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Sample records for affinity chromatographic purification

  1. Purification of urokinase by combined cation exchanger and affinity chromatographic cartridges.

    PubMed

    Hou, K C; Zaniewski, R

    1990-02-23

    Crude urokinase from human urine processed through foam flotation and ammonium sulfate precipitation containing 720 National Health Institute Committee on Thrombolytic Agents U/mg activity was purified by an SP cation exchanger followed by a zinc-chelated affinity chromatographic cartridge. The cartridges were of a radial-flow type formed by using acrylic and cellulose composite matrices. The high rigidity of the matrix structure permits fast flow of protein solutions (liters per minute) and thus allows processing of a large volume of crude urokinase under low operating pressures. A greater than six-fold increase in specific enzyme activity of urokinase was achieved by adsorbing and eluting 1 l of a 3 mg/ml crude urokinase solution on an SP cartridge. The eluent was further purified by passing through a zinc-chelated affinity cartridge to achieve greater than a eighteen-fold increase in urokinase specific activity. This report demonstrates the combined use of a cation exchanger with zinc-chelated chromatographic cartridges in purifying urokinase on a relatively large scale. The relationship between the amount of zinc chelated in the matrix to its effect on urokinase purification is also discussed. PMID:2329161

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures.

    PubMed Central

    Scopes, R K

    1977-01-01

    1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form. PMID:849261

  5. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

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

  6. 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. PMID:24751555

  7. Affinity purification of heme-tagged proteins.

    PubMed

    Asher, Wesley B; Bren, Kara L

    2014-01-01

    Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay. PMID:24943311

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

    PubMed

    Guan, Dongli; Chen, Zhilei

    2014-07-01

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

  9. Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification.

    PubMed

    Liu, Fang; Chen, Wilfred

    2013-01-01

    Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification. PMID:23801586

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

    PubMed

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

    2015-01-01

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

  11. Affinity purification of aprotinin from bovine lung.

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

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

  14. Improved native affinity purification of RNA.

    PubMed

    Batey, Robert T; Kieft, Jeffrey S

    2007-08-01

    RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable. PMID:17548432

  15. Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.

    PubMed

    Martínez-Ceron, María C; Marani, Mariela M; Taulés, Marta; Etcheverrigaray, Marina; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

    2011-05-01

    Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. PMID:21495625

  16. Chromatographic Purification of Highly Active Yeast Ribosomes

    PubMed Central

    Meskauskas, Arturas; Leshin, Jonathan A.; Dinman, Jonathan D.

    2011-01-01

    Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes. PMID:22042245

  17. Chymopapain. Chromatographic purification and immunological characterization.

    PubMed

    Buttle, D J; Barrett, A J

    1984-10-01

    Chymopapain (EC 3.4.22.6) was purified from commercially available spray-dried latex of papaya (Carica papaya) fruit by (NH4)2SO4 fractionation and fast protein chromatography on the Mono S cation-exchange column. Multiple forms of chymopapain separated chromatographically were shown to be immunologically identical. A major form was isolated and found to be homogeneous by several criteria, and fully active, and its N-terminal amino acid was identified as tyrosine. Latex from fresh unripe papaya fruit contained predominantly one form of chymopapain, and it is concluded that chymopapain is a single enzyme distinct from the other cysteine proteinases of C. papaya latex. PMID:6437389

  18. Heparin affinity purification of extracellular vesicles

    PubMed Central

    Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

    2015-01-01

    Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

  19. Characterization of methacrylate chromatographic monoliths bearing affinity ligands.

    PubMed

    Černigoj, Urh; Vidic, Urška; Nemec, Blaž; Gašperšič, Jernej; Vidič, Jana; Lendero Krajnc, Nika; Štrancar, Aleš; Podgornik, Aleš

    2016-09-16

    We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher. PMID:27554023

  20. Dye affinity cryogels for plasmid DNA purification.

    PubMed

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

    2015-11-01

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

  1. Host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification.

    PubMed

    Levy, Nicholas E; Valente, Kristin N; Lee, Kelvin H; Lenhoff, Abraham M

    2016-06-01

    Downstream purification of monoclonal antibodies (mAbs) is normally performed using a platform process that is empirically tuned to optimize impurity removal for each new product. A more fundamental understanding of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work examines the chromatographic properties of Chinese hamster ovary host cell protein (HCP) impurities in non-affinity chromatographic resins commonly used in polishing steps for monoclonal antibody purification: ion-exchange, hydrophobic interaction, and multimodal. Using proteomic analysis, the specific HCP impurities that elute close to mAb products are identified for these resins at typical downstream processing conditions. Additionally, the interactions of HCP impurities with mAb products are profiled to determine the total extent of product association and the specific HCP species that form associative complexes under conditions encountered in polishing columns. Product association and co-elution were both identified as viable mechanisms of HCP retention for the non-affinity resins tested here. A relatively large sub-population of HCP impurities was found to co-elute or associate with mAbs in each polishing column, but only a small population of HCPs-including lipoprotein lipase, chrondroitin sulfate proteoglycan 4, nidogen-1, and SPARC-were identified as difficult to remove across an entire downstream mAb process. Biotechnol. Bioeng. 2016;113: 1260-1272. © 2015 Wiley Periodicals, Inc. PMID:26550778

  2. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Sondek, John

    2004-09-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, and for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:18429272

  3. Overview of affinity tags for protein purification.

    PubMed

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

    2013-01-01

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

  4. Affinity based and molecularly imprinted cryogels: Applications in biomacromolecule purification.

    PubMed

    Andaç, Müge; Galaev, Igor Yu; Denizli, Adil

    2016-05-15

    The publications in macro-molecularly imprinted polymers have increased drastically in recent years with the development of water-based polymer systems. The macroporous structure of cryogels has allowed the use of these materials within different applications, particularly in affinity purification and molecular imprinting based methods. Due to their high selectivity, specificity, efficient mass transfer and good reproducibility, molecularly imprinted cryogels (MICs) have become attractive for researchers in the separation and purification of proteins. In this review, the recent developments in affinity based cryogels and molecularly imprinted cryogels in protein purification are reviewed comprehensively. PMID:26454622

  5. Protein Complex Purification by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  6. Development of a novel affinity membrane purification system for deoxyribonuclease.

    PubMed

    Landry, Kyle S; Levin, Robert E

    2014-02-01

    A membrane based affinity purification system was developed for the purification of the DNA specific nuclease, DNase I. Single stranded DNA was bound to unmodified polyvinylidene fluoride (PVDF) membranes which were used to purify DNase I from a solution of bovine serum albumin. Using coated membranes, a 6-fold increase in specific activity was achieved with 80 % enzyme recovery. This method provides a simple yet effective way to purify DNase I and can be very useful for the purification of other DNA specific enzymes. PMID:24318589

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

    PubMed

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

    2016-06-01

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

  8. 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)

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

  11. Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe.

    PubMed

    Ball, H L; Bertolini, G; Mascagni, P

    1995-01-01

    The purification of large synthetic peptides using conventional separation techniques often results in poor yields and homogeneity due to the accumulation of chromatographically similar deletion and truncated impurities. We have developed a highly effective synthetic strategy and one-step purification procedure that is based on (i) the application of single coupling using HBTU/HOBt activation to reduce incomplete couplings, (ii) the use of N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping agent to terminate deletion sequences and (iii) the N-terminal derivatization of the complete peptidyl-resin with a reversible Fmoc-based chromatographic probe possessing enhanced physico-chemical properties (i.e. hydrophobicity, charge or affinity label). We report the application of a biotinylated probe, activated as the succinimidyl carbonate, for the purification of a 101 residue chaperonin protein from Rattus norvegicus (rat cpn10), previously synthesized using an optimized synthetic protocol. Biotinylated rat cpn10 was separated from underivatized impurities on an immobilized monomeric avidin column. Free rat cpn10 was released from avidin-agarose column with 5% aqueous triethylamine and after desalting by RP-HPLC gave 9.9% recovery. Characterization and assessment of homogeneity was achieved using ESI-MS, CZE and RP-HPLC. PMID:9223007

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

    PubMed

    Starokadomskyy, Petro; Burstein, Ezra

    2014-01-01

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

  13. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

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

  14. A new affinity gel for the purification of α-carbonic anhdrases.

    PubMed

    Sahin, Aysegul; Isık, Semra; Arslan, Oktay; Supuran, Claudiu T; Guler, Ozen Ozensoy

    2015-04-01

    The new affinity gel reported in this study was prepared using EUPERGIT C250L as a chromatographic bed material, to which etylenediamine spacer arms were attached to prevent steric hindrance between the matrix and ligand, and to facilitate effective binding of the CA-specific ligand, of the aromatic sulfonamide type for the purification of α-carbonic anhydrases (Cas; EC 4.2.1.1). Indeed, the aminoethyl moieties of the affinity gel were derivatized by reaction with 4-isothiocyanatobenzenesulfonamide, with the formation of a thiourea-based gel, having inhibitory effects against CAs. Both bovine erythrocyte carbonic anhydrase BCA and human (h) erythrocyte CA isoforms I, II (hCA I and II) have been purified from hemolysates, by using this affinity gel. The greatest purification fold and column yields for BCA and for cytosolic (hCA I + II) enzymes were of 181-fold (21.07%) and 184-fold (9.49%), respectively. Maximum binding was achieved at 15 °C and I = 0.3 ionic strength for α-carbonic anhydrases. PMID:24936879

  15. An affinity-based strategy for the design of selective displacers for the chromatographic separation of proteins.

    PubMed

    Vutukuru, Srinavya; Kate, Sandesh D; McCallum, Scott A; Morrison, Christopher J; Cramer, Steven M; Kane, Ravi S

    2008-06-01

    We describe an affinity-based strategy for designing selective protein displacers for the chromatographic purification of proteins. To design a displacer that is selective for a target protein, we attached a component with affinity for the target protein to a resin-binding component; we then tested the ability of such displacers to selectively retain the target protein on a resin relative to another protein having a similar retention time. In particular, we synthesized displacers based on biotin, which selectively retained avidin as compared to aprotinin on SP Sepharose high performance resin. In addition, we have extended this approach to develop an affinity-peptide-based displacer that discriminates between lysozyme and cytochrome c. Here, a selective displacer was designed from a lysozyme-binding peptide that had been identified and optimized previously using phage-display technology. Our results suggest a general strategy for designing highly selective affinity-based displacers by identifying molecules (e.g., peptides) that bind to a protein of interest and using an appropriate linker to attach these molecules to a moiety that binds to the stationary phase. PMID:18512879

  16. A heme fusion tag for protein affinity purification and quantification

    PubMed Central

    Asher, Wesley B; Bren, Kara L

    2010-01-01

    We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an l-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination. PMID:20665691

  17. Affitins for protein purification by affinity magnetic fishing.

    PubMed

    Fernandes, Cláudia S M; Dos Santos, Raquel; Ottengy, Stella; Viecinski, Aline Canani; Béhar, Ghislaine; Mouratou, Barbara; Pecorari, Frédéric; Roque, A Cecília A

    2016-07-29

    Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing. PMID:27342136

  18. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

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

    2016-05-15

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

  19. Affinity purification of proteins binding to GST fusion proteins.

    PubMed

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

  20. 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).

  1. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

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

    1983-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

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

  3. Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

    PubMed

    Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

    2016-03-01

    Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. PMID:26786724

  4. Chromatographic purification and size separation of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Duesberg, G. S.; Muster, J.; Krstic, V.; Burghard, M.; Roth, S.

    1998-08-01

    The efficient purification of single-wall and multi-wall carbon nanotubes (NTs) by columnar size exclusion chromatography (SEC) is reported. In this process, carbon nanospheres (polyhedra), amorphous carbon and metal particles are removed from aqueous surfactant-stabilised dispersions of NT raw material. TEM and AFM investigations revealed that more than 40-50% of the purified material consists of individual tubes. In addition, length separation of the tubes is achieved.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2010-08-01

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

  8. 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

  9. Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

    PubMed Central

    Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry–based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes. PMID:18370112

  10. Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose.

    PubMed

    Chaga, G; Bochkariov, D E; Jokhadze, G G; Hopp, J; Nelson, P

    1999-12-24

    A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins. The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5alpha cells. Each protein was expressed, extracted and purified in one chromatographic step. The purification procedure for each protein can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent--Co2+-carboxymethylaspartate agarose Superflow--was utilized at linear flow-rates as high as 5 cm/min. The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis. Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity. The operational capacity of Co2+-carboxymethylaspartate agarose for each protein was determined. PMID:10669292

  11. Deep liquid-chromatographic purification of uranium extract from technetium

    SciTech Connect

    Volk, V.; Dvoeglazov, K; Podrezova, L.; Vidanov, V.; Pavlyukevich, E.

