fig. 3 PMID:7127911

  16. 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.

  17. 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

  18. Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography.

    PubMed

    Wang, Fei; Guo, Jinjin; Bai, Quan; Wang, Lili

    2014-01-01

    Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion-exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10(8) L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR.

  19. 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.

  20. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

    PubMed

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-16

    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction.

  1. 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.

  2. The antigenicity in guinea pigs and monkeys of three mycobacterial polysaccharides purified by affinity chromatography with concanavalin A.

    PubMed

    Daniel, T M

    1975-06-01

    The antigenicity of 3 polysaccharides purified from culture filtrates of Mycobacterim tuberculosis by affinity chromatography using a concanavalin A-agarose absorbent was studied. All 3 purified polysaccharides were found to be potent elicitors of delayed skin test reactions in sensitized guinea pigs and in a tuberculos monkey. This antigenicity could not be attributed to contaminating protein. Small dermal reactions were also observed in control guinea pigs. All 3 polysaccharides reacted with precipitating antibody in guinea pig sera, the antigenic specificity observed with the guinea pig sera differing from that demonstrated with reference goat antiserum. The 3 polysaccharides were also demonstrated to contain hemagglutination antigenic sites.

  3. 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.

  4. SAINT-MS1: protein-protein interaction scoring using label-free intensity data in affinity purification-mass spectrometry experiments.

    PubMed

    Choi, Hyungwon; Glatter, Timo; Gstaiger, Mathias; Nesvizhskii, Alexey I

    2012-04-06

    We present a statistical method SAINT-MS1 for scoring protein-protein interactions based on the label-free MS1 intensity data from affinity purification-mass spectrometry (AP-MS) experiments. The method is an extension of Significance Analysis of INTeractome (SAINT), a model-based method previously developed for spectral count data. We reformulated the statistical model for log-transformed intensity data, including adequate treatment of missing observations, that is, interactions identified in some but not all replicate purifications. We demonstrate the performance of SAINT-MS1 using two recently published data sets: a small LTQ-Orbitrap data set with three replicate purifications of single human bait protein and control purifications and a larger drosophila data set targeting insulin receptor/target of rapamycin signaling pathway generated using an LTQ-FT instrument. Using the drosophila data set, we also compare and discuss the performance of SAINT analysis based on spectral count and MS1 intensity data in terms of the recovery of orthologous and literature-curated interactions. Given rapid advances in high mass accuracy instrumentation and intensity-based label-free quantification software, we expect that SAINT-MS1 will become a useful tool allowing improved detection of protein interactions in label-free AP-MS data, especially in the low abundance range.

  5. SAINT-MS1: protein-protein interaction scoring using label-free intensity data in affinity purification – mass spectrometry experiments

    PubMed Central

    Choi, Hyungwon; Glatter, Timo; Gstaiger, Mathias; Nesvizhskii, Alexey I.

    2013-01-01

    We present a statistical method SAINT-MS1 for scoring protein-protein interactions based on the label-free MS1 intensity data from affinity purification - mass spectrometry (AP-MS) experiments. The method is an extension of Significance Analysis of INTeractome (SAINT), a model-based method previously developed for spectral count data. We reformulated the statistical model for the log-transformed intensity data, including adequate treatment of missing observations, i.e. interactions whose quantitative data are inconsistent over replicate purifications. We demonstrate the performance of SAINT-MS1 using two recently published datasets: a small LTQ-Orbitrap dataset with three replicate purifications of single human bait protein and control purifications, and a larger drosophila dataset targeting insulin receptor/target of rapamycin signaling pathway generated using an LTQ-FT instrument. Using the drosophila dataset, we also compare and discuss the performance of SAINT analysis based on spectral count and MS1 intensity data in terms of the recovery of orthologous and literature-curated interactions. Given rapid advances in high mass accuracy instrumentation and intensity-based label-free quantification software, we expect that SAINT-MS1 will become a useful tool allowing improved detection of protein interactions in label-free AP-MS data, especially in the low abundance range. PMID:22352807

  6. [Obtaining of ScFv-CBD fusion protein and its application for affinity purification of recombinant human interferon alpha2b].

    PubMed

    Hil'chuk, P V; Okuniev, O V; Pavlova, M V; Irodov, D M; Horbatiuk, O B

    2006-01-01

    The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.

  7. Analytical characterization of complex, biotechnological feedstocks by pH gradient ion exchange chromatography for purification process development.

    PubMed

    Kröner, Frieder; Hanke, Alexander T; Nfor, Beckley K; Pinkse, Martijn W H; Verhaert, Peter D E M; Ottens, Marcel; Hubbuch, Jürgen

    2013-10-11

    The accelerating growth of the market for proteins and the growing interest in new, more complex molecules are bringing new challenges to the downstream process development of these proteins. This results in a demand for faster, more cost efficient, and highly understood downstream processes. Screening procedures based on high-throughput methods are widely applied nowadays to develop purification processes for proteins. However, screening highly complex biotechnological feedstocks, such as complete cell lysates containing target proteins often expressed with a low titre, is still very challenging. In this work we demonstrate a multidimensional, analytical screening approach based on pH gradient ion exchange chromatography (IEC), gel electrophoresis and protein identification via mass spectrometry to rationally characterize a biotechnological feedstock for the purpose of purification process development. With this very simple characterization strategy a two-step purification based on consecutive IEC operations was rapidly laid out for the purification of a diagnostic protein from a cell lysate reaching a purity of ∼80%. The target protein was recombinantly produced using an insect cell expression system.

  8. 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.

  9. 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...

  10. Characterization and purification of iodinated bovine thyrotropin by high performance liquid chromatography

    SciTech Connect

    Stanton, P.G.; Hearn, M.T.

    1986-08-01

    Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of (/sup 125/I)bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact (/sup 125/I)bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

  11. 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

    2012-12-07

    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, 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.

  12. 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.

  13. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

    PubMed

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E; Yates, John R

    2011-11-04

    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.

  14. 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.

  15. Separation and analysis of cis-diol-containing compounds by boronate affinity-assisted micellar electrokinetic chromatography.

    PubMed

    Wang, Heye; Lü, Chenchen; Li, Hengye; Chen, Yang; Zhou, Min; Ouyang, Jian; Liu, Zhen

    2013-10-01

    Cis-diol-containing compounds (CDCCs) are usually highly hydrophilic compounds and are therefore difficult to separate by conventional reversed-phase-based micellar electrokinetic chromatography (MEKC) due to poor selectivity. Here, we report a new method, called boronate affinity-assisted micellar electrokinetic chromatography (BAA-MEKC), to solve this issue. A boronic acid with a hydrophobic alkyl chain was added to the background electrolyte, which acted as a modifier to adjust the selectivity. CDCCs can covalently react with the boronic acid to form negatively charged surfactant-like complexes, which can partition into micelles formed with a cationic surfactant. Thus, CDCCs can be separated according to the differential partition constants of their boronic acid complexes between the micellar phase and the surrounding aqueous phase. To verify this method, eight nucleosides were employed as the test compounds and their separation confirmed that the combination of boronate affinity interaction with MEKC can effectively enhance the separation of CDCCs. The effects of experimental conditions on the separation were investigated. Finally, the BAA-MEKC method was applied to the separation and analysis of nucleosides extracted from human urine. BAA-MEKC exhibited better selectivity and improved separation as compared with conventional MEKC and CZE. Successful quantitative analysis of urinary nucleosides by BAA-MEKC was demonstrated.

  16. 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%.

  17. 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.

  18. A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography.

    PubMed

    Passarinha, L A; Bonifácio, M J; Soares-da-Silva, P; Queiroz, J A

    2008-01-11

    Catechol-O-methyltransferase (COMT) is a significant target in protein engineering due to its role not only in normal brain function but also to its possible involvement in some human disorders. In this work, a new approach was employed for the purification of recombinant human soluble COMT (hSCOMT) using hydrophobic interaction chromatography, as the main isolation method, from an Escherichia coli culture broth. A simplified overall process flow is proposed. Indeed, with an optimized heterologous expression system for recombinant hSCOMT production, such as E. coli, it was possible to produce and recover the active monomeric enzyme directly from the cell crude culture broth either by a freeze/thaw or ultrasonication lysis step. The recombinant enzyme present in the bacterial soluble fraction, exhibited similar affinity for epinephrine (K(m) 276 [215; 337] microM) and the methyl donor (S-adenosyl-L-methionine, SAMe) (K(m) 36 [30; 41]microM) as human SCOMT. After the precipitation step by 55% of ammonium sulphate, a HIC step on the butyl-sepharose resin was found to be highly effective in selectively eluting a range of contaminating key proteins present in the concentrate soluble extract. Consequently, the partially purified eluate from HIC could then be loaded and polished by gel filtration in order to increase the process efficiency. The final product appeared as a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure resulted in a global 10.9-fold purification with a specific activity of 5500 nmol/h/mg of protein. The widespread applicability of the process, here described, to different COMT sources could make this protocol highly useful for all studies requiring purified and active COMT proteins.

  19. Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane.

    PubMed

    Zhao, Yingxin; Zhang, Wei; White, Michael A; Zhao, Yingming

    2003-08-01

    Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.

  20. Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH.

    PubMed

    Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Yamada, Atsushi; Endo, Mika; Koike, Tohru

    2006-10-01

    While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.

  1. Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

    PubMed Central

    Jergil, Bengt; Guilford, Hugh; Mosbach, Klaus

    1974-01-01

    1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3′:5′-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N6-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP–Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50–100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N6-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP–Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4374933

  2. Preparative isolation and purification of harpagoside from Scrophularia ningpoensis hemsley by high-speed counter-current chromatography.

    PubMed

    Tong, Shengqiang; Yan, Jizhong; Lou, Jianzhong

    2006-01-01

    The bioactive component harpagoside was successfully separated from the crude extract of Scrophularia ningpoensis Hemsley by one-step purification using high-speed counter-current chromatography (HSCCC). A two-phase solvent system containing n-butanol:ethyl acetate:water (1:9:10) was selected following consideration of the partition coefficient of the target compound. A 276 mg quantity of the crude extract was loaded onto a 250 mL HSCCC column and yielded 11 mg harpagoside at over 97% purity. The chemical structure of harpagoside was determined by HPLC-ESI/MS and 1H-NMR.

  3. 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.

  4. Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC).

    PubMed

    Orr, Valerie; Scharer, Jeno; Moo-Young, Murray; Honeyman, C Howie; Fenner, Drew; Crossley, Lisa; Suen, Shing-Yi; Chou, C Perry

    2012-07-01

    Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 24h and recombinant PAC with high purity (19 U/mg), yield (72% recovery), and productivity (41 mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins.

  5. Large scale purification of the SERCA inhibitor thapsigargin from Thapsia garganica L. roots using centrifugal partition chromatography.

    PubMed

    Ollivier, Anthony; Grougnet, Raphaël; Cachet, Xavier; Meriane, Djamila; Ardisson, Janick; Boutefnouchet, Sabrina; Deguin, Brigitte

    2013-05-01

    Thapsigargin (Tg) is a selective and irreversible inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-dependent pump at subnanomolecular concentrations. As such, it has become a powerful tool in the study of Ca(2+) signaling pathway. Purification of Tg from Thapsia species requires repeated chromatographic steps with normal-phase alumina or silica and reverse phase chromatography. We thus developed an innovative procedure coupling high pressure automatized extraction with centrifugal partition chromatography allowing a fast and safe large-scale isolation of highly pure Tg, in two steps from Thapsia garganica L. roots. Comparison of influence of extraction procedures, storage conditions and harvesting areas on Tg content in different Algerian specimens of Thapsia garganica L. roots has been precised by mean of HPLC quantification procedure. Highest Tg content were found in the fresh material of the sample from Setif area.

  6. 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.

  7. Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand.

    PubMed

    Stocker-Majd, Gisela; Hilbrig, Frank; Freitag, Ruth

    2008-06-13

    Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.

  8. [Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen].

    PubMed

    Wang, Yang-Mu; Bi, Jing-Xiu; Zhao, Lan; Zhou, Wei-Bin; Li, Yan; Huang, Yong-Dong; Zhang, Yan; Lin, Hai; Su, Zhi-Guo

    2006-03-01

    A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

  9. Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock.

    PubMed

    Davidoff, Andrew M; Ng, Catherine Y C; Sleep, Susan; Gray, John; Azam, Selina; Zhao, Yuan; McIntosh, Jenny H; Karimipoor, Morteza; Nathwani, Amit C

    2004-11-01

    Recombinant vectors based on the recently isolated AAV serotype 8 (rAAV-8) shows great promise for gene therapy, particularly for disorders affecting the liver. Transition of this vector system to the clinic, however, is limited by the lack of an efficient scaleable purification method. In this report, we describe a simple method for purification of rAAV-8 vector particles based on ion exchange chromatography that generates vector stocks with greater than 90% purity. The average yield of purified rAAV-8 from five different vector preparation was 41%. Electron microscopy of these purified stocks revealed typical icosohedral virions with less than 10% empty particles. Liver targeted delivery of ion-exchange purified rAAV-8 vector encoding the human factor IX (hFIX) gene, resulted in plasma hFIX levels approaching 30% of normal in immunocompetent mice, which is 20-fold higher than observed with an equivalent number of rAAV-5 ion exchange purified vector particles. The method takes less then 5 h to process and purify rAAV-8 vector from producer cells and represents a significant advance on the CsCl density centrifugation technique in current use for purification of rAAV-8 vector systems and will likely facilitate the transition of the rAAV-8 vector system to the clinic.

  10. A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

    PubMed

    Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

    2013-11-01

    Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHpurification step.

  11. 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.

  12. [Affinity chromatography and proteomic screening as the effective method for S100A4 new protein targets discovery].

    PubMed

    Koshelev, Iu A

    2014-01-01

    Affinity chromatography followed by a selective binding proteins identification can be using as effective method for a biological impotent interactions discovery. The molecular structure and their surface charge as and conformational regulation possibilities, which change their surface hydrophobic properties, all they should to taken in account during method optimization process. With the same' method we had identify some new S100A4 target proteins such as cytoskeleton proteins Sept2, Sept7, Sept11 and this interaction would can to highlight as S100A4 would regulate cell motility. Even we had identify the transcription cofactor Ddx5 and through such complex formation a S100A4 protein would can to regulate E-cadherin, p21 Waf1/Cip1), Bnip3 gene expression. The same protocol can be using for a target proteins search with another S100 protein family members, because their molecules demonstrate a high homology level in amino aside sequences and 3D structures.

  13. 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

  14. 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 %).

  15. Determination of soluble immunoglobulin G in bovine colostrum products by Protein G affinity chromatography-turbidity correction and method validation.

    PubMed

    Holland, Patrick T; Cargill, Anne; Selwood, Andrew I; Arnold, Kate; Krammer, Jacqueline L; Pearce, Kevin N

    2011-05-25

    Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.

  16. Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

    2007-04-10

    In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

  17. 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.

  18. Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.

    PubMed

    Teng, S F; Sproule, K; Husain, A; Lowe, C R

    2000-03-31

    A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.

  19. Ultra sensitive affinity chromatography on avidin-functionalized PMMA microchip for low abundant post-translational modified protein enrichment.

    PubMed

    Xia, Hui; Murray, Kermit; Soper, Steven; Feng, June

    2012-02-01

    Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. In this paper, we report a novel and sensitive affinity chromatography on the avidin-functionalized poly(methyl methacrylate) (PMMA) microchip for enrichment of nanogram (ng) amount of PTMs. The chemical modification of poly(methyl methacrylate) (PMMA) surfaces yield avidin-terminated PMMA surfaces after UV radiation and consecutive EDC mediated coupling (amide reaction). This functionalized PMMA micro-device was developed to identify and specifically trap biotinylated PTM proteins of low abundance from complex protein mixture. Here we selected carbonylated protein as a representative PTM to illustrate the wide application of this affinity microchip for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively by atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. Quantitative study of biotinlated carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this subproteome enrichment micro-device can be assembled with other lab-on-a-chip components for follow-up protein analysis.

  20. Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes.

    PubMed

    Ribeiro, Mariana Borsoi; Vijayalakshmi, Mookambesvaran; Todorova-Balvay, Daniele; Bueno, Sonia Maria Alves

    2008-01-01

    The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.

  1. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  2. Use and practice of achiral and chiral supercritical fluid chromatography in pharmaceutical analysis and purification.

    PubMed

    Lemasson, Elise; Bertin, Sophie; West, Caroline

    2016-01-01

    The interest of pharmaceutical companies for complementary high-performance chromatographic tools to assess a product's purity or enhance this purity is on the rise. The high-throughput capability and economic benefits of supercritical fluid chromatography, but also the "green" aspect of CO2 as the principal solvent, render supercritical fluid chromatography very attractive for a wide range of pharmaceutical applications. The recent reintroduction of new robust instruments dedicated to supercritical fluid chromatography and the progress in stationary phase technology have also greatly benefited supercritical fluid chromatography. Additionally, it was shown several times that supercritical fluid chromatography could be orthogonal to reversed-phase high-performance liquid chromatography and could efficiently compete with it. Supercritical fluid chromatography is an adequate tool for small molecules of pharmaceutical interest: synthetic intermediates, active pharmaceutical ingredients, impurities, or degradation products. In this review, we first discuss about general chromatographic conditions for supercritical fluid chromatography analysis to better suit compounds of pharmaceutical interest. We also discuss about the use of achiral and chiral supercritical fluid chromatography for analytical purposes and the recent applications in these areas. The use of preparative supercritical fluid chromatography by pharmaceutical companies is also covered.

  3. A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

    PubMed

    Gianazza, E; Arnaud, P

    1982-01-01

    The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.

  4. An illustration of the clinical relevance of detecting human antimouse antibody interference by affinity chromatography.

    PubMed

    Koper, N P; Massuger, L F; Thomas, C M; Beyer, C; Crooy, M J

    1999-10-01

    Elevated Cancer antigen 125 (CA 125) serum concentrations (up to 221 kU/1) were measured in a 39 year old woman with a positive family history of breast cancer. The serum determinations were performed with the automated Immulite OM-MA chemiluminescent enzyme immunoassay system (Diagnostic Products). Laparoscopic evaluation of the ovaries did not reveal any abnormalities. CA 125 measurements in the same patient using the automated IMx immunoassay system (Abbott) demonstrated normal serum levels. Using a previously reported chromatography procedure IgG type human antimouse antibody activity was found to be present in the serum samples explaining the falsely elevated levels. To prevent this interference the manufacturer modified the assay system by replacing the monoclonal M11 detection antibody with a rabbit polyclonal antibody. Using the modified OM-MA CA 125 assay results were comparable with the IMx values.

  5. Purification of high value proteins from particle containing potato fruit juice via direct capture membrane adsorption chromatography.