    2013-07-01

    The recycling of uranium in the nuclear fuel cycle requires the removal of a number of radioactive and stable impurities like {sup 99}Tc from spent fuels. In order to improve the grade of uranium extract purification from technetium the method of liquid chromatography and the apparatus for its performance have been developed. Process of technetium extraction and concentrating in aqueous solution containing reducing agent has been studied on simulated solutions (U-Tc-HNO{sub 3}-30% TBP-isoparM). The dynamic tests of the method have been carried out on the laboratory unit. Solution of diformyl-hydrazine in nitric acid was used as a stationary phase. Silica gel with specific surface of 186 m{sup 2}/g was used as a carrier of the stationary phase. It is shown that the volume of purified extract increases as the solution temperature increases, concentration of reducing agent increases and extract flow rate decreases. It is established that the technetium content in uranium by this method could achieve a value below 0.3 ppm. Some variants of overload and composition of the stationary phase containing the extracted technetium have been offered and tested. It is defined that the method provides reduction of processing medium-active wastes by more than 10 times during finish refining process. (authors)

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    2010-01-01

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

  14. Affinity Purification Strategies for Proteomic Analysis of Transcription Factor Complexes

    PubMed Central

    2013-01-01

    Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein–protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities. PMID:23937658

  15. 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.

  16. Production and Purification of Streptokinase by Protected Affinity Chromatography

    PubMed Central

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

    2009-01-01

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

  17. 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.

  18. Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.

    PubMed

    Gupta, Munishwar N; Jain, Sulakshana; Roy, Ipsita

    2002-01-01

    Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu(2+), Zn(2+), and Ni(2+), which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn(2+) directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL(-1), as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. PMID:11822903

  19. A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

    PubMed

    Kim, Heejae; Chen, Wilfred

    2016-09-20

    Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. PMID:27457699

  20. 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

  1. Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies.

    PubMed

    Ishihara, Takashi; Hosono, Mareto

    2015-07-15

    The performance of amino acids in Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography for monoclonal antibody purification was investigated. Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies. Improved clearance of impurity, high molecular weight species (HMW) and host cell proteins (HCP) was observed in the purification processes when using the amino acids as base-buffer constituents, additives or eluents compared with that of buffers without these amino acids. In addition, we designed a buffer system in which the mobile phases were composed of only a single amino acid, histidine, and applied it to the above three chromatographies. Effective HMW and HCP clearance was also obtained in this manner. These results suggest that amino acids may enhance impurity clearance during the purification of monoclonal antibodies. PMID:26057847

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

    PubMed Central

    2011-01-01

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

  3. Functional chromatographic technique for natural product isolation†

    PubMed Central

    Lau, Eric C.; Mason, Damian J.; Eichhorst, Nicole; Engelder, Pearce; Mesa, Celestina; Kithsiri Wijeratne, E. M.; Gunaherath, G. M. Kamal B.; Leslie Gunatilaka, A. A.

    2015-01-01

    Natural product discovery arises through a unique interplay between chromatographic purification and biological assays. Currently, most techniques used for natural product purification deliver leads without a defined biological action. We now describe a technique, referred to herein as functional chromatography, that deploys biological affinity as the matrix for compound isolation. PMID:25588099

  4. 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. PMID:9889081

  5. Unusual chromatographic behaviour and one-step purification of a novel membrane proteinase from Bacillus cereus.

    PubMed

    Fricke, B; Buchmann, T; Friebe, S

    1995-11-01

    Cell envelopes of Bacillus cereus contain a casein-cleaving membrane proteinase (CCMP) and an insulin-cleaving membrane proteinase (ICMP), which differ in their substrate and inhibitor specificity from all Bacillus proteinases described previously. They remained localized in the cytoplasmic membrane after treatment with lysozyme and mutanolysin and they are strongly attached to the membrane compared with other known membrane proteinases. Only high a concentration of the Zwitterionic detergent sulfobetain SB-12 enabled an effective solubilization of both membrane proteinases. The usual conventional purification methods, such as chromatofocusing, ion-exchange chromatography and hydrophobic interaction chromatography in the presence of detergent concentrations beyond their critical micelle concentration, could not be applied to the purification, because the solubilized membrane proteinases bound strongly and irreversibly to the chromatographic matrix. In the search for other purification methods, we used a tentacle ion-exchanger (EMD trimethylaminoethyl-Fractogel) to reduce the hydrophobic interactions between the proteinases and the matrix. All contaminating proteins could be removed by a first gradient of sodium chloride without elution of CCMP; a second gradient with isopropanol and a decreasing salt concentration resulted in an efficiently purified CCMP. The ICMP was irreversibly denaturated. Purified CCMP is a member of the metalloproteinase family with a pH optimum in the neutral range and a temperature optimum of 40 degrees C, whose properties differ from the serine-type membrane proteinase of Bacillus subtilis described by Shimizu et al. [Agric. Biol. Chem., 47 (1983) 1775]. It consists of two subunits in sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Mr 53,000 and 65,000); however, the molecular mass of the purified enzyme could not be determined by size exclusion or SDS-PAGE, because the purified enzyme

  6. Chromatographic purification of equine immunoglobulin G F(ab)2 from plasma.

    PubMed

    Fernandes, Aldon; Kaundinya, John Oswald; Daftary, Gautam; Saxena, Lalit; Banerjee, Subhasis; Pattnaik, Priyabrata

    2008-12-01

    The antibody fragments generated from hyperimmune equine IgG is widely used as anti-snake venom, anti-scorpion venom, anti-diphtheria, anti-tetanus, anti-gangrene and anti-rabies agents. Antibody fragments, F(ab)(2), because of their specificity and absence of undesired reactivity are preferred over complete IgG. This paper discusses a novel purification technique for chromatographic purification of anti-rabies immunoglobulin G (IgG) fragment F(ab)(2) from horse serum. F(ab)(2) was purified by two successive chromatography steps using Cellufine A-200 and ProSep-vA Ultra media. The purified F(ab)(2) was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified F(ab)(2) was reactive to rabies antigen in immuno-electrophoresis and diffusion tests. The purified F(ab)(2) was biologically functional and was found to show a potency of 1500 IU ml(-1). Comparative analysis of the purity with commercially available F(ab)(2) by HPLC analysis and SDS-PAGE indicated that the present product is better in purity. To our knowledge, this is the first report providing evidence on purification of equine antibody fragment using controlled pore glass based protein A chromatography media. PMID:19008160

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

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

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

  8. 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.

  9. 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.

  10. A comparison of protein A chromatographic stationary phases: performance characteristics for monoclonal antibody purification.

    PubMed

    Liu, Zhuo; Mostafa, Sigma S; Shukla, Abhinav A

    2015-01-01

    Protein A chromatography remains the dominant capture step used during the downstream purification of monoclonal antibodies (mAbs). With the recent expiry of the Repligen patent on recombinant Protein A, a variety of new Protein A resins have been introduced in the market. Given productivity limitations during downstream processing that have come into sharper focus with the recent increase in cell culture titers for mAbs, the selection of an appropriate Protein A resin has direct implications on the overall process economics of mAb production. The performance of seven different Protein A chromatographic resins was compared with respect to static binding capacity and dynamic binding capacity as a function of flow rate. This data was translated into a comparison of productivity (g mAb purified per unit resin volume per unit time) for the seven stationary phases. In addition, elution pH and host cell protein impurity levels after product capture on each of these resins were determined. The current article provides an effective methodology and dataset for the selection of the optimal Protein A chromatographic resin. PMID:24823474

  11. Preparation of group I introns for biochemical studies and crystallization assays by native affinity purification.

    PubMed

    Vicens, Quentin; Gooding, Anne R; Duarte, Luis F; Batey, Robert T

    2009-01-01

    The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides -- some containing exons -- were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3' overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules. PMID:19710925

  12. One-step chromatographic procedure for purification of B-phycoerythrin from Porphyridium cruentum.

    PubMed

    Tang, Zhihong; Jilu Zhao; Ju, Bao; Li, Wenjun; Wen, Shaohong; Pu, Yang; Qin, Song

    2016-07-01

    B-phycoerythrin (B-PE) was separated and purified from microalga Porphyridium cruentum using one-step chromatographic method. Phycobiliproteins in P. cruentum was extracted by osmotic shock and initially purified by ultrafiltration. Further purification was carried out with a SOURCE 15Q exchange column and analytical grade B-PE was obtained with a purity ratio (A545/A280) of 5.1 and a yield of 68.5%. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and β and a low intensity band of 27 kDa assigned to γ subunit. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level. PMID:26851659

  13. 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

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

    PubMed

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

    2001-02-01

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

  15. 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.

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

    NASA Astrophysics Data System (ADS)

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

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

  17. 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

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

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

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

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

    PubMed

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

    2016-04-01

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

  20. Control of an affinity purification procedure using a thermal biosensor.

    PubMed

    Flygare, L; Larsson, P O; Danielsson, B

    1990-10-01

    Lactate dehydrogenase (LDH) was recovered from a solution by affinity binding to an N(6)-(6-aminohexyl)-AMP-Sepharose gel. An enzyme thermistor unit was employed to continously measure the activity of the unbound LDH. The enzyme activity signal from the enzyme thermistor was used in a PID controller to regulate the addition of AMP-Sepharose gel to the LDH solution. In another type of experiment, a desktop computer was utilized to control the addition of the adsorbent. Both systems worked satisfactorily, and enabled a rapid and accurate assessment of correct addition of adsorbent. PMID:18597264

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

    PubMed

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

    2005-03-01

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

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

    PubMed

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

    2009-06-24

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

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

    PubMed

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

    2016-07-27

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

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

    PubMed

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

    2015-01-01

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

  5. GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.

    PubMed

    Maity, Ranjan; Pauty, Joris; Krietsch, Jana; Buisson, Rémi; Genois, Marie-Michelle; Masson, Jean-Yves

    2013-01-01

    Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest. PMID:24193370

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

    PubMed

    Ahirwar, Rajesh; Nahar, Pradip

    2015-08-01

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

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

    PubMed

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

    2014-01-01

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

  8. One-step purification of glucoamylase by affinity precipitation with alginate.

    PubMed

    Teotia, S; Lata, R; Khare, S K; Gupta, M N

    2001-01-01

    It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens. PMID:11746949

  9. 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. PMID:24777569

  10. A dual protease approach for expression and affinity purification of recombinant proteins.

    PubMed

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. PMID:27105777

  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

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column. PMID:21194702

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

    SciTech Connect

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

    2000-02-01

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

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

    PubMed

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

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

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

    PubMed

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

    2014-05-01

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

  15. Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.

    PubMed

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

    2014-06-01

    We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports. PMID:24756322

  16. Affinity purification and mass spectrometry: an attractive choice to investigate protein-protein interactions in plant immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Affinity purification of protein complexes from biological tissues, followed by liquid chromatography- tandem mass spectrometry (AP-MS/MS), has ballooned in recent years due to sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, improvements in affinity purifica...

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-02-01

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

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

    SciTech Connect

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

    1990-09-25

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

  20. 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

  1. SnAvi--a new tandem tag for high-affinity protein-complex purification.

    PubMed

    Schäffer, Ursula; Schlosser, Andreas; Müller, Kristian M; Schäfer, Angelika; Katava, Nenad; Baumeister, Ralf; Schulze, Ekkehard

    2010-04-01

    Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins. PMID:20047968

  2. Solubilization and purification of the alpha 1-adrenergic receptor using a novel affinity resin.

    PubMed Central

    Graham, R M; Hess, H J; Homcy, C J

    1982-01-01

    The highly selective alpha 1-adrenergic receptor antagonist prazosin was used to identify binding sites having alpha-adrenergic specificity in rat hepatic plasma membranes. Solubilization of the membrane-bound receptors was achieved by incubation with the nonionic detergent digitonin, and binding activity was assayed by using [3H]prazosin and a polyethylene glycol precipitation technique. Only 20-30% of the total receptor pool was released by the solubilization procedure. However, binding of [3H]prazosin was saturable [maximal value, 206 +/- 8 fmol/mg of protein (membrane) vs. 74 +/- 4 fmol/mg of protein (soluble)] and of high affinity [Kd, 0.6 +/- 0.2 nM (membrane) vs. 0.8 +/- 0.2 nM (soluble)]. To aid in purification of the receptors, an affinity resin was developed using an analog of prazosin, 2-(4-succinoylpiperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (CP 57,609; Kd 2.7 X 10(-7) M) immobilized via an amide linkage to agarose. The resulting resin demonstrated high affinity (Kd 3.2 X 10(-7) M) for the solubilized receptors, as determined by competitive inhibition assay. The degree of substitution to the resin was determined by a direct radioimmunoassay using antibodies against albumin-complexed CP 57,609 and found to be 0.1 to 0.2 mumol/ml of agarose. Affinity chromatography using the resin resulted in 513-fold purification in a single step. Moreover, the specificity of the purified binding sites was similar to that of membrane-bound receptors. This novel affinity resin should thus provide a powerful tool for isolating the receptor protein in quantities sufficient for detailed biochemical characterization. PMID:6285370

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2015-04-01

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

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

    PubMed

    Amarasinghe, Chinthaka; Jin, Jian-Ping

    2015-01-01

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

  6. 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.

  7. 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.

  8. Efficient and rapid purification of lentil alpha-galactosidase by affinity precipitation with alginate.

    PubMed

    Celem, Evran Biçak; Bolle, Sharon Sibel; Onal, Seçil

    2009-10-01

    Alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified (26-fold) from the germinating seeds of lentil (Lens culinaris) by affinity precipitation with alginate. The purified enzyme gave a single band corresponding to molecular mass of 40 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were determined as 40 degrees C and 5.5, respectively. The enzyme was very stable at a temperature range of 4-65 degrees C and at a pH range of 4-7. The values of kinetic constants Km and Vmax using p-nitrophenyl-alpha-D-galactopyranoside (PNPG) as substrate were 0.191 mM and 0.73 U, respectively. Results suggest that affinity precipitation is an attractive process for the purification of alpha-galactosidase. PMID:20027865

  9. Affinity purification of antibodies using immobilized FB domain of protein A.

    PubMed

    Solomon, B; Raviv, O; Leibman, E; Fleminger, G

    1992-04-24

    A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A. PMID:1517325

  10. Evaluation of Quantitative Performance of Sequential Immobilized Metal Affinity Chromatographic Enrichment for Phosphopeptides

    PubMed Central

    Sun, Zeyu; Hamilton, Karyn L.; Reardon, Kenneth F.

    2014-01-01

    We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium-dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. While our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semi-quantitative method when used in large-scale phosphoproteomics studies in complex backgrounds. PMID:24096195

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

    PubMed

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

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed. PMID:24630982

  12. A Robust and Fully-Automated Chromatographic Method for the Quantitative Purification of Ca and Sr for Isotopic Analysis

    NASA Astrophysics Data System (ADS)

    Smith, H. B.; Kim, H.; Romaniello, S. J.; Field, P.; Anbar, A. D.

    2014-12-01

    High throughput methods for sample purification are required to effectively exploit new opportunities in the study of non-traditional stable isotopes. Many geochemical isotopic studies would benefit from larger data sets, but these are often impractical with manual drip chromatography techniques, which can be time-consuming and demand the attention of skilled laboratory staff. Here we present a new, fully-automated single-column method suitable for the purification of both Ca and Sr for stable and radiogenic isotopic analysis. The method can accommodate a wide variety of sample types, including carbonates, bones, and teeth; silicate rocks and sediments; fresh and marine waters; and biological samples such as blood and urine. Protocols for these isotopic analyses are being developed for use on the new prepFAST-MCTM system from Elemental Scientific (ESI). The system is highly adaptable and processes up to 24-60 samples per day by reusing a single chromatographic column. Efficient column cleaning between samples and an all Teflon flow path ensures that sample carryover is maintained at the level of background laboratory blanks typical for manual drip chromatography. This method is part of a family of new fully-automated chromatographic methods being developed to address many different isotopic systems including B, Ca, Fe, Cu, Zn, Sr, Cd, Pb, and U. These methods are designed to be rugged and transferrable, and to allow the preparation of large, diverse sample sets via a highly repeatable process with minimal effort.

  13. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    PubMed Central

    Kaake, Robyn M.; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis. Affinity purification coupled with quantitative mass spectrometry has become the primary method for studying in vivo protein interactions of protein complexes and whole organism proteomes. Recent developments in sample preparation and affinity purification strategies allow the capture, identification, and quantification of protein interactions of protein complexes that are stable, dynamic, transient, and/or weak. Current efforts have mainly focused on generating reliable, reproducible, and high confidence protein interaction data sets for functional characterization. The availability of increasing amounts of information on protein interactions in eukaryotic systems and new bioinformatics tools allow functional analysis of quantitative protein interaction data to unravel the biological significance of the identified protein interactions. Existing studies in this area have laid a solid foundation toward generating a complete map of in vivo protein interaction networks of protein complexes in cells or tissues. PMID:20445003

  14. 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.

  15. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2014-01-01

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

  18. Chromatographic extraction with di(2-ethylhexyl)orthophosphoric acid for production and purification of promethium-147

    DOEpatents

    Boll, Rose A [Knoxville, TN

    2008-10-14

    A method of producing and purifying promethium-147 including the steps of: irradiating a target material including neodymium-146 with neutrons to produce promethium-147 within the irradiated target material; dissolving the irradiated target material to form an acidic solution; loading the acidic solution onto a chromatographic separation apparatus containing HDEHP; and eluting the apparatus to chromatographically separate the promethium-147 from the neodymium-146.

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

    PubMed

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

    2012-11-01

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

  20. 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.

  1. Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry.

    PubMed

    Mirzaei, Hamid; Regnier, Fred

    2005-04-15

    It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of a wide variety of routes by which proteins are oxidized is the generation of carbonyl groups. This paper reports a method for the isolation of oxidized proteins, which involves (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. The selectivity of the method was validated by adding in vitro oxidized biotinylated BSA to a yeast lysate and showing that the predominant protein recovered was BSA. This method was applied to the question of whether large doses of 2-nitropropane produce oxidized proteins. A study of rat liver homogenates showed that animals dosed with 2-nitropropane produced 17 times more oxidized protein than controls in 6 h. Tryptic digestion of these oxidized proteins followed by reversed-phase chromatography and tandem mass spectrometry led to the identification of 14 peptides and their parent proteins. Nine of the 14 identified peptides were found to carry 1 or 2 oxidation sites and 5 of the 9 peptides were biotinylated. The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification. In some cases, it is even possible to identify the site of oxidation. PMID:15828771

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

    NASA Astrophysics Data System (ADS)

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

    1998-08-01

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

  3. Linker peptide and affinity tag for detection and purification of single-chain Fv fragments.

    PubMed

    Küttner, Gabriele; Giessmann, Elke; Wessner, Helga; Scholz, Christa; Reinhardt, Dina; Winkler, Karsten; Marx, Uwe; Höhne, Wolfgang

    2004-05-01

    The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs. PMID:15152607

  4. 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. PMID:25086199

  5. 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

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

    PubMed

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

    2014-01-01

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

  7. 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. PMID:25569629

  8. Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

    PubMed

    Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong

    2014-11-15

    The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). PMID:25261834

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

    PubMed

    Novick, Daniela; Rubinstein, Menachem

    2012-01-01

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

  10. Identification of novel interacting protein partners of SMN using tandem affinity purification.

    PubMed

    Shafey, Dina; Boyer, Justin G; Bhanot, Kunal; Kothary, Rashmi

    2010-04-01

    Mutations in the survival motor neuron (SMN) gene cause spinal muscular atrophy (SMA), a neuromuscular disease associated with muscle weakness that progresses to paralysis, respiratory distress, and ultimately death. Both neurons and muscle are severely affected in this disease. Tandem affinity purification (TAP) has emerged as a useful tool for studying protein complexes in vitro. We have used this purification system to discover novel SMN interacting partners in C2C12 muscle and PC12 neuronal cells. To do so, we fused a TAP-tag, consisting of a HIS hexamer and FLAG epitope separated by the tobacco etch virus (TEV) protease cleavage site, to either the N- or C-terminal region of SMN. Interestingly, the profile of SMN interacting proteins varies depending on the cell type and stage. We have identified a number of novel SMN interacting proteins in both C2C12 and PC12 cells, and from among these we have validated Annexin II and myosin regulatory light chain (MRLC). The discovery of these proteins will lead to a better understanding of the mechanisms underlying the pathophysiology of SMA. PMID:20201562

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

    PubMed

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

    2016-03-01

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

  12. Analyzing protein-protein interactions from affinity purification-mass spectrometry data with SAINT

    PubMed Central

    Choi, Hyungwon; Liu, Guomin; Mellacheruvu, Dattatreya; Tyers, Mike; Gingras, Anne-Claude; Nesvizhskii, Alexey I.

    2012-01-01

    Significance Analysis of INTeractome (SAINT) is a software package for scoring protein-protein interactions based on label-free quantitative proteomics data (e.g. spectral count or intensity) in affinity purification – mass spectrometry (AP-MS) experiments. SAINT allows bench scientists to select bona fide interactions and remove non-specific interactions in an unbiased manner. However, there is no `one-size-fits-all' statistical model for every dataset, since the experimental design varies across studies. Key variables include the number of baits, the number of biological replicates per bait, and control purifications. Here we give a detailed account of input data format, control data, selection of high confidence interactions, and visualization of filtered data. We explain additional options for customizing the statistical model for optimal filtering in specific datasets. We also discuss a graphical user interface of SAINT in connection to the LIMS system ProHits which can be installed as a virtual machine on Mac OSX or PC Windows computers. PMID:22948729

  13. Rapid purification of cytosolic epoxide hydrolase from normal and clofibrate-treated animals by affinity chromatography.

    PubMed Central

    Prestwich, G D; Hammock, B D

    1985-01-01

    Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively adsorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I50, approximately equal to 3 X 10(-4) M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I50, 1-50 X 10(-7) M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO4/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO4/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction. Images PMID:3856846

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

    PubMed

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

    2015-01-01

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

  15. 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.

  16. FastTrack to supercritical fluid chromatographic purification: Implementation of a walk-up analytical supercritical fluid chromatography/mass spectrometry screening system in the medicinal chemistry laboratory.

    PubMed

    Aurigemma, Christine; Farrell, William

    2010-09-24

    Medicinal chemists often depend on analytical instrumentation for reaction monitoring and product confirmation at all stages of pharmaceutical discovery and development. To obtain pure compounds for biological assays, the removal of side products and final compounds through purification is often necessary. Prior to purification, chemists often utilize open-access analytical LC/MS instruments because mass confirmation is fast and reliable, and the chromatographic separation of most sample constituents is sufficient. Supercritical fluid chromatography (SFC) is often used as an orthogonal technique to HPLC or when isolation of the free base of a compound is desired. In laboratories where SFC is the predominant technique for analysis and purification of compounds, a reasonable approach for quickly determining suitable purification conditions is to screen the sample against different columns. This can be a bottleneck to the purification process. To commission SFC for open-access use, a walk-up analytical SFC/MS screening system was implemented in the medicinal chemistry laboratory. Each sample is automatically screened through six column/method conditions, and on-demand data processing occurs for the chromatographers after each screening method is complete. This paper highlights the "FastTrack" approach to expediting samples through purification. PMID:20728893

  17. Affinity purification and characterization of (2'-5')oligo(adenylate)-dependent RNase from mouse spleen.

    PubMed

    Bayard, B; Bette-Bobillo, P; Aliau, S

    1994-07-15

    Murine (2'-5')An-dependent RNase, a key enzyme of the interferon system, was purified from mouse spleen by affinity chromatography to immobilized (2'-5')An. Since the ribonuclease has high affinity to (2'-5')An, optimal non-denaturing conditions were obtained to disrupt the (2'-5')An-nuclease complex. Low-pH buffers in the presence of 0.1% Triton X-100 removed almost 80% of the enzyme from the (2'-5')An-agarose, preserving its (2'-5')An binding activity and RNA cleavage function. Purification was monitored using a classical radiobinding assay, ultraviolet covalent crosslinking method and denaturing-renaturing affinity blotting assay. The purified enzyme was a 160-kDa dimer that migrated with an apparent molecular mass of 78 kDa and was > 80% pure, as assessed by silver-stained SDS gels. Both a 160-kDa dimer and 78-kDa monomer were found in the cellular extract at a 5:1 ratio. Binding of radiolabeled (2'-5')An to (2'-5')An-dependent RNase either in crude extract or in purified form reached equilibrium by 5 h at 4 degrees C. 2-Mercaptoethanol was required to obtain (2-'5')An-binding activity but, interestingly, in the absence of this reducing agent, (2'-5')An-binding activity was initiated by preincubation with poly(U), a synthetic substrate of the nuclease. This new mechanistic feature indicates that interaction of poly(U) with nuclease induced a conformational modification allowing, in a second step, the binding of (2'-5')An. Furthermore, when activated by low amounts of (2'-5')An, the eluted purified enzyme degraded mRNA but there was still degradation in the absence of (2'-5')An. This suggested a loss of regulatory protein(s) during the purification step. Scatchard analysis showed that the purified enzyme had a Kd of 106 pM for (2'-5')An, similar to estimates obtained using crude spleen extracts (Kd 112 pM), indicating that the purified nuclease had almost identical (2'-5')An-binding properties to those identified in spleen extracts. PMID:8055909