    PubMed

    Schoenbeck, I; Graf, A M; Leuthold, M; Pastor, A; Beutel, S; Scheper, T

    2013-12-01

    Potato fruit juice (PFJ) is a by-product from industrial starch production. It still contains several valuable components such as amino acids, minerals and proteins. An economic technology for the isolation and purification of different native potato proteins is the ion exchange chromatography, which can be performed either by classical bed chromatography or by membrane adsorption chromatography (MA-IEX). An already published MA-IEX process for the downstreaming of PFJ is based on the following steps: prefiltration/microfiltration, fractionation with MA-IEX, ultra-/diafiltration and finally drying. In order to further minimize process complexity and costs, new MA-IEX-modules were designed and tested in this research project to facilitate the processing of crude, particle-containing solutions using a tangential flow through the membranes. Modules with fleece polymer spacers and extruded polymer spacers, as well as different spacer channel sizes were tested for their binding capacities and their long-term stability. An optimized setup was found for the technical scale. Modules with extruded polymer spacers channel size 250 μm show the highest binding capacities (anion exchanger approx. 0.34 mg/cm(2), cation exchanger approx. 0.16 mg/cm(2)), while the modules with extruded polymer spacers channel size 480 μm show the best long-term stability with 23 passes without intermediary cleaning.

  6. Ion-exchange chromatography: basic principles and application to the partial purification of soluble mammalian prolyl oligopeptidase.

    PubMed

    Cummins, Philip M; Dowling, Oonagh; O'Connor, Brendan F

    2011-01-01

    Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

  7. Isolation and purification of two antioxidant isomers of resveratrol dimer from the wine grape by counter-current chromatography.

    PubMed

    Kong, Qingjun; Ren, Xueyan; Hu, Ruilin; Yin, Xuefeng; Jiang, Guoshan; Pan, Yuanjiang

    2016-06-01

    Resveratrol dimers belong to a group of compounds called stilbenes, which along with proanthocyanidins, anthocyanins, catechins, and flavonols are natural phenolic compounds found in grapes and red wine. Stilbenes have a variety of structural isomers, all of which exhibit various biological properties. Counter-current chromatography with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (2:5:4:5, v/v/v/v) was applied to isolate and purify stilbene from the stems of wine grape. Two isomers of resveratrol dimers trans-ε-viniferin and trans-δ-viniferin were obtained from the crude sample in a one-step separation, with purities of 93.2 and 97.5%, respectively, as determined by high-performance liquid chromatography. The structures of these two compounds were identified by (1) H and (13) C NMR spectroscopy. In addition, their antioxidant activities were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The antioxidant activities of trans-δ-viniferin were higher than that of trans-ε-viniferin in this model. This work demonstrated that counter-current chromatography is a powerful and effective method for the isolation and purification of polyphenols from wine grape. Additionally, the DPPH radical assay showed that the isolated component trans-δ-viniferin exhibited stronger antioxidant activities than trans-ε-viniferin and a little bit weaker than vitamin E at the same concentration.

  8. [Purification of ovalbumin from hen egg white by high-speed counter-current aqueous two-phase chromatography].

    PubMed

    Zhi, Wen-Bo; Deng, Qiu-Yun; Song, Jiang-Nan; Ouyang, Fan

    2005-01-01

    High-speed counte-recurrent chromatography (HSCCC) is a continuous liquid-liquid partition chromatography without solid matrix, which has the significant features of high resolution and high recovery. The separation of bio-macromolecule in aqueous two-phase systems (ATPs) with HSCCC is still under research, and the establishment of high-speed counter-current aqueous two-phase chromatography (HSCCC-ATP) relies on the improvement of equipment structure and optimization of operation parameters. By using a multi-column high-speed counter-current chromatograph, the separation of protein mixture and the purification of ovalbumin from hen egg white were studied. The effects of pH and PEG concentration on the partition coefficients of proteins were tested in PEG1000-phosphate ATPs, and distinct differences among partition coefficients of proteins were found at pH 9.2 and 15.0% (W/W) PEG concentration in said system. The separation of protein mixture, consisting of cytochrome C, lysozyme and myoglobin was successfully performed in 15.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 0.8mL/min, using upper phase as stationary phase. pH and PEG concentration also had distinct effects on the partition coefficients of the major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme. The optimal pH value and PEG concentration for the purification of ovalbumin by HSCCC-ATP were found to be 9.2 and 16.0% (W/W) respectively. Ovalbumin was successfully purified to homogeneity from the hen egg white sample in 16.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 1.8mL/min, using upper phase as stationary phase. The purification recovery of ovalbumin was around 95%.

  9. Use of a tandem affinity purification assay to detect interactions between West Nile and dengue viral proteins and proteins of the mosquito vector.

    PubMed

    Colpitts, Tonya M; Cox, Jonathan; Nguyen, Annie; Feitosa, Fabiana; Krishnan, Manoj N; Fikrig, Erol

    2011-08-15

    West Nile and dengue viruses are (re)emerging mosquito-borne flaviviruses that cause significant morbidity and mortality in man. The identification of mosquito proteins that associate with flaviviruses may provide novel targets to inhibit infection of the vector or block transmission to humans. Here, a tandem affinity purification (TAP) assay was used to identify 18 mosquito proteins that interact with dengue and West Nile capsid, envelope, NS2A or NS2B proteins. We further analyzed the interaction of mosquito cadherin with dengue and West Nile virus envelope protein using co-immunoprecipitation and immunofluorescence. Blocking the function of select mosquito factors, including actin, myosin, PI3-kinase and myosin light chain kinase, reduced both dengue and West Nile virus infection in mosquito cells. We show that the TAP method may be used in insect cells to accurately identify flaviviral-host protein interactions. Our data also provides several targets for interrupting flavivirus infection in mosquito vectors.

  10. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes

    PubMed Central

    Cloutier, Philippe; Al-Khoury, Racha; Lavallée-Adam, Mathieu; Faubert, Denis; Jiang, Heng; Poitras, Christian; Bouchard, Annie; Forget, Diane; Blanchette, Mathieu; Coulombe, Benoit

    2015-01-01

    Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex. PMID:19450687

  11. One-pot glyco-affinity precipitation purification for enhanced proteomics: the flexible alignment of solution-phase capture/release and solid-phase separation.

    PubMed

    Sun, Xue-Long; Haller, Carolyn A; Wu, XiaoYi; Conticello, Vincent P; Chaikof, Elliot L

    2005-01-01

    A one-pot affinity precipitation purification of carbohydrate-binding protein was demonstrated by designing thermally responsive glyco-polypeptide polymers, which were synthesized by selective coupling of pendant carbohydrate groups to a recombinant elastin-like triblock protein copolymer (ELP). The thermally driven inverse transition temperature of the ELP-based triblock polymer is maintained upon incorporation of carbohydrate ligands, which was confirmed by differential scanning calorimetry and (1)H NMR spectroscopy experiments. As a test system, lactose derivatized ELP was used to selectively purify a galactose-specific binding lectin through simple temperature-triggered precipitation in a high level of efficiency. Potential opportunities might be provided for enhanced proteomic, cell isolation as well as pathogen detection applications.

  12. Affinity separation of human plasma gelsolin on Affi-Gel Blue.

    PubMed

    Yamamoto, H; Terabayashi, M; Egawa, T; Hayashi, E; Nakamura, H; Kishimoto, S

    1989-05-01

    Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.

  13. Recovery and purification process development for monoclonal antibody production

    PubMed Central

    Ma, Junfen; Winter, Charles; Bayer, Robert

    2010-01-01

    Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

  14. Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti-HIV mAb 4E10 from transgenic tobacco.

    PubMed

    Platis, Dimitris; Maltezos, Anastasios; Ma, Julian K-C; Labrou, Nikolaos E

    2009-01-01

    Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.

  15. Isolation of two molecular populations of human complement factor H by hydrophobic affinity chromatography.

    PubMed Central

    Ripoche, J; Al Salihi, A; Rousseaux, J; Fontaine, M

    1984-01-01

    Human complement factor H was prepared in highly purified form from fresh serum by euglobulin precipitation, DEAE-Sephacel chromatography and Sephacryl S-300 gel filtration. This preparation allowed the recovery of 37% of the initial factor H. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that factor H was homogeneous both in reduced and non-reduced media and exhibited a molecular mass of 150 kDa. Charge-shift experiments clearly showed the presence of hydrophobic sites in the factor H molecule. Charge shifts were observed with two detergent systems (Triton/sodium deoxycholate and Triton/cetyltrimethylammonium bromide). Factor H was able to bind to phenyl-Sepharose. This property allowed us to study two populations of factor H. These two populations exhibited the same physicochemical parameters, but revealed differences in their ability to aggregate in low- and iso-ionic-strength media. The molecular basis and biological significance of this heterogeneity are discussed. Images Fig. 3. Fig. 4. Fig. 5. PMID:6235808

  16. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  17. Rapid identification of regulatory microRNAs by miTRAP (miRNA trapping by RNA in vitro affinity purification).

    PubMed

    Braun, Juliane; Misiak, Danny; Busch, Bianca; Krohn, Knut; Hüttelmaier, Stefan

    2014-04-01

    MicroRNAs (miRNAs) control gene expression at the post-transcriptional level. However, the identification of miRNAs regulating the fate of a specific messenger RNA remains limited due to the imperfect complementarity of miRNAs and targeted transcripts. Here, we describe miTRAP (miRNA trapping by RNA in vitro affinity purification), an advanced protocol of previously reported MS2-tethering approaches. MiTRAP allows the rapid identification of miRNAs targeting an in vitro transcribed RNA in cell lysates. Selective co-purification of regulatory miRNAs was confirmed for the MYC- as well as ZEB2-3'UTR, two well-established miRNA targets in vivo. Combined with miRNA-sequencing, miTRAP identified in addition to miRNAs reported to control MYC expression, 18 novel candidates including not in silico predictable miRNAs. The evaluation of 10 novel candidate miRNAs confirmed 3'UTR-dependent regulation of MYC expression as well as putative non-canonical targeting sites for the not in silico predictable candidates. In conclusion, miTRAP provides a rapid, cost-effective and easy-to-handle protocol allowing the identification of regulatory miRNAs for RNAs of choice in a cellular context of interest. Most notably, miTRAP not only identifies in silico predictable but also unpredictable miRNAs regulating the expression of a specific target RNA.