  18. 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. PMID:8183950

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

    PubMed

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

    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

  20. 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

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

    PubMed

    Salehi, Nasrin; Peng, Ching-An

    2016-07-01

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

  2. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

    PubMed Central

    Agbanyo, F R; Vijayalakshmi, D; Craik, J D; Gati, W P; McAdam, D P; Asakura, J; Robins, M J; Paterson, A R; Cass, C E

    1990-01-01

    Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups. Images Fig. 5. Fig. 6. Fig. 7. PMID:2241896

  3. miRNA Tagging and Affinity-purification (miRAP)

    PubMed Central

    He, Miao

    2016-01-01

    MicroRNAs(miRNAs) are a group of endogenously expressed 20~23 nt small noncoding RNAs, which can directly regulate mRNA stability or translation in a sequence specific manner by incomplete base pairing at the 3′UTR of target mRNA, or indirectly affect transcriptional network by regulating transcription factors. As key regulators of gene expression, miRNAs are involved in the control of diverse developmental and physiological processes, including embryogenesis, differentiation, developmental timing, organogenesis, growth control, and programmed cell death. Aberrant miRNA expression profiles have been observed in many pathological conditions, including cancers, psychiatric diseases, virus infection, etc. However, the underlying mechanisms have been difficult to study in part due to the cellular heterogeneity of complex tissue. To systematically analyze miRNA expression in complex tissue, we present here a novel miRNA tagging and Affinity Purification method, miRAP, which can be applied to genetically defined cell types in any complex tissues in mice. This method is based on the fact that mature miRNAs are incorporated into RNA-induced silencing complex (RISC), in which the Argonaute protein AGO2 directly binds miRNAs and their mRNA targets. We demonstrate that epitope tagging of AGO2 protein allows direct purification of miRNAs from tissue homogenates using antibodies against the engineered molecular tag. We further established a Cre-loxP binary expression system to deliver epitope-tagged AGO2 (tAGO2) to genetically defined cell types.

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

    PubMed Central

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

    2009-01-01

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

  5. Novel flavonol 2-oxoglutarate dependent dioxygenase: affinity purification, characterization, and kinetic properties.

    PubMed

    Anzellotti, D; Ibrahim, R K

    2000-10-15

    A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11-] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as well as by affinity chromatography on 2-oxoglutarate-Sepharose and immunoaffinity columns. The specific activity of the 6-hydroxylase eluted from Mono Q (97.1 pkat/mg) was enriched 538-fold, with a 0.63% recovery. Both affinity chromatography steps resulted in the elimination of most contaminating proteins, but not without loss of enzyme activity and stability. The molecular mass of both the native and denatured enzyme was found to be 42 and 45 kDa, respectively, suggesting a monomeric protein. The enzyme exhibits strict specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polymethylated flavonols in this plant. The cofactor dependence of the enzyme is similar to that of other plant dioxygenases, particularly its dependence on ferrous ions for catalytic activity and reactivation. Internal amino acid sequence information indicated its relatedness to other plant flavonoid dioxygenases. The results of substrate interaction kinetics and product inhibition studies suggest an ordered, sequential reaction mechanism (TerTer), where 2-oxoglutarate is the first substrate to bind, followed by O2 and the flavonol substrate. Product release occurs in the reverse order where the hydroxylated flavonol is the first to be released, followed by CO2 and succinate. To our knowledge, this is the first reported 2-oxoglutarate-dependent dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid compound. PMID:11068865

  6. One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode.

    PubMed

    Kuo, Ting-Yu; Hong, Zhi-Wei; Tsai, Chung-Che; Yang, Yu-Chi; Fu, Hua-Wen

    2016-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established. In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin. This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy. PMID:27404433

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

    PubMed

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

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d. PMID:26766115

  8. GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice*

    PubMed Central

    Bartoi, Tudor; Rigbolt, Kristoffer T. G.; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-01-01

    GABAB receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABAB1, are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABAB receptors. The transgenic mice express GABAB1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABAB1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABAB receptors via the GABAB2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABAB2. The mice equipped with tags on GABAB1 facilitate validation and identification of native binding partners of GABAB receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  9. GABAB receptor constituents revealed by tandem affinity purification from transgenic mice.

    PubMed

    Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-07-01

    GABA(B) receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA(B1), are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA(B2). The mice equipped with tags on GABA(B1) facilitate validation and identification of native binding partners of GABA(B) receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  10. Mapping differential interactomes by affinity purification coupled with data independent mass spectrometry acquisition

    PubMed Central

    Lambert, Jean-Philippe; Ivosev, Gordana; Couzens, Amber L.; Larsen, Brett; Taipale, Mikko; Lin, Zhen-Yuan; Zhong, Quan; Lindquist, Susan; Vidal, Marc; Aebersold, Ruedi; Pawson, Tony; Bonner, Ron; Tate, Stephen; Gingras, Anne-Claude

    2013-01-01

    Characterizing changes in protein-protein interactions associated with sequence variants (e.g. disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies where cost and time are major considerations. To this end, we have coupled AP to data-independent mass spectrometric acquisition (SWATH), and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. Here, we use AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes, and propose a scalable pipeline for systems biology studies. PMID:24162924

  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

    2015-01-01

    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. PMID:26404184

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

    PubMed Central

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

    2015-01-01

    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. PMID:26404184

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-05-15

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

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

    PubMed

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

    2015-10-30

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

  16. Affinity chromatography of trypsin and related enzymes. III. Purification of Streptomyces griseus trypsin using an affinity adsorbent containing a tryptic digest of protamine as a ligand.

    PubMed

    Yokosawa, H; Hanba, T; Ishii, S

    1976-04-01

    A new, simple method has been developed for the purification of Streptomyces griseus trypsin [EC 3.4.21.4] from Pronase. Only a single operation of affinity chromatography on an agarose derivative, which was easily prepared by coupling a tryptic digest of salmine to cyanogen bromide-activated Sepharose 4B, was required. A high degree of homogeneity was demonstrated for the purified enzyme by disc electrophoresis, SDS-polyacrylamide gel electrophoresis and gel filtration, as well as by active-site titration. The behavior of a carboxypeptides B [EC 3.4.12.3]-like enzyme present in Pronase is also discussed. PMID:819428

  17. Magnetic Parkia pendula seed gum as matrix for Concanavalin A lectin immobilization and its application in affinity purification.

    PubMed

    Rêgo, Moacyr J B M; Almeida, Sinara M; Bezerra, Sérgio A; Carvalho Júnior, Luiz B; Beltrão, Eduardo I C

    2014-09-01

    The present work aimed to magnetize Parkia pendula seeds gum and use it as a matrix for Concanavalin A covalent immobilization. This composite was applied in affinity purification of glycoconjugates. Parkia pendula seeds were hydrated and the gum provenient from the supernatant was precipitated and washed with ethanol and dried. The gum was magnetized in co-precipitation using solutions of Fe+2 and Fe+3. Matrix activation was accomplished with NaIO4. Magnetized Parkia pendula seeds gum with covalently immobilized Concanavalin A was used as an affinity matrix for the recognition of bovine serum fetuin glycoprotein. Fetuin elution was carried out with a solution of glucose (300mM) and evaluated through SDS-PAGE. The efficiency of lectin immobilization and fetuin purification were 63% and 14%, respectively. These results indicate that the composite produced is a promising magnetic polysaccharide matrix for lectins immobilization. Thus, such system can be applied for affinity purification allowing an easy recovery by magnetic field. PMID:25140501

  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. Chromatographic speciation of Cr(III)-species, inter-species equilibrium isotope fractionation and improved chemical purification strategies for high-precision isotope analysis

    PubMed Central

    Larsen, K.K.; Wielandt, D.; Schiller, M.; Bizzarro, M.

    2016-01-01

    Chromatographic purification of chromium (Cr), which is required for high-precision isotope analysis, is complicated by the presence of multiple Cr-species with different effective charges in the acid digested sample aliquots. The differing ion exchange selectivity and sluggish reaction rates of these species can result in incomplete Cr recovery during chromatographic purification. Because of large mass-dependent inter-species isotope fractionation, incomplete recovery can affect the accuracy of high-precision Cr isotope analysis. Here, we demonstrate widely differing cation distribution coefficients of Cr(III)-species (Cr3+, CrCl2+ and CrCl2+) with equilibrium mass-dependent isotope fractionation spanning a range of ~1‰/amu and consistent with theory. The heaviest isotopes partition into Cr3+, intermediates in CrCl2+ and the lightest in CrCl2+/CrCl3°. Thus, for a typical reported loss of ~25% Cr (in the form of Cr3+) through chromatographic purification, this translates into 185 ppm/amu offset in the stable Cr isotope ratio of the residual sample. Depending on the validity of the mass-bias correction during isotope analysis, this further results in artificial mass-independent effects in the mass-bias corrected 53Cr/52Cr (μ53 Cr* of 5.2 ppm) and 54Cr/52Cr (μ54Cr* of 13.5 ppm) components used to infer chronometric and nucleosynthetic information in meteorites. To mitigate these fractionation effects, we developed strategic chemical sample pre-treatment procedures that ensure high and reproducible Cr recovery. This is achieved either through 1) effective promotion of Cr3+ by >5 days exposure to HNO3 —H2O2 solutions at room temperature, resulting in >~98% Cr recovery for most types of sample matrices tested using a cationic chromatographic retention strategy, or 2) formation of Cr(III)-Cl complexes through exposure to concentrated HCl at high temperature (>120 °C) for several hours, resulting in >97.5% Cr recovery using a chromatographic elution strategy that

  20. Chromatographic speciation of Cr(III)-species, inter-species equilibrium isotope fractionation and improved chemical purification strategies for high-precision isotope analysis.

    PubMed

    Larsen, K K; Wielandt, D; Schiller, M; Bizzarro, M

    2016-04-22

    Chromatographic purification of chromium (Cr), which is required for high-precision isotope analysis, is complicated by the presence of multiple Cr-species with different effective charges in the acid digested sample aliquots. The differing ion exchange selectivity and sluggish reaction rates of these species can result in incomplete Cr recovery during chromatographic purification. Because of large mass-dependent inter-species isotope fractionation, incomplete recovery can affect the accuracy of high-precision Cr isotope analysis. Here, we demonstrate widely differing cation distribution coefficients of Cr(III)-species (Cr(3+), CrCl(2+) and CrCl2(+)) with equilibrium mass-dependent isotope fractionation spanning a range of ∼1‰/amu and consistent with theory. The heaviest isotopes partition into Cr(3+), intermediates in CrCl(2+) and the lightest in CrCl2(+)/CrCl3°. Thus, for a typical reported loss of ∼25% Cr (in the form of Cr(3+)) through chromatographic purification, this translates into 185ppm/amu offset in the stable Cr isotope ratio of the residual sample. Depending on the validity of the mass-bias correction during isotope analysis, this further results in artificial mass-independent effects in the mass-bias corrected (53)Cr/(52)Cr (μ(53)Cr* of 5.2ppm) and (54)Cr/(52)Cr (μ(54)Cr* of 13.5ppm) components used to infer chronometric and nucleosynthetic information in meteorites. To mitigate these fractionation effects, we developed strategic chemical sample pre-treatment procedures that ensure high and reproducible Cr recovery. This is achieved either through 1) effective promotion of Cr(3+) by >5 days exposure to HNO3H2O2 solutions at room temperature, resulting in >∼98% Cr recovery for most types of sample matrices tested using a cationic chromatographic retention strategy, or 2) formation of Cr(III)-Cl complexes through exposure to concentrated HCl at high temperature (>120°C) for several hours, resulting in >97.5% Cr recovery using a

  1. Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

    PubMed Central

    2010-01-01

    Background Poly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions. Results PARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1. Conclusions This study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism. PMID:20388209

  2. Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry.