  18. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  19. Model-based process development for the purification of a modified human growth hormone using multimodal chromatography.

    PubMed

    Sejergaard, Lars; Karkov, Hanne Sophie; Krarup, Janus Kristian; Hagel, Anne Birgitte Bagge; Cramer, Steven M

    2014-01-01

    This study demonstrates how the multimodal Capto adhere resin can be used in concert with calcium chloride or arginine hydrochloride as mobile phase modifiers to create a highly selective purification process for a modified human growth hormone. Importantly, these processes are shown to result in significant clearance of product related aggregates and host cell proteins. Furthermore, the steric mass action model is shown to be capable of accurately describing the chromatographic process and the aggregate removal. Finally, justification of the selected operating ranges is evaluated using the model together with Latin hypercube sampling. The results in this article establish the utility of multimodal chromatography when used with appropriate mobile phase modifiers for the downstream bioprocessing of a modified human growth hormone and offer new approaches for bioprocess verification.

  20. Isolation and purification of orientin and vitexin from Trollius chinensis Bunge by high-speed counter-current chromatography.

    PubMed

    Yu, Xiao-Xue; Huang, Jie-Yun; Xu, Dan; Xie, Zhi-Yong; Xie, Zhi-Sheng; Xu, Xin-Jun

    2014-01-01

    Orientin and vitexin are the two main bioactive compounds in Trollius chinensis Bunge. In this study, a rapid method was established for the isolation and purification of orientin and vitexin from T. chinensis Bunge using high-speed counter-current chromatography in one step, with a solvent system of ethyl acetate-ethanol-water (4:1:5, v/v/v). A total of 9.8 mg orientin and 2.1 mg vitexin were obtained from 100 mg of the ethyl acetate extract, with purities of 99.2% and 96.0%, respectively. Their structures were identified by UV, MS and NMR. The method was efficient and convenient, which could be used for the preparative separation of orientin and vitexin from T. chinensis Bunge.

  1. Steric exclusion chromatography for purification of cell culture-derived influenza A virus using regenerated cellulose membranes and polyethylene glycol.

    PubMed

    Marichal-Gallardo, Pavel; Pieler, Michael M; Wolff, Michael W; Reichl, Udo

    2017-02-03

    Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration. In this study, a 75cm(2) cellulose membrane adsorber was used for the purification of a clarified and inactivated influenza A virus broth produced in a 5L bioreactor using suspension Madin Darby canine kidney cells. Product recovery was above 95% based on hemagglutination activity and single radial immunodiffusion assays. Maximum depletion of double stranded host cell DNA and total protein was 99.7% and 92.4%, respectively. Purified virus particles showed no aggregation with a monodisperse peak around 84nm. 250mL of the clarified inactivated virus broth was purified within 40min. The surface area productivity based on the recovery of the viral hemagglutinin antigen was 28-50mgm(-2)h(-1) depending on the feed and loading conditions.

  2. Purification method development for chiral separation in supercritical fluid chromatography with the solubilities in supercritical fluid chromatographic mobile phases.

    PubMed

    Gahm, Kyung H; Tan, Helming; Liu, Jodi; Barnhart, Wesley; Eschelbach, John; Notari, Steve; Thomas, Samuel; Semin, David; Cheetham, Janet

    2008-04-14

    A comprehensive approach was applied to develop a chiral purification method for an analyte that was found to be unusually difficult to scale-up in supercritical fluid chromatography (SFC). This was performed by studying major factors such as the solubility of an analyte in SFC mobile phases, impurity profiles, and cycle time. For this case study, the solubility in SFC mobile phase was measured by a packed column technique, coupled with a novel trapping mechanism to enhance measurement precision in SFC conditions. The solubility studies in SFC mobile phases suggested a couple of possible SFC mobile phases, in which the analyte would potentially be most soluble. The SFC methods were developed to purify a sample containing 15% of an impurity, after considering impurity profiles and cycle times of several potential methods in addition to SFC mobile phase solubility. An equal volume mixture of acetonitrile and ethanol was chosen for the final purification method, since this mixture demonstrated the relatively high SFC solubility among all solvent combinations with enhanced resolution between the analyte and the impurity as well as the shortest run time. The solubility of the compound was also determined in various organic solvents using a high throughput solubility screening system to better understand relative change of solubility from neat solution to SFC mobile phases.

  3. Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography.

    PubMed

    Bermejo, Ruperto; Acién, F Gabriel; Ibáñez, M José; Fernández, José M; Molina, Emilio; Alvarez-Pez, José M

    2003-06-25

    B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae. Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics. Tedious methodologies for B-PE purification have been published. In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum. Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid-sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h(-1). After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer. In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid-sodium acetate buffer, pH 5.5. With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies. The B-PE purity was tested using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopic characterization.

  4. Alternative purification method for recombinant measles viral nucleoprotein expressed in insect cells by ion-exchange chromatography.

    PubMed

    Lee, Han Saem; Kim, You-Jin; Yang, Jeongsun; Yoon, Hee Sook; Kim, Seung Tae; Kim, Kisoon

    2014-03-01

    Recombinant measles virus nucleoproteins (rMeV N) and fusion (F) proteins were characterized as major antigenic proteins expressed in insect cells mediated by recombinant baculoviruses (rBVs). Band intensities were analyzed by Western blotting to recognize IgG and IgM antibodies against the rMeV N and F proteins in human sera and cerebrospinal fluids (CSFs) from patients with measles infections. Positive results from the blots using the rMeV N were consistent with the results of enzyme-linked immunosorbent assays (ELISAs) in which whole viral proteins were used as antigens. Human sera and CSFs reacted more strongly with the rMeV N than with the rMeV F proteins prepared in an identical expression system. For efficient and reliable purification, ion-exchange chromatography using Source Q anion resin was applied, and high-purity rMeV N protein was harvested. To characterize the similarity with the native viral protein to purified N protein, structural mimicry of purified recombinant proteins with intact rMeV N was shown through transmission electron microscopy, and the truncation and the phosphorylation status of the expressed protein were analyzed. These results suggest that the rMeV N purified by ion-exchange chromatography has features similar to those of naïve N including a self-assembled structure, phosphorylation and antigenic function. Thus, these expression and purification methods can be applied to the large-scale production of the rMeV N, which is essential for the development of new diagnostic tools and vaccines for acute and chronic MeV infections.

  5. Comparative study of glycated hemoglobin by ion exchange chromatography and affinity binding nycocard reader in type 2 diabetes mellitus.

    PubMed

    Gautam, N; Dubey, R K; Jayan, A; Nepaune, Y; Padmavathi, P; Chaudhary, S; Jha, S K; Sinha, A K

    2014-12-01

    The aim of this study was to compare the level of glycated hemoglobin (HbA1c) in type 2 Diabetes Mellitus (DM) patients by two different methods namely Ion Exchange Chromatography and Affinity Binding Nycocard Reader. This is a cross-sectional study conducted on confirmed type 2 diabetes mellitus patients (n = 100) who visited Out Patients Department of the Universal College of Medical Sciences Teaching hospital, Bhairahawa, Nepal from November 2012 to March 2013. The diagnosis of diabetes mellitus was done on the basis of their fasting (164.46 ± 45.33 mg/dl) and random (187.93 ± 78.02 mg/dl) serum glucose level along with clinical history highly suggestive of type 2 DM. The HbA1c values of (7.8 ± 1.9%) and (8.0 ± 2.2%) were found in DM patients as estimated by those two different methods respectively. The highest frequency was observed in HbA1c > 8.0% indicating maximum cases were under very poor glycemic control. However, there were no significant differences observed in HbA1c value showing both methods are comparable in nature and can be used in lab for ease of estimation. The significant raised in HbA1c indicates complications associated with DM and monitoring of therapy become hard for those patients. Despite having standard reference method for HbA1c determination, the availability of report at the time of the patient visit can be made easy by using Nycocard Reader and Ion Exchange Chromatography techniques without any delay in communicating glycemic control, clinical decision-making and changes in treatment regimen.

  6. RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments

    ERIC Educational Resources Information Center

    Bailey, Cheryl P.

    2009-01-01

    This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

  7. Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: A study using the hydatid vaccine EG95

    PubMed Central

    Read, A.J.; Gauci, C.G.; Lightowlers, M.W.

    2009-01-01

    The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95. PMID:19349218

  8. Chromatography.

    ERIC Educational Resources Information Center

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  9. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography.

    PubMed

    Machado, Gleyce Alves; Oliveira, Heliana Batista de; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-05-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

  10. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST.

  11. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

    SciTech Connect

    Niture, Suryakant K.; Doneanu, Catalin E.; Velu, Chinavenmeni S.; Bailey, Nathan I.; Srivenugopal, Kalkunte S. . E-mail: Kalkunte.srivenugopal@ttuhsc.edu

    2005-12-02

    Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

  12. Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer

    PubMed Central

    Ambrose, Sarah R.; Gordon, Naheema S.; Goldsmith, James C.; Wei, Wenbin; Zeegers, Maurice P.; James, Nicholas D.; Knowles, Margaret A.; Bryan, Richard T.; Ward, Douglas G.