    PubMed

    Boyarchuk, Ekaterina; Robin, Philippe; Fritsch, Lauriane; Joliot, Véronique; Ait-Si-Ali, Slimane

    2016-01-01

    Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular

  3. One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

    PubMed Central

    Shih, Kuo-Shun; Lin, Chih-Chang; Hung, Hsiao-Fang; Yang, Yu-Chi; Wang, Chung-An; Jeng, Kee-Ching; Fu, Hua-Wen

    2013-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection. PMID:23577158

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags.

    PubMed

    Salvail-Lacoste, Alix; Di Tomasso, Geneviève; Piette, Benjamin L; Legault, Pascale

    2013-07-01

    Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3' homogeneity. Here, we explored strategies to also ensure 5' homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5' heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II 2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5'-sequence heterogeneity in several cases, significant levels of 5' heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5' homogeneity, we tested the suitability of using a small CRISPR RNA stem-loop at the 5' end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5'-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR-SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5'-CRISPR-based strategy can be combined with affinity purification using the 3'-ARiBo tag for quick purification of RNA with both 5' and 3' homogeneity. PMID:23657939

  8. Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags

    PubMed Central

    Salvail-Lacoste, Alix; Di Tomasso, Geneviève; Piette, Benjamin L.; Legault, Pascale

    2013-01-01

    Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3′ homogeneity. Here, we explored strategies to also ensure 5′ homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5′ heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II ϕ2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5′-sequence heterogeneity in several cases, significant levels of 5′ heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5′ homogeneity, we tested the suitability of using a small CRISPR RNA stem–loop at the 5′ end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5′-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR–SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5′-CRISPR-based strategy can be combined with affinity purification using the 3′-ARiBo tag for quick purification of RNA with both 5′ and 3′ homogeneity. PMID:23657939

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

    PubMed

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

    2010-01-01

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

  10. 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. PMID:24840788

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

    PubMed Central

    2015-01-01

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

  12. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction.

    PubMed

    Karpol, Alon; Kantorovich, Lia; Demishtein, Alik; Barak, Yoav; Morag, Ely; Lamed, Raphael; Bayer, Edward A

    2009-01-01

    Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. PMID:18979459

  13. 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

  14. Ribonucleic acid purification.

    PubMed

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

    2014-08-15

    Research on RNA has led to many important biological discoveries and improvement of therapeutic technologies. From basic to applied research, many procedures employ pure and intact RNA molecules; however their isolation and purification are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality. The current techniques to isolate and purify RNA molecules still have several limitations and the requirement for new methods able to improve RNA quality to meet regulatory demands is growing. In fact, as basic research improves the understanding of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the purification of biological compounds, requiring high purity degree. In addition, its application in biopharmaceutical manufacturing is well established. This paper discusses the importance and the progress of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In particular and in view of the high specificity, affinity chromatography has been recently applied to RNA purification processes. Accordingly, recent chromatographic investigations based on biorecognition phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential contribution of these methods to overcome the challenges of RNA purification. PMID:24951289

  15. 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.

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

    PubMed

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

    2015-09-01

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

  17. Purification of rat liver plasma membranes by wheat-germ-agglutinin affinity partitioning.

    PubMed Central

    Persson, A; Johansson, B; Olsson, H; Jergil, B

    1991-01-01

    Rat liver plasma membranes were separated from other cellular membranes by affinity partitioning in an aqueous polymer two-phase system by using the lectin wheat-germ agglutinin covalently bound to dextran as the affinity ligand. In borate buffer the bulk of membranes partitioned in the poly(ethylene glycol)-rich top phase, whereas plasma membranes were pulled selectively into the dextran-rich bottom phase in the presence of ligand. The purity and yield of plasma membranes prepared by lectin affinity partitioning and by conventional sucrose-density-gradient centrifugation was similar, as judged from marker-enzyme activities. The affinity procedure, not dependent on lengthy centrifugations, is fast and gentle and will be advantageous when studying labile components. PMID:1703408

  18. Heparin Affinity: Purification of a Tumor-Derived Capillary Endothelial Cell Growth Factor

    NASA Astrophysics Data System (ADS)

    Shing, Y.; Folkman, J.; Sullivan, R.; Butterfield, C.; Murray, J.; Klagsbrun, M.

    1984-03-01

    A tumor-derived growth factor that stimulates the proliferation of capillary endothelial cells has a very strong affinity for heparin. This heparin affinity makes it possible to purify the growth factor to a single-band preparation in a rapid two-step procedure. The purified growth factor is a cationic polypeptide, has a molecular weight of about 18,000, and stimulates capillary endothelial cell proliferation at a concentration of about 1 nanogram per milliliter.

  19. Coupling isotachophoresis with affinity chromatography for rapid and selective purification with high column utilization, part 2: experimental study.

    PubMed

    Shkolnikov, Viktor; Santiago, Juan G

    2014-07-01

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

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

    PubMed Central

    2015-01-01

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

  1. Isolation and purification of cat albumin from cat serum by copper ion affinity chromatography: further analysis of its primary structure.

    PubMed

    Dandeu, J P; Rabillon, J; Guillaume, J L; Camoin, L; Lux, M; David, B

    1991-02-22

    Proteins, regardless of their origin, have to be highly purified, particularly from the immunochemical point of view, if they are to be used to study their allergenicity. It is shown that cat albumin, a highly potent allergen for cat-sensitive humans, can be isolated and purified from cat serum using immobilized metal ion affinity chromatography (copper ions) instead of a salting-out process or precipitation with alcohol, techniques generally used for the preparation of serum proteins. During the process described, immunoglobulins are concomitantly isolated in a relatively pure form. Cat albumin amino acid composition and sequence were analysed after an ultimate purification by ion-exchange chromatography. The highest homology (greater than 80%) was found with the rat serum albumin. PMID:2045457

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

    PubMed Central

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

    2011-01-01

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

  3. 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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. 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.

  7. 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.

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

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

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

  9. Application of superparamagnetic microspheres for affinity adsorption and purification of glutathione

    NASA Astrophysics Data System (ADS)

    Wang, Qiang; Guan, Yueping; Yang, Mingzhu

    2012-10-01

    The superparamagnetic poly-(MA-DVB) microspheres with micron size were synthesized by the modified suspension polymerization method. Adsorption of glutathione by magnetic poly-(MA-DVB) microspheres with IDA-copper was investigated. The effect of solution pH value, affinity adsorption and desorption of glutathione was studied. The results showed that the optimum pH value for glutathione adsorption was found at pH=3.5, the maximum capacity for glutathione of magnetic poly-(MA-DVB) microspheres was estimated at 42.4 mg/g by fitting the experimental data to the Langmuir equation. The adsorption equilibrium of glutathione was obtained in about 10 min and the adsorbed glutathione was desorbed from the magnetic microspheres in about 30 min using NaCl buffer solution. The magnetic microspheres could be repeatedly utilized for the affinity adsorption of glutathione.

  10. Design and optimization of a chromatographic purification process for Streptococcus pneumoniae serotype 23F capsular polysaccharide by a Design of Experiments approach.

    PubMed

    Ji, Yu; Tian, Yang; Ahnfelt, Mattias; Sui, Lili

    2014-06-27

    Multivalent pneumococcal vaccines were used worldwide to protect human beings from pneumococcal diseases. In order to eliminate the toxic organic solutions used in the traditional vaccine purification process, an alternative chromatographic process for Streptococcus pneumoniae serotype 23F capsular polysaccharide (CPS) was proposed in this study. The strategy of Design of Experiments (DoE) was introduced into the process development to solve the complicated design procedure. An initial process analysis was given to review the whole flowchart, identify the critical factors of chromatography through FMEA and chose the flowthrough mode due to the property of the feed. A resin screening study was then followed to select candidate resins. DoE was utilized to generate a resolution IV fractional factorial design to further compare candidates and narrow down the design space. After Capto Adhere was selected, the Box-Behnken DoE was executed to model the process and characterize all effects of factors on the responses. Finally, Monte Carlo simulation was used to optimize the process, test the chosen optimal conditions and define the control limit. The results of three scale-up runs at set points verified the DoE and simulation predictions. The final results were well in accordance with the EU pharmacopeia requirements: Protein/CPS (w/w) 1.08%; DNA/CPS (w/w) 0.61%; the phosphorus content 3.1%; the nitrogen 0.315% and the Methyl-pentose percentage 47.9%. Other tests of final pure CPS also met the pharmacopeia specifications. This alternative chromatographic purification process for pneumococcal vaccine without toxic organic solvents was successfully developed by the DoE approach and proved scalability, robustness and suitability for large scale manufacturing. PMID:24845825

  11. Aryl thioglycoside-based affinity purification of exo-acting cellulases.

    PubMed

    Piyachomkwan, K; Penner, M H

    1998-01-15

    The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters. PMID:9451508

  12. 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.

  13. Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture

    SciTech Connect

    Ji, H.; Smith, L.M. )

    1993-05-15

    A simple and rapid method for the preparation of highly pure plasmid DNA has been developed. The DNA is directly captured from bacterial cell lysates by formation of a triple-helical structure between the plasmid dsDNA and a 20-base biotinylated oligonucleotide attached to streptavidin-coated magnetic beads and then eluted from the beads in pH 9 buffer solution. No phenol extraction, ethanol precipitation, RNase digestion, or CsCl gradient centrifugation is required. A general purpose cloning vector, pHJ19, was constructed for this application from pUC19 DNA by insertion of a 40-base sequence suitable for triple-helix formation. The approach was also found suitable for the purification of [lambda] bacteriophage DNA. 32 refs., 6 figs., 1 tab.

  14. Affinity tag for protein purification and detection based on the disulfide-linked complex of InaD and NorpA.

    PubMed

    Kimple, Michelle E; Sondek, John

    2002-09-01

    Affinity tags are not only used for the expression and purification of recombinant proteins but also for the detection of protein-protein interactions. Common problems with many affinity tags are excessive length, which may interfere with the structure and function of tagged proteins, and low affinity and/or specificity for primary detection and purification agents. Preliminary results suggest that the C-terminalfive residues of the Drosophila protein NorpA, based on the short, covalent interaction they make with the N-terminal PDZ domain (PDZI) of InaD, are useful as a general affinity tag. First, a PDZI-alkaline phosphatase fusion protein specifically detects both its physiological ligand and a heterologous protein expressing the NorpA C-terminal five residues. The interaction of PDZI with a NorpA-tagged protein is reversible by a reducing agent, which allows nitrocellulose membranes to be stripped completely and reused. In addition, a NorpA-tagged protein can specifically bind to immobilized PDZI resin, while other cellular proteins are washed through. After washing, the NorpA-tagged protein is eluted by a reducing buffer. The NorpA tag's short length makes it the smallest affinity tag available, and its specific and high-affinity interaction with PDZI could yield a powerful system that improves on currently available technology. PMID:12238768

  15. 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.

  16. Affinity purification of specific chromatin segments from chromosomal loci in yeast.

    PubMed

    Griesenbeck, Joachim; Boeger, Hinrich; Strattan, J Seth; Kornberg, Roger D

    2003-12-01

    Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods. Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures. Gel filtration analysis establishes the uniformity of the transcriptionally activated state. Activator proteins interact in the manner anticipated from previous studies in vivo. This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states. PMID:14645537

  17. Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography.

    PubMed Central

    Fifis, T; Scopes, R K

    1978-01-01

    1. Affinity elution chromatography was used to purify phosphoglycerate kinase from a variety of sources. The choice of buffer pH for the chromatography was made according to the relative electrophoretic mobility of the enzyme from the species concerned. 2. Outlines of the methods used to isolate the enzyme from over 20 sources are presented. The enzyme was purified from the muscle tissue of a variety of mammals, fish and birds, from liver of several animals, from yeast, Escherichia coli, and plant leaves. The more acidic varieties of the enzymes were purified by conventional gradient elution from ion-exchangers as affinity elution procedures were not applicable. 3. The structural and kinetic parameters investigated show that phosphoglycerate kinase is evolutionarily a highly conservative enzyme; there were few differences in properties regardless of source or function (glycolytic, gluconeogenic or photosynthetic). 4. A detailed comparison of the enzyme preparations purified from bovine muscle and bovine liver failed to detect any significant differences between them; the evidence indicates that they are genetically identical. PMID:367367

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

    PubMed

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

    1991-03-01

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

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

    PubMed

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

    2015-05-22

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

  20. A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris

    PubMed Central

    ALLERSON, CHARLES R.; MARTINEZ, ALAN; YIKILMAZ, EMINE; ROUAULT, TRACEY A.