    2015-01-01

    Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun proteomics. None of the lectins showed a strong preference for bladder cancer cell lines over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over those from non-cancer controls. However, several lectins showed a strong preference for bladder cell line glycans over serum glycans and are potentially useful for enriching glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1 as proteins warranting further investigation as urinary biomarkers for low-grade bladder cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring lectin binding to unfractionated proteomes, but it is possible that more specific reagents and/or a focus on individual proteins may produce clinically useful biomarkers. PMID:28248271

  13. Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques

    NASA Astrophysics Data System (ADS)

    Zhu, Feifei; Trinidad, Jonathan C.; Clemmer, David E.

    2015-07-01

    Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.

  14. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

    PubMed Central

    Machado, Gleyce Alves; de Oliveira, Heliana Batista; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-01-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (Junbound) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJunbound) and aqueous (AJunbound) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for Junbound, 92.5% and 93.5% for DJunboundand 82.5% and 82.6% for AJunbound. By immunoblot, the DJunboundfraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJunboundfraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot. PMID:23778661

  15. LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.

    PubMed

    Song, Ehwang; Zhu, Rui; Hammoud, Zane T; Mechref, Yehia

    2014-11-07

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC-MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC-ESI-MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts.

  16. In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles.

    PubMed

    Gädke, Johannes; Kleinfeldt, Lennart; Schubert, Chris; Rohde, Manfred; Biedendieck, Rebekka; Garnweitner, Georg; Krull, Rainer

    2017-01-20

    This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time, >85% of the product was adsorbed onto the particles. The particles were magnetically separated using handheld neodymium magnets and the product was eluted. The GNTA-SPION were successfully regenerated and reused in five consecutive cycles. In the one-step purification, the purity of the product reached >99.9% regarding protein A. A very low particle concentration of 0.5g/L was sufficient for effective product separation. Bacterial growth was not influenced negatively by this concentration. Particle analysis showed similar properties between freshly synthesized and regenerated GNTA-SPION. The overall process efficiency was however influenced by partial disintegration of particle agglomerates and thus loss of particles. The demonstration of very fast in situ product removal from growing bacterial culture combined with a very high product purity within one step shows possibilities for automated large scale purification combined with recycling of biomass.

  17. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  18. Microwave-Assisted Extraction and Purification of Arctiin and Arctigenin from Fructus Arctii by High-Speed Countercurrent Chromatography.

    PubMed

    Lü, Haitao; Sun, Zhaoyun; Shan, Hu; Song, Jiying

    2016-03-01

    An efficient method for the rapid extraction, separation and purification of bioactive lignans, arctiin and arctigenin, from Fructus arctii by microwave-assisted extraction coupled with high-speed countercurrent chromatography was developed. The optimal extraction conditions of arctiin and arctigenin were evaluated by orthogonal array. Arctigenin could be converted from arctiin by hydrochloric acid hydrolysis. The separations were performed at a preparative scale with two-phase solvents composed of ethyl acetate-ethanol-water (5 : 1 : 5, v/v/v) for arctiin, and n-hexane-ethyl acetate-ethanol-water (4 : 4 : 3 : 4, v/v/v/v) for arctigenin. From 500 mg of crude extract sample, 122.3 mg of arctiin and 45.7 mg of arctigenin were obtained with the purity of 98.46 and 96.57%, and the recovery of 94.3 and 81.6%, respectively. Their structures were determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as UV, FT-IR, electrospray ion source (ESI)-MS, (1)H-NMR and (13)C-NMR spectrum. According to the antioxidant activity assay, arctigenin had stronger 1,1-diphenyl-2-picrylhydrazyl free radicals scavenging activity.

  19. Preparative isolation and purification of phlorotannins from Ecklonia cava using centrifugal partition chromatography by one-step.

    PubMed

    Lee, Ji-Hyeok; Ko, Ju-Young; Oh, Jae-Young; Kim, Chul-Young; Lee, Hee-Ju; Kim, Jaeil; Jeon, You-Jin

    2014-09-01

    Various bioactive phlorotannins of Ecklonia cava (e.g., dieckol, eckol, 6,6-bieckol, phloroglucinol, phloroeckol, and phlorofucofuroeckol-A) are reported. However, their isolation and purification are not easy. Centrifugal partition chromatography (CPC) can be used to efficiently purify the various bioactive-compounds efficiently from E. cava. Phlorotannins are successfully isolated from the ethyl acetate (EtOAc) fraction of E. cava by CPC with a two-phase solvent system comprising n-hexane:EtOAc:methanol:water (2:7:3:7, v/v) solution. The dieckol (fraction I, 40.2mg), phlorofucofuroeckol-A (fraction III, 31.1mg), and fraction II (34.1mg) with 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are isolated from the crude extract (500 mg) by a one-step CPC system. The purities of the isolated dieckol and phlorofucofuroeckol-A are ⩾90% according to high performance liquid chromatography (HPLC) and electrospray ionization multi stage tandem mass spectrometry analyses. The purified 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are collected from fraction II by recycle-HPLC. Thus, the CPC system is useful for easy and simple isolation of phlorotannins from E. cava.

  20. Isolation and purification of prenylated phenolics from Amorpha fruticosa by high-speed counter-current chromatography.

    PubMed

    Chen, Chu; Wu, Yan; Chen, Yang; Du, Leilei

    2015-08-01

    Prenylated phenolics such as amorfrutins are recently identified potent anti-inflammatory and antidiabetic natural products. In this work, high-speed counter-current chromatography was investigated for the isolation and purification of prenylated phenolics from the fruits of Amorpha fruticosa by using a two-phase solvent system composed of n-hexane/ethanol/water (5:4:1, v/v). As a result, 14.2 mg of 5,7-dihydroxy-8-geranylflavanone, 10.7 mg of amorfrutin A and 17.4 mg of amorfrutin B were obtained from 200 mg of n-hexane-soluble crude extract in one step within 250 min. The purities of 5,7-dihydroxy-8-geranylflavanone, amorfrutins A and B were 95.2, 96.7 and 97.1%, respectively, as determined by ultra high performance liquid chromatography. The structural identification was performed by mass spectrometry and (1) H and (13) C NMR spectroscopy. The results indicated that the established method is an efficient and convenient way to purified prenylated phenolics from A. fruticosa extract.

  1. Isolation and purification of diastereoisomeric flavonolignans from silymarin by binary-column recycling preparative high-performance liquid chromatography.

    PubMed

    Zhao, Weiquan; Yang, Guang; Zhong, Fanyi; Yang, Nan; Zhao, Xin; Qi, Yunpeng; Fan, Guorong

    2014-09-01

    Silymarin extracted from Silybum marianum (L.) Gaertn consists of a large number of flavonolignans, of which diastereoisomeric flavonolignans including silybin A and silybin B, and isosilybin A and isosilybin B are the main bioactive components, whose preparation from the crude extracts is still a difficult task. In this work, binary-column recycling preparative high-performance liquid chromatography systems without sample loop trapping, where two columns were switched alternately via one or two six-port switching valves, were established and successfully applied to the isolation and purification of the four diastereoisomeric flavonolignans from silymarin. The proposed system showed significant advantages over conventional preparative high-performance liquid chromatography with a single column in increasing efficiency and reducing the cost. To obtain the same amounts of products, the proposed system spends only one tenth of the time that the conventional system spends, and needs only one eleventh of the solvent that the conventional system consumes. Using the proposed system, the four diastereoisomers were successfully isolated from silymarin with purities over 98%.

  2. Isolation and purification of closely related citrus limonoid glucosides by flash chromatography.

    PubMed

    Raman, Girija; Cho, Minhee; Brodbelt, Jennifer S; Patil, Bhimanagouda S

    2005-01-01

    Several citrus limonoid glycosides have proved to be particularly difficult to purify using conventional techniques. A reversed-phase flash chromatographic technique has been developed for the separation and isolation of the closely related limonoid glucosides, nomilin 17-beta-D-glucopyranoside and nomilinic acid 17-beta-D-glucopyranoside, with confirmation of their identities by electrospray ionisation mass spectrometry. Furthermore, the semi-purification of the mixture of glucosides enriched with flavanone glucosides such as naringin, narirutin and other limonoid glucosides was obtained. The closely eluting glucosides were successfully separated to achieve a good yield and purity of 93%.

  3. Extraction and Purification of Glucoraphanin by Preparative High-Performance Liquid Chromatography (HPLC)

    ERIC Educational Resources Information Center

    Lee, Iris; Boyce, Mary C.

    2011-01-01

    A student activity that focuses on the isolation of glucoraphanin from broccoli using preparative high-performance liquid chromatography (HPLC) is presented here. Glucoraphanin is a glucosinolate, whose byproducts are known to possess anticancer properties. It is present naturally at high levels in broccoli and other "Brassica" vegetables. This…

  4. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  5. Scalable chromatography-based purification of virus-like particle carrier for epitope based influenza A vaccine produced in Escherichia coli.

    PubMed

    Lagoutte, Priscillia; Mignon, Charlotte; Donnat, Stéphanie; Stadthagen, Gustavo; Mast, Jan; Sodoyer, Régis; Lugari, Adrien; Werle, Bettina

    2016-06-01

    Virus-like particles (VLPs) are promising molecular structures for the design and construction of novel vaccines, diagnostic tools, and gene therapy vectors. Size, oligomer assembly and repetitiveness of epitopes are optimal features to induce strong immune responses. Several VLP-based vaccines are currently licensed and commercialized, and many vaccine candidates are now under preclinical and clinical studies. In recent years, the development of genetically engineered recombinant VLPs has accelerated the need for new, improved downstream processes. In particular, a rapid low cost purification process has been identified as a remaining key challenge in manufacturing process development. In the present study we set up a size-exclusion chromatography-based, scalable purification protocol for the purification of a VLP-based influenza A vaccine produced in Escherichia coli. Recombinant VLPs derived from the RNA bacteriophage MS2 displaying an epitope from the ectodomain of Matrix 2 protein from influenza A virus were produced and purified. The 3 steps purification protocol uses a recently developed multimodal size-exclusion chromatography medium (Capto™ Core 700) in combination with detergent extraction and size-exclusion polishing to reach a 89% VLP purity with a 19% yield. The combination of this downstream strategy following production in E. coli would be suited for production of VLP-based veterinary vaccines targeting livestock and companion animals where large amounts of doses must be produced at an affordable price.

  6. Preparative Isolation and Purification of Flavone C-Glycosides from the Leaves of Ficus microcarpa L. f by Medium-Pressure Liquid Chromatography, High-Speed Countercurrent Chromatography, and Preparative Liquid Chromatography.

    PubMed

    Wang, Xiaohong; Liang, Yong; Zhu, Licai; Xie, Huichun; Li, Hang; He, Junting; Pan, Man; Zhang, Tianyou; Ito, Yoichiro

    2010-01-01

    Combined with medium-pressure liquid chromatography (MPLC) and preparative high-performance liquid chromatography (perp-HPLC), high-speed countercurrent chromatography (HSCCC) was applied for separation and purification of flavone C-glycosides from the crude extract of leaves of Ficus microcarpae L. f. HSCCC separation was performed on a two-phase solvent system composed of methyl tert- butyl ether - ethyl acetate - 1-butanol - acetonitrile - 0.1% aqueous trifluoroacetic acid at a volume ratio of 1:3:1:1:5. Partially resolved peak fractions from HSCCC separation were further purified by preparative HPLC. Four well-separated compounds were obtained and their purities were determined by HPLC. The purities of these peaks were 97.28%, 97.20%, 92.23%, and 98.40%.. These peaks were characterized by ESI-MS(n). According to the reference, they were identified as orientin (peak I), isovitexin-3″-O-glucopyranoside (peak II), isovitexin (peak III), and vitexin (peak IV), yielded 1.2 mg, 4.5 mg, 3.3 mg, and 1.8 mg, respectively.

  7. Caprylic acid-induced impurity precipitation from protein A capture column elution pool to enable a two-chromatography-step process for monoclonal antibody purification.

    PubMed

    Zheng, Ji; Wang, Lu; Twarowska, Barbara; Laino, Sarah; Sparks, Colleen; Smith, Timothy; Russell, Reb; Wang, Michelle

    2015-01-01

    This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA-induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high-molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host-cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15-25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5-1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA-based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography.

  8. RNase one gene isolation, expression, and affinity purification models research experimental progression and culminates with guided inquiry-based experiments.

    PubMed

    Bailey, Cheryl P

    2009-01-01

    This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations of materials and methods and the semester culminates in a poster session. Experimental plans take into account the expense and time required to move from gene isolation to enzyme assays. This combination of instructor-guided and student-designed experiments is a manageable foray into guided inquiry-based learning in a biochemistry laboratory course, while providing a cohesive story and context for individual experiments.

  9. Isolation and purification of heroin from heroin street samples by preparative high performance liquid chromatography.

    PubMed

    Guo, Zhen; Zheng, Hui; Lu, Yanzhen; Wei, Yun

    2012-09-10

    The present study established a novel method using preparative high performance liquid chromatography to isolate and purify heroin·HCl from heroin street samples to be used as a reference standard. Different kinds of mobile phases and columns were used, ultimately the mobile phase consisting of hexane-isopropanol-methanol (65:28:7, v/v) and the SIL preparative column prepared in laboratory were selected as the final condition. Heroin was further purified by the drowning-out crystallization method using isopropanol-methanol (50:1, v/v) and hexane as drowning-out anti-solvents and salting-out agents, respectively. The purity was assessed by analytical high performance liquid chromatography and the confirmation of the chemical structure was performed by IR and NMR. About 110.7mg of heroin·HCl at a purity of over 99.52% was obtained from 180mg of heroin street samples which contained 156.15mg of heroin·HCl component by preparative high performance liquid chromatography. This method is suitable for preparing heroin standards in forensic science area.

  10. Atom transfer radical polymerization to fabricate monodisperse poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres and its application for protein affinity purification.

    PubMed

    Yu, Ling; Shi, Zhuan Zhuan; Li, Chang Ming

    2015-09-01

    Poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres for the first time were successfully synthesized by atom transfer radical polymerization (ATRP) method at room temperature. The co-polymerization approach was investigated to delicately control the microsphere morphology and size-distribution by reaction conditions including solvent percentage, monomer loading and rotation speed. The results show that the average size of the microspheres is ∼5.7 μm with coexistence of epoxy, hydroxyl and ether groups, which provide plentiful functional sites for protein anchoring. The mechanism of the microsphere formation is proposed. The microsphere successfully demonstrates its unique application for affinity purification of proteins, in which the functional epoxy group facilitates a simple and efficient protein covalent immobilization to purify immunoglobulin G on the microspheres, while the hydrophilic poly (ethylene glycol) motif can repulse nonspecific protein adsorption for good specificity. This microspheres can be used in broad protein biosensors due to their abundant functional groups and high surface to volume ratio.

  11. Using iTRAQ® Combined with Tandem Affinity Purification to Enhance Low-abundance Proteins Associated with Somatically-mutated EGFR Core Complexes in Lung Cancer

    PubMed Central

    Haura, Eric B.; Müller, André; Brietwieser, Florian P.; Li, Jiannong; Grebien, Florian; Colinge, Jacques; Bennett, Keiryn L.

    2010-01-01

    In this study we report a novel use for the iTRAQ® reagent combined with a peptide mass inclusion list to enhance the signal of low-abundance proteins during analysis by mass spectrometry. C-tagged-SH-EGFR was retrovirally-transduced into two mutant lung cancer cell lines (HCC827 and PC9) and the core protein complexes enriched by tandem affinity purification. Tryptically-digested peptides were derivatised with iTRAQ® and analysed by higher-energy collision-induced dissociation mass spectrometry. The data revealed that UBS3B is a member of the EGFR core complex in the HCC827 cell line, that was not apparent by standard, unbiased one-dimensional shotgun analysis and collision-induced dissociation. The expression level of UBS3B, however, was 6 to 10 times lower than that observed in the PC9 cell line. Thus, using iTRAQ® in this fashion allows the identification of low-abundance interactors when combined with samples where the same protein has a higher abundance. Ultimately, this approach may uncover proteins that were previously unknown or only suspected as members of core protein complexes. PMID:20945942

  12. Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry.

    PubMed

    Moon, Sungyoon; Han, Dohyun; Kim, Yikwon; Jin, Jonghwa; Ho, Won-Kyung; Kim, Youngsoo

    2014-03-14

    The heterotrimeric enzyme AMP-activated protein kinase (AMPK) is a major metabolic factor that regulates the homeostasis of cellular energy. In particular, AMPK mediates the insulin resistance that is associated with type 2 diabetes. Generally, cellular processes require tight regulation of protein kinases, which is effected through their formation of complex with other proteins and substrates. Despite their critical function in regulation and pathogenesis, there are limited data on the interaction of protein kinases. To identify proteins that interact with AMPK, we performed large-scale affinity purification (AP)-mass spectrometry (MS) of the AMPK-α1 and -β1 subunits. Through a comprehensive analysis, using a combination of immunoprecipitaion and ion trap mass spectrometry, we identified 381 unique proteins in the AMPKα/β interactomes: 325 partners of AMPK-α1 and 243 for AMPK-β1. Further, we identified 196 novel protein-protein interactions with AMPK-α1 and AMPK-β1. Notably, in our bioinformatics analysis, the novel interaction partners mediated functions that are related to the regulation of actin organization. Specifically, several such proteins were linked to pancreatic beta-cell functions, including glucose-stimulated insulin secretion, beta-cell development, beta-cell differentiation, and cell-cell communication.

  13. Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis.

    PubMed

    Suzuki, Ken; Sakamoto, Hironori; Shinozaki, Yukiko; Tabata, Jun; Watanabe, Takashi; Mochizuki, Atsushi; Koitabashi, Motoo; Fujii, Takeshi; Tsushima, Seiya; Kitamoto, Hiroko K

    2013-09-01

    Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).

  14. Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

    PubMed

    Li, Xu; Gao, Min; Choi, Jong Min; Kim, Beom-Jun; Zhou, Mao-Tian; Chen, Zhen; Jain, Antrix N; Jung, Sung Yun; Yuan, Jingsong; Wang, Wenqi; Wang, Yi; Chen, Junjie

    2017-04-01

    Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors.

  15. Purification of two triterpenoids from Schisandra chinensis by macroporous resin combined with high-speed counter-current chromatography.

    PubMed

    Zhu, Lijie; Li, Bin; Liu, Xiuying; Meng, Xianjun

    2014-10-01

    A method for preparative purification of corosolic acid and nigranoic acid from Schisandra chinensis (SC) was established using a combination of macroporous absorption resin column separation and high-speed counter-current chromatography (HSCCC). The crude extracts obtained from SC using 70% ethanol were separated on a macroporous resin column and then eluted with a graded ethanol series. The 70% ethanol fraction was used as the sample for separation of the two triterpenoids by HSCCC. The two-phase solvent system used for HSCCC separation was chloroform-n-butanol-methanol-water (10:0.5:7:4, v/v/v/v). The upper phase was used as the stationary phase of HSCCC. Corosolic acid (16.4 mg) of 96.3% purity and nigranoic acid (9.5 mg) of 98.9% purity were obtained in a one-step HSCCC separation from 100 mg of the sample. The structures of corosolic acid and nigranoic acid were identified by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR.