    2003-01-01

    Regulated expression of proteins involved in mammalian iron metabolism is achieved in part through the interaction of the iron regulatory proteins IRP1 and IRP2 with highly conserved RNA stem-loop structures, known as iron-responsive elements (IREs), that are located within the 5′ or 3′ untranslated regions of regulated transcripts. As part of an effort to determine the structures of the IRP–IRE complexes using crystallographic methods, we have developed an efficient process for obtaining functionally pure IRP1 and IRP2 that relies upon the improved overexpression (>10 mg of soluble IRP per liter of culture) of each human IRP in the yeast Pichia pastoris and large-scale purification using RNA affinity chromatography. Despite the utility of RNA affinity chromatography in the isolation of RNA-binding proteins, current methods for preparing RNA affinity matrices produce columns of low capacity and limited stability. To address these limitations, we have devised a simple method for preparing stable, reusable, high-capacity RNA affinity columns. This method utilizes a bifunctional linker to covalently join a 5′-amino tethered RNA with a thiol-modified Sepharose, and can be used to load 150 nmole or more of RNA per milliliter of solid support. We demonstrate here the use of an IRE affinity column in the large-scale purification of IRP1 and IRP2, and suggest that the convenience of this approach will prove attractive in the analysis of other RNA-binding proteins. PMID:12592010

  1. From pathways to networks: connecting dots by establishing protein-protein interaction networks in signaling pathways using affinity purification and mass spectrometry

    PubMed Central

    Li, Xu; Wang, Wenqi; Chen, Junjie

    2015-01-01

    Signal transductions are the basis of biological activities in all living organisms. Studying the signaling pathways, especially under physiological conditions, has become one of the most important facets of modern biological research. During the last decade, mass spectrometry has been used extensively in biological research and is proven to be effective in addressing important biological questions. Here, we review the current progress in the understanding of signaling networks using mass spectrometry approaches. We will focus on studies of protein-protein interactions that use affinity purification followed by mass spectrometry approach. We discuss obstacles to affinity purification, data processing, functional validation, and identification of transient interactions and provide potential solutions for pathway-specific proteomics analysis, which we hope one day will lead to a comprehensive understanding of signaling networks in humans. PMID:25137225

  2. From pathways to networks: connecting dots by establishing protein-protein interaction networks in signaling pathways using affinity purification and mass spectrometry.

    PubMed

    Li, Xu; Wang, Wenqi; Chen, Junjie

    2015-01-01

    Signal transductions are the basis of biological activities in all living organisms. Studying the signaling pathways, especially under physiological conditions, has become one of the most important facets of modern biological research. During the last decade, MS has been used extensively in biological research and is proven to be effective in addressing important biological questions. Here, we review the current progress in the understanding of signaling networks using MS approaches. We will focus on studies of protein-protein interactions that use affinity purification followed by MS approach. We discuss obstacles to affinity purification, data processing, functional validation, and identification of transient interactions and provide potential solutions for pathway-specific proteomics analysis, which we hope one day will lead to a comprehensive understanding of signaling networks in humans. PMID:25137225

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

    PubMed Central

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

    2012-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is a robust technique used to identify protein-protein interactions. With recent improvements in sample preparation, and dramatic advances in MS instrumentation speed and sensitivity, this technique is becoming more widely used throughout the scientific community. To meet the needs of research groups both large and small, we have developed software solutions for tracking, scoring and analyzing AP-MS data. Here, we provide details for the installation and utilization of ProHits, a Laboratory Information Management System designed specifically for AP-MS interaction proteomics. This protocol explains: (i) how to install the complete ProHits system, including modules for the management of mass spectrometry files and the analysis of interaction data, and (ii) alternative options for the use of pre-existing search results in simpler versions of ProHits, including a virtual machine implementation of our ProHits Lite software. We also describe how to use the main features of the software to analyze AP-MS data. PMID:22948730

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

    PubMed

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

    2006-05-01

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

  6. Systematic analyses of the ultraviolet radiation resistance-associated gene product (UVRAG) protein interactome by tandem affinity purification.

    PubMed

    Son, Ji-Hye; Hwang, Eurim C; Kim, Joungmok

    2016-03-01

    Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions. PMID:26590968

  7. SAINTq: Scoring protein-protein interactions in affinity purification - mass spectrometry experiments with fragment or peptide intensity data.

    PubMed

    Teo, Guoci; Koh, Hiromi; Fermin, Damian; Lambert, Jean-Philippe; Knight, James D R; Gingras, Anne-Claude; Choi, Hyungwon

    2016-08-01

    SAINT (Significance Analysis of INTeractome) is a probabilistic method for scoring bait-prey interactions against negative controls in affinity purification - mass spectrometry (AP-MS) experiments. Our published SAINT algorithms use spectral counts or protein intensities as the input for calculating the probability of true interaction, which enables objective selection of high-confidence interactions with false discovery control. With the advent of new protein quantification methods such as Data Independent Acquisition (DIA), we redeveloped the scoring method to utilize the reproducibility information embedded in the peptide or fragment intensity data as a key scoring criterion, bypassing protein intensity summarization required in the previous SAINT workflow. The new software package, SAINTq, addresses key issues in the interaction scoring based on intensity data, including treatment of missing values and selection of peptides and fragments for scoring each prey protein. We applied SAINTq to two independent DIA AP-MS data sets profiling the interactome of MEPCE and EIF4A2 and that of 14-3-3β, and benchmarked the performance in terms of recovering previously reported literature interactions in the iRefIndex database. In both data sets, the SAINTq analysis using the fragment-level intensity data led to the most sensitive detection of literature interactions at the same level of specificity. This analysis outperforms the analysis using protein intensity data summed from fragment intensity data that is equivalent to the model in SAINTexpress. PMID:27119218

  8. Dual affinity method for plasmid DNA purification in aqueous two-phase systems.

    PubMed

    Barbosa, H S C; Hine, A V; Brocchini, S; Slater, N K H; Marcos, J C

    2010-02-26

    The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate. PMID:20083249

  9. Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli.

    PubMed

    Beshay, Usama; Miksch, Gerhard; Friehs, Karl; Flaschel, Erwin

    2009-02-01

    In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a beta-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space. Its export into the medium was achieved by means of co-expression of a bacteriocin-release protein, the Kil protein from pColE1. Bioreactors were modified so that special devices containing metal chelate pentadentate chelator PDC resins were located within the bioreactor. Using the bioreactor with an internal device the Zn2+-PDC had a 4.3-fold higher binding capacity than metal-free PDC (12.3 and 2.6 kU ml(-1) PDC, respectively. Using the bioreactor with charged PDC in an external circuit revealed even higher beta-glucanase concentration (65.6 kU ml(-1)), i.e. 1.5-fold compared to the internal adsorbent system. PMID:18481103

  10. Preparation and Affinity-Purification of Supervillin Isoform 4 (SV4) Specific Polyclonal Antibodies.

    PubMed

    Chen, Xueran; Li, Hao; Wang, Hongzhi; Yang, Haoran; Ye, Fang; Liang, Chaozhao; Fang, Zhiyou

    2016-04-01

    Human Supervillin isoform 4 (SV4), a bigger splicing isoform of Supervillin, contains extra coding exons 3, 4 and 5 (E345), compared to Supervillin isoform 1. Although previous studies have shown that SV4 associated with membrane and cytoskeleton, regulated cell migration and cell survival, its functions are still largely unknown. To broaden our understanding, SV4 specific antibody is important for further study in signaling pathway. The His-SV4 (E345) and GST-SV4 (E345) fusion proteins, which contained SV4 specific domain E345, were purified from bacteria. The His-SV4 (E345) proteins were injected in rabbits as immunogen to produce anti-SV4 serum, and SV4 antibodies were purified by GST-SV4 (E345) proteins cross-linked to affinity resins. SV4 antibodies exclusively recognized SV4 protein both in vitro and in vivo through multi-step testing by ELISA, western blot, immunoprecipitation, and immunofluorescence. Taken together, our data demonstrate a novel SV4-specific polyclonal antibody which will provide a useful tool for further characterization of SV4 function. PMID:27015936

  11. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS).

    PubMed

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A

    2016-09-01

    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1). PMID:27274533

  12. Affinity column for purification of the human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor

    SciTech Connect

    Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

    1987-05-01

    The TXA/sub 2//PGH/sub 2/ receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific /sup 3/H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA/sub 2//PGH/sub 2/ receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor.

  13. Recombinant expression and affinity purification of snake venom gland parvalbumin in Escherichia coli.

    PubMed

    Jia, Ying; Pérez, John C

    2009-07-01

    Parvalbumins (PV) are small, acidic, water soluble and calcium-binding proteins generally present in muscular and nervous tissues. In the present study, we identified and characterized a cDNA clone encoding PV, named AplPV, from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. AplPV belongs to EF-hand proteins with six alpha-helices constituting three EF-hand domains. The deduced amino acid sequence of AplPV is 91% and 68% identical to the previously characterized PVs of Boa constrictor and Cyprinus carpio, respectively. The full-length cDNA was subcloned into the expression vector pGEX and transformed into Escherichia coli (E.coli) to produce recombinant protein. The bacterially expressed GST-AplPV fusion protein was highly expressed, and effectively purified by Glutathione-Sepharose affinity chromatography. A high concentration of thrombin protease specifically cleaved and removed the GST tag from fusion protein, and further purified by Benzamidine column for removal of thrombin protease. As a result, the 12 kDa AplPV recombinant protein alone was purified. To investigate the tissue-specific biological occurrence of AplPV, a polyclonal antibody (anti-AplPV-antibody) was raised against GST-AplPV fusion protein in rabbit. Western blot analysis revealed that immunoreactive bands were exhibited in both recombinant protein and samples of venom glands, but not in any crude venom. This specific occurrence indicates a specialized function of AplPV in snake venom glands. PMID:19275943

  14. 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

  15. Development and validation study for the chromatographic purification process for tetanus anatoxin on Sephacryl S-200 High Resolution.

    PubMed

    Prado, S M; Vancetto, M D; de Oliveira, J M; Fratelli, F; Higashi, H G

    1999-01-01

    The tetanus purified anatoxin is used in the preparation of multiple immunoprophylactics. WHO (World Health Organization) specifies that the tetanus anatoxin must exhibit a degree of purity greater than or equal to 1,000 Lf/mg protein nitrogen (PN). Today liquid chromatography is a well established technique for the purification of tetanus anatoxin and several different methods are used in production scale. On a small scale, we purified tetanus anatoxin on Sephacryl S-200 High Resolution (gel filtration) and we obtained a successful high-yield purification. On the basis of these results, by combining conventional tangential flow filtration (TFF) at 50,000 N.M.W.L. (Nominal Molecular Weight Limit) ultrafiltration membrane with gel filtration on Sephacryl S-200 High Resolution, we have been able to purify 14 lots of tetanus anatoxin using the Bioprocess System (Amersham Pharmacia Biotech) to a large scale operation. Using this method, 77,401,332 doses of tetanus toxoid were prepared in 14 consecutive lots, supporting the reproducibility and reliability of the method presented here. PMID:10597658

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

    PubMed

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

    2013-01-31

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

  17. Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.

    PubMed

    Bonturi, Nemailla; Radke, Vanessa Soraia Cortez Oliveira; Bueno, Sônia Maria Alves; Freitas, Sindélia; Azzoni, Adriano Rodrigues; Miranda, Everson Alves

    2013-03-01

    The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity. PMID:23411021

  18. A Novel Humanized GLP-1 Receptor Model Enables Both Affinity Purification and Cre-LoxP Deletion of the Receptor

    PubMed Central

    Jun, Lucy S.; Showalter, Aaron D.; Ali, Nosher; Dai, Feihan; Ma, Wenzhen; Coskun, Tamer; Ficorilli, James V.; Wheeler, Michael B.; Michael, M. Dodson; Sloop, Kyle W.

    2014-01-01

    Class B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R), a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r−/− animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential GLP-1R partner

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

    PubMed

    Caramelo-Nunes, Catarina; Tomaz, Cândida T

    2015-01-01

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

  20. Application of high-performance chromatographic and electrophoretic methods to the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum.

    PubMed

    Eriksson, K O; Kourteva, I; Yao, K Q; Liao, J L; Kilár, F; Hjertén, S; Chaga, G

    1987-06-26

    The high resolving power of the preparative and analytical high-performance chromatographic and electrophoretic methods recently developed in this laboratory for the separation of biopolymers has been demonstrated by the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum. Crude glucose oxidase was purified to homogeneity in one step by high-performance hydrophobic-interaction chromatography (HIC) on a pentylagarose column. Crude catalase was purified by a combination of HIC and high-performance anion-exchange chromatography on 3-diethylamino-2-hydroxypropylagarose. The homogeneity of the enzymes was monitored by high-performance electrophoresis and free zone electrophoresis. The pI values of these two enzymes determined by isoelectric focusing in the high-performance electrophoresis apparatus were 4.2 and 6.5, respectively. Their molecular weights were determined by high-performance molecular sieve chromatography on an agarose column. Glucose oxidase has a molecular weight of 175,000 and probably consists of two identical subunits, as sodium dodecyl sulphate polyacrylamide gel electrophoresis gave a molecular weight of around 72,000. The molecular weight of catalase, which is probably composed of non-identical subunits, as indicated by sodium dodecyl sulphate electrophoresis, is around 320,000. Some other characteristics of these two enzymes were also investigated, e.g., electrophoretic mobility, pH stability and optimum pH. PMID:3116021

  1. Chromatographic elution process design space development for the purification of saponins in Panax notoginseng extract using a probability-based approach.

    PubMed

    Chen, Teng; Gong, Xingchu; Chen, Huali; Zhang, Ying; Qu, Haibin

    2016-01-01

    A Monte Carlo method was used to develop the design space of a chromatographic elution process for the purification of saponins in Panax notoginseng extract. During this process, saponin recovery ratios, saponin purity, and elution productivity are determined as process critical quality attributes, and ethanol concentration, elution rate, and elution volume are identified as critical process parameters. Quadratic equations between process critical quality attributes and critical process parameters were established using response surface methodology. Then probability-based design space was computed by calculating the prediction errors using Monte Carlo simulations. The influences of calculation parameters on computation results were investigated. The optimized calculation condition was as follows: calculation step length of 0.02, simulation times of 10 000, and a significance level value of 0.15 for adding or removing terms in a stepwise regression. Recommended normal operation region is located in ethanol concentration of 65.0-70.0%, elution rate of 1.7-2.0 bed volumes (BV)/h and elution volume of 3.0-3.6 BV. Verification experiments were carried out and the experimental values were in a good agreement with the predicted values. The application of present method is promising to develop a probability-based design space for other botanical drug manufacturing process. PMID:26549198

  2. 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. PMID:26778144

  3. Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

    PubMed

    Kollmar, Martin

    2006-08-15

    The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. PMID:16516959

  4. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    PubMed

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. PMID:26616099

  5. 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...

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

    PubMed Central

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

    2013-01-01

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

  7. Purification of recombinant poly(ADP-ribose) polymerases.

    PubMed

    Amé, Jean-Christophe; Kalisch, Thomas; Dantzer, Françoise; Schreiber, Valérie

    2011-01-01

    The purification of Poly(ADP-ribose) polymerases from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible three chromatographic steps protocol. After cell lysis, proteins from the crude extract are separated on a Heparine Sepharose™ column. The PARP-containing fractions are then affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute the PARP from the previous affinity column are removed on the high-performance strong cations exchanger Source™ 15S matrix. The columns connected to an ÄKTA™ purifier system allow the purification of PARPs in 3 days with a high-yield recovery. As described in the protocol, more than 11 mg of pure and highly active mouse PARP-2 can be obtained from 1 L of Sf9 insect cell culture. PMID:21870259

  8. A Generic Tool for Transcription Factor Target Gene Discovery in Arabidopsis Cell Suspension Cultures Based on Tandem Chromatin Affinity Purification1[W][OPEN

    PubMed Central

    Verkest, Aurine; Abeel, Thomas; Heyndrickx, Ken S.; Van Leene, Jelle; Lanz, Christa; Van De Slijke, Eveline; De Winne, Nancy; Eeckhout, Dominique; Persiau, Geert; Van Breusegem, Frank; Inzé, Dirk; Vandepoele, Klaas; De Jaeger, Geert

    2014-01-01

    Genome-wide identification of transcription factor (TF) binding sites is pivotal to our understanding of gene expression regulation. Although much progress has been made in the determination of potential binding regions of proteins by chromatin immunoprecipitation, this method has some inherent limitations regarding DNA enrichment efficiency and antibody necessity. Here, we report an alternative strategy for assaying in vivo TF-DNA binding in Arabidopsis (Arabidopsis thaliana) cells by tandem chromatin affinity purification (TChAP). Evaluation of TChAP using the E2Fa TF and comparison with traditional chromatin immunoprecipitation and single chromatin affinity purification illustrates the suitability of TChAP and provides a resource for exploring the E2Fa transcriptional network. Integration with transcriptome, cis-regulatory element, functional enrichment, and coexpression network analyses demonstrates the quality of the E2Fa TChAP sequencing data and validates the identification of new direct E2Fa targets. TChAP enhances both TF target mapping throughput, by circumventing issues related to antibody availability, and output, by improving DNA enrichment efficiency. PMID:24453163

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

    PubMed

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

    2014-08-15

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

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

    PubMed

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

    2015-01-01

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

  11. Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

    PubMed Central

    Hubbard, B R; Ulrich, M M; Jacobs, M; Vermeer, C; Walsh, C; Furie, B; Furie, B C

    1989-01-01

    The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form gamma-carboxyglutamic acid residues within the vitamin K-dependent proteins. This enzyme recognizes the gamma-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood coagulation proteins. To purify this enzyme to homogeneity, the carboxylase from bovine liver microsomes was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the protein was fractionated with ammonium sulfate, and then the enzyme was isolated by affinity chromatography using a synthetic peptide based upon the structure of the prothrombin propeptide. Elution with 10 mM propeptide yielded a single major band on SDS gel electrophoresis with a molecular weight of 77,000. In the presence of high concentrations of propeptide, only minimal carboxylase activity was measurable. Antibodies to the protein inhibited the carboxylase activity in crude preparations. In an alternative affinity purification strategy the propeptide was coupled through an NH2-terminal cysteine to an activated thiol-Sepharose column. The carboxylase-propeptide complex was eluted at 25 degrees C by reductive cleavage of the enzyme-propeptide complex in the presence of detergent and phospholipids. The eluted protein (Mr, 77,000) contained both stable vitamin K-dependent carboxylase and vitamin K epoxidase activity. The protein, purified by either method, was detected as a single band (Mr, 77,000) in a Western blot using anti-carboxylase antibodies. A 10,000-fold purification of carboxylase activity from crude microsomes was estimated. Purified bovine liver vitamin K-dependent carboxylase should facilitate the study of its structure and of the mechanism of action of vitamin K as a cofactor in the reaction catalyzed by this enzyme. Images PMID:2780546

  12. 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…

  13. ( sup 3 H)phenamil binding protein of the renal epithelium Na+ channel. Purification, affinity labeling, and functional reconstitution

    SciTech Connect

    Barbry, P.; Chassande, O.; Marsault, R.; Lazdunski, M.; Frelin, C. )

    1990-01-30

    This paper describes a large-scale purification procedure of the amiloride binding component of the epithelium Na+ channel. (3H)Phenamil was used as a labeled ligand to follow the purification. The first two steps are identical with those previously described. A third step was a hydroxyapatite column. The purified material consisted of a homodimer of two 88-kDa proteins that migrated anomalously in SDS-PAGE to give an apparent Mr of 105,000. Deglycosylation by treatment with neuraminidase and endoglycosidase F or with neuraminidase and glycopeptidase F indicated that less than 5% of the mass of the native receptor was carbohydrate. Sedimentation analysis of the purified Na+ channel in H2O and D2O sucrose gradients and gel filtration experiments led to an estimated molecular weight of the (3H)phenamil receptor protein-detergent-phospholipid complex of 288,000 and of the native (3H)phenamil receptor protein of 158,000. (3H)Br-benzamil is another labeled derivative of amiloride that recognized binding sites that had the same pharmacological properties as (3H)phenamil binding sites and that copurified with them. Upon irradiation of kidney membranes, (3H)Br-benzamil incorporated specifically into a 185-kDa polypeptide chain under nonreducing electrophoretic conditions and a 105-kDa protein under reducing conditions. The same labeling pattern was observed at the different steps of the purification. Reconstitution of the purified phenamil receptor into large unilamellar vesicles was carried out. A low but significant phenamil- and amiloride-sensitive electrogenic Na+ transport was observed.

  14. Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.

    PubMed

    Ahrends, Robert; Kosinski, Jan; Kirsch, Dieter; Manelyte, Laura; Giron-Monzon, Luis; Hummerich, Lars; Schulz, Oliver; Spengler, Bernhard; Friedhoff, Peter

    2006-01-01

    To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex. PMID:16772401

  15. 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

  16. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    SciTech Connect

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. )

    1990-11-15

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of {sup 125}I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  17. Lectin affinity chromatography of glycolipids

    SciTech Connect

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

    1987-05-01

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

  18. Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 6-phosphogluconate dehydratase from Zymomonas mobilis.

    PubMed

    Scopes, R K; Griffiths-Smith, K

    1984-02-01

    Using differential dye-ligand chromatography and affinity elution with a substrate analog, 6-phosphogluconate dehydratase (EC 4.2.1.12) has been isolated from extracts of Zymomonas mobilis in a one-step procedure with 50% recovery. The specific activity of freshly isolated enzyme was 245 units mg-1. The enzyme contains iron, and it is rapidly inactivated in oxidizing conditions. It is inhibited by glycerophosphates, most strongly by the D-alpha-isomer which structurally corresponds to half of the substrate molecule. PMID:6326623

  19. Overview of the Purification of Recombinant Proteins

    PubMed Central

    Wingfield, Paul T.

    2015-01-01

    When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

  20. Purification of a thermostable chitinase from Bacillus cereus by chitin affinity and its application in microbial community changes in soil.

    PubMed

    Liang, Tzu-Wen; Hsieh, Tung-Yen; Wang, San-Lang

    2014-06-01

    A thermostable chitinase was purified by chitin affinity from the culture supernatant of Bacillus cereus TKU028 with shrimp head powder (SHP) as the sole carbon/nitrogen source. TKU028 chitinase was purified using a one-step affinity adsorbent system, and the molecular mass of TKU028 chitinase (approximately 40 kDa) was then determined using SDS-PAGE. The enzyme was stable for 60 min at temperatures below 60 °C and stable over a broad pH range of 4-9 for 60 min. In addition, the temporal changes of a bacterial community in mangrove river sediment of the Tamsui River with added SHP were also analysed by PCR-denaturing gradient gel electrophoresis to investigate the effects of B. cereus TKU028 on the degradation of SHP. The 6-week incubation sample of SHP and B. cereus TKU028-amended mangrove river sediment displayed the highest amount of biomass, reducing sugar and total sugar, and some variance of bacterial community composition existed in the soils. PMID:24342954

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

    PubMed

    Islam, Tuhidul; Fernández-Lahore, Marcelo

    2015-03-01

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

  2. Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Kakeya, Hideaki

    2016-01-01

    A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe. PMID:26831701

  3. Identification of sRNA interacting with a transcript of interest using MS2-affinity purification coupled with RNA sequencing (MAPS) technology

    PubMed Central

    Lalaouna, David; Massé, Eric

    2015-01-01

    RNA sequencing (RNAseq) technology recently allowed the identification of thousands of small RNAs (sRNAs) within the prokaryotic kingdom. However, drawing the comprehensive interaction map of a sRNA remains a challenging task. To address this problem, we recently developed a method called MAPS (MS2 affinity purification coupled with RNA sequencing) to characterize the full targetome of specific sRNAs. This method enabled the identification of target RNAs interacting with sRNAs, regardless of the type of regulation (positive or negative), type of targets (mRNA, tRNA, sRNA) or their abundance. We also demonstrated that we can use this technology to perform a reverse MAPS experiment, where an RNA fragment of interest is used as bait to identify interacting sRNAs. Here, we demonstrated that RybB and MicF sRNAs co-purified with internal transcribed spacers (ITS) of metZ–metW–metV tRNA transcript, confirming results obtained with MS2-RybB MAPS. Both raw and analyzed RNAseq data are available in GEO database (GSE66517). PMID:26484242

  4. 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). PMID:23224497

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

    PubMed

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

    2016-05-15

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2016-07-01

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

  8. Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

    PubMed

    Cooper, A D; Stubbings, G W; Kelly, M; Tarbin, J A; Farrington, W H; Shearer, G

    1998-07-01

    An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up. PMID:9691328

  9. Discovery of highly soluble antibodies prior to purification using affinity-capture self-interaction nanoparticle spectroscopy.