  16. Preparative isolation and purification of coumarins from Peucedanum praeruptorum Dunn by high-speed counter-current chromatography.

    PubMed

    Liu, Renmin; Feng, Lei; Sun, Ailing; Kong, Lingyi

    2004-11-19

    A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of coumarins from Peucedanum praeruptorum Dunn (Baihuaqianhu in Chinese) was successfully established by using light petroleum-ethyl acetate-methanol-water as the two-phase solvent system in gradient elution mode. The upper phase of light petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v) was used as the stationary phase of HSCCC. The mobile phase used in HSCCC was the lower phase of light petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v) and light petroleum-ethyl acetate-methanol-water (5:5:6.5:3.5, v/v) that was changed in gradient. Four kinds of coumarins and another unknown compound were obtained and yielded 5.3 mg of qianhucoumarin D, 7.7 mg of Pd-Ib, 35.8 mg of (+)-praeruptorin A, 31.9 mg of (+)-praeruptorin B and 6.4 mg of unknown compound with the purity of 98.6%, 92.8%, 99.5%, 99.4% and 99.8% in one-step separation, respectively. The structures of the coumarins were identified by 1H NMR and 13C NMR.

  17. Development of supercritical fluid extraction and supercritical fluid chromatography purification methods using rapid solubility screening with multiple solubility chambers.

    PubMed

    Gahm, Kyung H; Huang, Ke; Barnhart, Wesley W; Goetzinger, Wolfgang

    2011-01-01

    Rapid solubility screening in diverse supercritical fluids (SCFs) was carried out via multiple solubility chambers with a trapping device and online ultraviolet (UV) detection. With this device, it was possible to rapidly study the solubility variations of multiple components in a mixture. Results from solubility studies have been used to develop efficient supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) methods. After the investigation of solubilities of theophylline and caffeine in several neat organic solvents and SCFs, advantages of SFE over conventional organic solvent extraction were demonstrated with a model mixture of theophylline and caffeine. The highest solubility ratio of 1:40 (theophylline:caffeine) was observed in the SCF with 20% acetonitrile (MeCN), where a ratio of 1:11 was the highest in the neat organic solvents. A model mixture of theophylline:caffeine (85:15 w/w, caffeine as an impurity) was successfully purified by SFE by leveraging the highest solubility difference. The SCF with 20% MeCN selectively removed caffeine and left theophylline largely intact. Rapid SCF solubility screening was applied to development of SFE and SFC methods in a drug discovery environment. Two successful applications were demonstrated with proprietary Amgen compounds to either remove an achiral impurity before chiral purification or enhance chiral chromatographic throughput.

  18. Selection of operating parameters on the basis of hydrodynamics in centrifugal partition chromatography for the purification of nybomycin derivatives.

    PubMed

    Adelmann, S; Baldhoff, T; Koepcke, B; Schembecker, G

    2013-01-25

    The selection of solvent systems in centrifugal partition chromatography (CPC) is the most critical point in setting up a separation. Therefore, lots of research was done on the topic in the last decades. But the selection of suitable operating parameters (mobile phase flow rate, rotational speed and mode of operation) with respect to hydrodynamics and pressure drop limit in CPC is still mainly driven by experience of the chromatographer. In this work we used hydrodynamic analysis for the prediction of most suitable operating parameters. After selection of different solvent systems with respect to partition coefficients for the target compound the hydrodynamics were visualized. Based on flow pattern and retention the operating parameters were selected for the purification runs of nybomycin derivatives that were carried out with a 200 ml FCPC(®) rotor. The results have proven that the selection of optimized operating parameters by analysis of hydrodynamics only is possible. As the hydrodynamics are predictable by the physical properties of the solvent system the optimized operating parameters can be estimated, too. Additionally, we found that dispersion and especially retention are improved if the less viscous phase is mobile.

  19. Separation and purification of glucosinolates from crude plant homogenates by high-speed counter-current chromatography.

    PubMed

    Fahey, Jed W; Wade, Kristina L; Stephenson, Katherine K; Chou, F Edward

    2003-05-09

    Glucosinolates are anionic, hydrophilic plant secondary metabolites which are of particular interest due to their role in the prevention of cancer and other chronic and degenerative diseases. The separation and purification of glucosinolates from a variety of plant sources (e.g. seeds of broccoli, arugula and the horseradish tree), was achieved using high-speed counter-current chromatography (HSCCC). A high-salt, highly polar system containing 1-propanol-acetonitrile-saturated aqueous ammonium sulfate-water (1:0.5:1.2:1), was run on a semi-preparative scale and then transferred directly to preparative scale. Up to 7 g of a concentrated methanolic syrup containing about 10% glucosinolates was loaded on an 850-ml HSCCC column, and good separation and recovery were demonstrated for 4-methylsulfinylbutyl, 3-methylsulfinylpropyl, 4-methylthiobutyl, 2-propenyl and 4-(rhamnopyranosyloxy)benzyl glucosinolates. Multiple injections (5 to 6 times) were performed with well-preserved liquid stationary phase under centrifugal force. Pooled sequential runs with broccoli seed extract yielded about 20 g of its predominant glucosinolate, glucoraphanin, which was produced at > 95% purity and reduced to powdered form.

  20. Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography.

    PubMed

    Nestola, Piergiuseppe; Silva, Ricardo J S; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

    2014-06-20

    Adenovirus serotype 5 (Ad5) was successfully separated by size-exclusion chromatography (SEC) using a simple, yet efficient, two-column, quasi-continuous, simulated moving-bed process operated in an open-loop configuration. The operating cycle is divided into two identical half-cycles, each of them consisting of the following sequence of sub-steps: (i) elution of the upstream column and direction of the effluent of the downstream column to waste; (ii) elution of the upstream column and redirection of its effluent to waste while the downstream column is fed with the clarified bioreaction bulk and its effluent collected as purified product; (iii) operation of the system as in step (i) but collecting the effluent of the downstream column as product; (iv) elution of the upstream column and direction of its effluent to waste while the flow through the downstream column is temporarily halted. Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The pilot-scale run yielded a virus recovery of 86%, and a clearance of 90% and 89% for DNA and HCP, respectively, without any fine tunning of the predetermined operating parameters. These figures compare very favorably against single-column batch chromatography for the same volume of size-exclusion resin. However, and most importantly, the virus yield was increased from 57% for the batch system to 86% for the two-column SEC process because of internal recycling of the mixed fractions of contaminated Ad5, even though the two-column process was operated strictly in an open-loop configuration. And last, but not least, the productivity was increased by 6-fold with the two-column process. In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were overcome to a considerable extent by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.

  1. Preparative isolation and purification of sinomenine from Sinomenium acutum by centrifugal partition chromatography.

    PubMed

    Cheong, Jong Hye; Kim, Chul Young; Kim, Jinwoong

    2007-08-01

    Preparative centrifugal partition chromatography (CPC) was successfully carried out for the separation of sinomenine from the methanolic extract of Sinomenium acutum stems and rhizomes. The optimum two-phase solvent system of CPC was composed of n-hexane/ethyl acetate/methanol/water at a volume ratio of 1:6:2:8 (v/v/v/v) with 0.1% triethylamine (TEA). Preparative CPC yielded 44.3 mg of sinomenine from 400 mg of MeOH extract with a purity of 96.9%.

  2. Analysis of Free Drug Fractions in Serum by Ultrafast Affinity Extraction and Two-Dimensional Affinity Chromatography using α1-Acid Glycoprotein Microcolumns

    PubMed Central

    Bi, Cong; Zheng, Xiwei; Hage, David S.

    2016-01-01

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110–830 ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10–20 min when using only 3–10 µL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. PMID:26797422

  3. Purification of glycocalicin from human plasma.

    PubMed

    HadjKacem, Basma; Mkaouar, Héla; Ben Amor, Ikram; Gargouri, Jalel; Gargouri, Ali

    2016-01-01

    Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product.

  4. Dynamic pH junction high-speed counter-current chromatography coupled with microwave-assisted extraction for online separation and purification of alkaloids from Stephania cepharantha.

    PubMed

    Yuan, Zhiquan; Xiao, Xiaohua; Li, Gongke

    2013-11-22

    A simple and efficient dynamic pH junction high-speed counter-current chromatography method was developed and further applied to the online extraction, separation and purification of alkaloids from Stephania cepharantha by coupling with microwave-assisted extraction. Mineral acid and organic base were added into the mobile phase and the sample solution, respectively, leading to the formation of a dynamic pH junction in the column and causing focus of alkaloids. Selective focus of analytes can be achieved on the basis of velocity changes of the pH junction through appropriate selection of solvent systems and optimization of additive concentrations. The extract can be directly introduced into the HSCCC for the online extraction, separation and purification of alkaloids from S. cepharantha. Continuous separation can be easily achieved with the same solvent system. Under the optimum conditions, 6.0 g original sample was extracted with 60 mL of the upper phase of hexane-ethyl acetate-methanol-water (1:1:1:1, v/v/v/v) containing 10% triethylamine under 50 °C and 400 W irradiation power for 10 min, the extracts were directly separated and purified by high-speed counter-current chromatography. A total of 5.7 mg sinomenine, 8.3mg 6,7-di-O-acetylsinococuline, 17.9 mg berbamine, 12.7 mg isotetrandrine and 14.6 mg cepharanthine were obtained with purities of 96.7%, 93.7%, 98.7%, 97.3% and 99.3%, respectively. The online method provides good selectivity to ionizable compounds and improves the separation and purification efficiency of the high-speed counter-current chromatography technique. It has good potential for separation and purification of effective compounds from natural products.

  5. Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for omega-aminocarboxylic acids.

    PubMed

    Marti, D; Schaller, J; Ochensberger, B; Rickli, E E

    1994-01-15

    The kringle 2 (E161T/C162S/EEE[K2HPg/C169S]TT) and the kringle 3 (TYQ[K3HPg]DS) domains of human plasminogen (HPg) were expressed in Escherichia coli in an expression vector with the phage T5 promotor/operator element N250PSN250P29 and the cDNA sequence for a hexahistidine tail to facilitate the isolation of the recombinant protein. A coagulation factor Xa (FXa)-sensitive cleavage site was introduced to remove the N-terminal histidine tag. In r-K2, mutations E161T and C162S were introduced to enhance the FXa cleavage yield and C169S to replace the cysteine residue, participating in the inter-kringle disulfide bridge between kringles 2 and 3. Recombinant proteins were isolated by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose and refolded under denaturing and reducing conditions followed by a non-denaturing and oxidising environment. The free thiol group in position 297 in r-K3 was selectively alkylated with iodoacetamide. The hexahistidine tail was successfully removed with FXa. The N-terminal sequence, the amino acid composition and the molecular mass analyses are in agreement with the expected data. The correct arrangement of the disulfide bonds was verified by sequence analysis of the corresponding thermolytic and subtilisin fragments. r-K2 exhibits weak binding to lysine-Bio-Gel. The weak binding affinity of r-K2 for omega-aminocarboxylic acids is confirmed by intrinsic fluorescence titration with 6-aminohexanoic acid (NH2C5COOH) indicating a Kd of approximately 401 microM. In contrast, r-K3 seems to be devoid of a binding affinity for omega-aminocarboxylic acids. Considering earlier determined Kd values of kringle 1, kringle 4 and kringle 5, the binding affinity of HPg kringle domains for NH2C5COOH is proposed to decrease in the following order, kringle 1 > kringle 4 > kringle 5 > kringle 2 > kringle 3.

  6. Combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the direct extraction and purification of pseudohypericin and hypericin from St. John's wort (Hypericum perforatum L.).

    PubMed

    Cai, Fanfan; Li, Yang; Zhang, Min; Zhang, Hongyang; Wang, Yuerong; Hu, Ping

    2015-08-01

    St. John's wort has attracted particular attention because of its beneficial effects as an antidepressant, antiviral, and anticancer agent. A method for the combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the simultaneous extraction and purification of pseudohypericin and hypericin from the herb is presented in this paper. Firstly, the constituents were extracted and directly adsorbed by expanded bed adsorption chromatography under optimal conditions. The stepwise elution was then performed by expanded bed adsorption chromatography that enriched the targets with higher purities and recoveries compared to other methods. Secondly, the eluent fractions from expanded bed adsorption chromatography were further separated by two-step high-speed countercurrent chromatography. A two-step high-speed countercurrent chromatography method with a biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water with a volume ratio of 1:2:1:2 was performed by stepwise changing the flow rate of the mobile phase. Consequently, 5.6 mg of pseudohypericin and 2.2 mg of hypericin with purities of 95.5 and 95.0%, respectively, were successfully obtained from 40 mg of crude sample.

  7. One-step purification of proteins from chicken egg white using counter-current chromatography.

    PubMed

    Shibusawa, Y; Kihira, S; Ito, Y

    1998-05-29

    Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.

  8. Use of Escherichia coli for the production and purification of membrane proteins.

    PubMed

    Postis, Vincent G L; Rawlings, Andrea E; Lesiuk, Amelia; Baldwin, Stephen A

    2013-01-01

    Individual types of ion channels and other membrane proteins are typically expressed only at low levels in their native membranes, rendering their isolation by conventional purification techniques difficult. The heterologous over-expression of such proteins is therefore usually a prerequisite for their purification in amounts suitable for structural and for many functional investigations. The most straightforward expression host, suitable for prokaryote membrane proteins and some proteins from eukaryotes, is the bacterium Escherichia coli. Here we describe the use of this expression system for production of functionally active polytopic membrane proteins and methods for their purification by affinity chromatography in amounts up to tens of milligrams.

  9. Purification of berry flavonol glycosides by long-bed gel permeation chromatography.

    PubMed

    Riihinen, Kaisu R; Gödecke, Tanja; Pauli, Guido F

    2012-06-29

    While Sephadex LH-20 gel is frequently employed as a stationary phase during pre-separations and in open column chromatography systems, its separation power in long-bed gel permeation chromatography (GPC) applications is much less prevalent. Aimed at the characterization of bioactive constituents, a long-bed GPC protocol was established for lingonberry juice concentrate. The method included pre-fractionation over HP-20 resin to eliminate sugars and organic acids as well as a major part of other predominant berry flavonoids (anthocyanins, flavan-3-ols and proanthocyanidins), prior to the elution of the fraction containing 10% (w/w) of quercetin glycosides (QGs). Subsequently, seven major QGs were purified using a 10-m Sephadex LH-20 system and isocratic elution with methanol. The total mass recovery was 99.3±1.4%, after eluting the highly-retained compounds from the employed pre-column with 70% acetone. Injecting 1070 mg per run, the yield of purified QGs ranged from 2 to 6 mg per collected single fraction. The LC-UV/PDA purities of isolated Q-3-O-α-rhamnoside and Q-3-O-β-galactoside were 82 and 94 area% at 250 nm, while the three Q-pentosides showed purities of 59, 30, and 57 area%. By comparison, purity assessment of these isolates by quantitative ¹H NMR (total integral and modified 100% method) led to significantly lower purities of 70 and 52% for Q-rha and Q-gal and 38, 25 and 46% for Q-pentosides, respectively. This can be explained by the presence of hidden residual complexity (RC), which is revealed by the quantitative NMR method. This finding has potentially broader implication as it reveals an unexpected degree of RC in GPC fractions. Despite remarkable separation power for congeneric flavonoids, long-bed GPC on Sephadex LH-20 produces materials, which require careful analysis of purity before interpreting bioassay results.

  10. High-throughput Protein Purification and Quality Assessment for Crystallization

    PubMed Central

    Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, Catherine; Gu, Minyi; Makowska-Grzyska, Magdalena; Wu, Ruiying; An, Hao; Chhor, Gekleng; Joachimiak, Andrzej

    2012-01-01

    The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are

  11. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  12. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

    PubMed

    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

  13. Chromatography

    MedlinePlus

    Chromatography is a way of separating two or more chemical compounds. Chemical compounds are chemicals that are ... of chemical compound. There are different kinds of chromatography. These include gas, high pressure liquid, or ion ...

  14. Isolation of a Trypanosoma cruzi antigen by affinity chromatography with a monoclonal antibody. Preliminary evaluation of its possible applications in serological tests.

    PubMed Central

    Carbonetto, C H; Malchiodi, E L; Chiaramonte, M; Durante de Isola, E; Fossati, C A; Margni, R A

    1990-01-01

    By affinity chromatography with a monoclonal antibody (163B6), obtained in our laboratory, we have isolated a T. cruzi antigen which could be useful for differential diagnosis of Chagas' disease from leishmaniasis. This antigen, a 52-kD protein, reacted with all sera from Chagas' disease patients tested but not with sera from patients with leishmania, in ELISA. The 52-kD antigen is widely distributed in the Trypanosoma genus since the 163B6 monoclonal antibody reacts with T. rangeli and 8 strains and a clone of T. cruzi epimastigotes. Images Fig. 1 Fig. 2 PMID:2119921

  15. Purification of pre-miR-29 by a new O-phospho-l-tyrosine affinity chromatographic strategy optimized using design of experiments.

    PubMed

    Afonso, Adriana; Pereira, Patrícia; Queiroz, João A; Sousa, Ângela; Sousa, Fani

    2014-05-23

    MicroRNAs are the most studied small non-coding RNA molecules that are involved in post-transcriptional regulation of target genes. Their role in Alzheimer's disease is being studied and explored in order to develop a new therapeutic strategy based on specific gene silencing. This disease is characterized by protein deposits, mainly deposits of extracellular Aβ plaques, produced upon endoproteolytic cleavage of APP by ß-site APP-cleaving enzyme 1 (BACE1). Recent studies have shown that particularly miR-29 cluster can be involved in the decrease of Aβ plaques production, by acting on BACE1 expression silencing. In order to use this microRNA as potential therapeutic it is essential to guarantee its purity, stability and integrity. Hence, the main purpose of this study was the development of a new affinity chromatographic strategy by using an O-phospho-l-tyrosine matrix and applying Box-Behnken design (BBD) to obtain pre-miR-29 with high purity degree and yield, envisioning its application in gene therapy. Thus, after process optimization the best results were achieved with a decreasing ammonium sulfate gradient in 10mM Tris buffer, pH 8 (1.6M (NH4)2SO4, 1.11M (NH4)2SO4 and 0M (NH4)2SO4), at 16°C. These experimental conditions allowed the recovery of pre-miR-29 with 52% of purity and 71% of recovery yield. The O-phospho-l-tyrosine matrix was initially chosen to mimic the natural interactions that occur inside the cell, and in fact it was proved a satisfactory selectivity for pre-miR-29. Also the innovative application of BBD for this strategy was efficient (R(2)=0.98 for % relative recovery and R(2)=0.93 for % relative purity) and essential to achieve best purification results in short time, saving lab resources.

  16. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

    PubMed

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-05-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length