    PubMed

    Wu, Jiemin; Schultz, Jason S; Weldon, Caroline L; Sule, Shantanu V; Chai, Qing; Geng, Steven B; Dickinson, Craig D; Tessier, Peter M

    2015-10-01

    Self-association of monoclonal antibodies (mAbs) at high concentrations can result in developability challenges such as poor solubility, aggregation, opalescence and high viscosity. There is a significant unmet need for methods that can evaluate self-association propensities of concentrated mAbs at the earliest stages in antibody discovery to avoid downstream issues. We have previously developed a method (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) that is capable of detecting weak antibody self-interactions using unusually dilute mAb solutions (tens of µg/ml). Here we optimize and implement this assay for characterization of unpurified and highly dilute mAbs directly in cell culture media. This assay was applied to screen 87 mAbs obtained via immunization. Our measurements reveal a wide range of self-associative propensities for mAbs that bind to the same antigen and which differ mainly in their complementarity-determining regions. The least associative mAbs identified by AC-SINS were confirmed to be highly soluble when purified and concentrated by three to five orders of magnitude. This approach represents a key advance in screening mAb variants using unpurified antibody samples, and it holds significant potential to both improve initial candidate selection as well as to guide protein engineering efforts to improve the properties of specific mAb candidates. PMID:26363633

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

    PubMed

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

    2008-01-01

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

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

    PubMed Central

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

    1984-01-01

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

  12. Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with alpha 2-macroglobulin.

    PubMed Central

    Sutcliffe, R G; Kukulska-Langlands, B M; Coggins, J R; Hunter, J B; Gore, C H

    1980-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000--820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A. Images Fig. 1. Fig. 4. Fig. 6. PMID:6169340

  13. Rapid screening method for quinolone residues in livestock and fishery products using immobilised metal chelate affinity chromatographic clean-up and liquid chromatography-fluorescence detection.

    PubMed

    Takeda, N; Gotoh, M; Matsuoka, T

    2011-09-01

    An efficient LC method was developed for screening the presence of quinolones (QLs)--comprising fluoroquinolones (FQs) and acidic quinolones (AQs)--residues in various livestock and fishery products. Targeted analytes were for nine FQs of marbofloxacin (MAR), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), danofloxacin (DAN), orbifloxacin (ORB), difloxacin (DIF) and sarafloxacin (SAR), and three AQs of oxolinic acid (OXA), nalidixic acid (NAL) and flumequine (FMQ). Samples comprised ten different food products covering five matrices: muscle (cattle, swine and chicken), liver (chicken), raw fish (shrimp and salmon), egg (chicken), and processed food (ham, sausage and fish sausage). This method involved a simple extraction with (1:1) acetonitrile-methanol, a highly selective clean-up with an immobilised metal chelate affinity column charged with Fe(3+), a fast isocratic LC analysis using a short column (20 mm × 4.6 mm, 3 µm) with a mobile phase of (15:85:0.1) methanol/water/formic acid, and fluorescence detection (excitation/emission wavelengths of 295 nm/455 nm for FQs (495 nm for MAR), and 320 nm/365 nm for AQs). Among FQs, pairs of NOR/OFL, ORB/DIF and ENR/DAN were incompletely resolved. A confirmatory LC run with a Mg(2+) containing methanolic mobile phase was also proposed for the samples suspected of being positive. The optimised method gave satisfactory recoveries of 88.5% (56.1-108.6%) and 78.7% (44.1-99.5%) for intra- and inter-day assays with relative standard deviations of 7.2% (0.7-18.4%) and 6.8% (1.4-16.6%), respectively. Limits of quantitation ranged from 0.8 µg kg(-1) (DAN) to 6.5 µg kg(-1) (SAR). This method was successfully employed to analyse 113 real samples and two positive samples were found: fish sausage (CIP 990 µg kg(-1)) and shrimp (ENR 20 µg kg(-1)). PMID:21749230

  14. Recombinant protein purification using gradient-assisted simulated moving bed hydrophobic interaction chromatography. Part I: selection of chromatographic system and estimation of adsorption isotherms.

    PubMed

    Palani, Sivakumar; Gueorguieva, Ludmila; Rinas, Ursula; Seidel-Morgenstern, Andreas; Jayaraman, Guhan

    2011-09-16

    The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate. PMID:21816402

  15. Continuous countercurrent chromatographic separator for the purification of sugars from biomass hydrolyzate. Final project report, July 1, 1996--September 30, 1997

    SciTech Connect

    Wooley, R J

    1997-12-01

    Production of pure sugars is required to enable production of fuels and chemicals from biomass feedstocks. Hydrolysis of cellulose and hemicellulose (principal constituents of biomass) produces sugars that can be utilized in various fermentation process to produce valuable chemicals. Unfortunately, the hydrolysis process also liberates chemicals from the biomass that can be toxic to the fermenting organisms. The two primary toxic components of biomass hydrolyzate are sulfuric acid (catalyst used in the hydrolysis) and acetic acid (a component of the feed biomass). In the standard batch chromatographic separation of these three components, sugar elutes in the middle. Batch chromatographic separations are not practical on a commercial scale, because of excess dilution and high capital costs. Because sugar is the {open_quotes}center product,{close_quotes} a continuous separation would require two costly binary separators. However, a single, slightly larger separator, configured to produce three products, would be more economical. This FIRST project develops a cost-effective method for purifying biomass hydrolyzate into fermentable sugars using a single continuous countercurrent separator to separate this ternary mixture.

  16. Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation.

    PubMed

    Fields, Conor; Li, Peng; O'Mahony, James J; Lee, Gil U

    2016-01-01

    The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future. PMID:26032605

  17. Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

    PubMed

    Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L; Connolly, Shawn; Little, Randie R; Stoyanov, Alexander V

    2016-02-01

    A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed. PMID:26717885

  18. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  19. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors

    PubMed Central

    Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  20. Revealing novel telomere proteins using in vivo cross-linking, tandem affinity purification, and label-free quantitative LC-FTICR-MS.

    PubMed

    Nittis, Thalia; Guittat, Lionel; LeDuc, Richard D; Dao, Ben; Duxin, Julien P; Rohrs, Henry; Townsend, R Reid; Stewart, Sheila A

    2010-06-01

    Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be

  1. Modified normal-phase ion-pair chromatographic methods for the facile separation and purification of imidazolium-based ionic compounds

    SciTech Connect

    Urban, ND; Schenkel, MR; Robertson, LA; Noble, RD; Gin, DL

    2012-07-04

    lmidazolium- and oligo(imidazolium)-based ionic organic compounds are important in the design of room-temperature ionic liquid materials; however, the chromatographic analysis and separation of such compounds are often difficult. A convenient and inexpensive method for effective thin-layer chromatography (TLC) analysis and column chromatography separation of imidazolium-based ionic compounds is presented. Normal-phase ion-pair TLC is used to effectively analyze homologous mixtures of these ionic compounds. Subsequent separation of the mixtures is performed using ion-pair flash chromatography on normal-phase silica gel, yielding high levels of recovery. This method also results in a complete exchange of the counter anion on the imidazolium compounds to the anion of the ion-pair reagent. (C) 2012 Elsevier Ltd. All rights reserved.

  2. Accelerated solvent extraction and pH-zone-refining counter-current chromatographic purification of yunaconitine and 8-deacetylyunaconitine from Aconitum vilmorinianum Kom.

    PubMed

    Shu, Xi-Kai; Li, Jia; Liu, Feng; Lin, Xiao-Jing; Wang, Xiao; Song, Chun-Xia

    2013-08-01

    This study aimed to seek an efficient method to extract and purify yunaconitine and 8-deacetylyunaconitine from Aconitum vilmorinianum Kom. by accelerated solvent extraction combined with pH-zone-refining counter-current chromatography. The major extraction parameters for accelerated solvent extraction were optimized by an orthogonal test design L9 (3)(4). Then a separation and purification method was established using pH-zone-refining counter-current chromatography with a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:2:8, v/v) with 10 mM triethylamine in the upper phase and 10 mM HCl in the lower phase. From 2 g crude extract, 224 mg of 8-deacetylyunaconitine (I) and 841 mg of yunaconitine (II) were obtained with a purity of over 98.0%. The chemical structures were identified by ESI-MS and (1)H and (13)C NMR spectroscopy. PMID:23784883

  3. Surface plasmon resonance spectroscopy-based high-throughput screening of ligands for use in affinity and displacement chromatography.

    PubMed

    Vutukuru, Srinavya; Kane, Ravi S

    2008-10-21

    We describe an approach that uses surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) for the high-throughput screening of ligands for use in displacement and affinity chromatographic processes. We identified a set of commercially available organic amines and allowed them to react with SAMs presenting interchain carboxylic anhydride groups; the resulting surfaces presented ligands of interest in a background of carboxylic acid groups. We used SPR spectroscopy to determine the extent of adsorption of two model proteinslysozyme and cytochrome conto these "multimodal" surfaces and to select promising "affinity" ligands for further characterization. The attachment of selected ligands to UltraLink Biosupport resulted in beads with a significantly greater affinity for lysozyme than for cytochrome c that would be suitable for use in affinity chromatographic processes. Furthermore, we also used the screens to design "affinity displacers"small molecules that selectively retain lysozyme on chromatographic resins, while displacing cytochrome c. The combination of SPR spectroscopy and SAMs represents a powerful technique for identifying novel ligands that enable the purification of complex protein mixtures. PMID:18788766

  4. Purification of phosphatidylinositol kinase from bovine brain myelin.

    PubMed Central

    Saltiel, A R; Fox, J A; Sherline, P; Sahyoun, N; Cuatrecasas, P

    1987-01-01

    A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme. PMID:3036072

  5. Reversed-phase liquid chromatographic purification and isolation of a radio-iodinated selective probe for mu opioid receptors in the brain.

    PubMed

    Miller, M M; Gould, B E; Joshi, D; Bennett, H P; James, S; Billiar, R B

    1992-02-01

    A Guard-PAK precolumn system was used for the reversed-phase liquid chromatography purification of a small, synthetic radiolabeled opioid peptide, FK 33-824 (D-Ala2, methyl-phe4, Met (O)ol5 enkephalin) (FK). This procedure involves trace enrichment of iodinated peptide onto the precolumn while iodination reagents are not retained. Radioactive contamination of high-performance liquid chromatography columns and injectors is thus avoided. Precolumn chromatography has sufficient resolving power to separate not only labeled from unlabeled peptide but also mono- from di-iodinated peptide. Purified 125I-labeled FK (estimated specific activity 85.9-153.7 Ci/mmol) showed high specific binding to mouse corpus striatum, neocortex, cingulate cortex, nucleus accumbens septi, diagonal band of Broca, nucleus medialis septi, area preopticus magnocellularis, and the nucleus of the caudate/putamen. Radioligand binding was inhibited by both antagonists (naloxone and naltrexone); and agonists D-Ala2, N-methyl-phe4, gly-ol5-enkephalin [DAGO]; FK; and beta-endorphin at all concentrations tested (1 x 10(-8) to 1 x 10(-4) M). Adrenocorticotropin hormone (ACTH) did not block ligand binding at any concentration tested. Distribution of mu opioid receptors was analyzed by light microscopic autoradiography. Sections incubated with 125I-labeled FK in the presence of agonists and antagonists demonstrated decreasing ligand binding with increasing doses of competitor. ACTH did not block ligand binding at any concentration tested. HPLC analyses of ligand which had been iodinated 1.5 half lives before the date of the experiment demonstrated a single peak similar to that of freshly iodinated ligand. Similar binding kinetics and autoradiographic labeling patterns were observed as compared to those obtained with freshly iodinated peptide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1314320

  6. Virus elimination during the recycling of chromatographic columns used during the manufacture of coagulation factors.

    PubMed

    Roberts, Peter L

    2014-07-01

    Various chromatographic procedures are used during the purification and manufacture of plasma products such as coagulation factors. These steps contribute to the overall safety of such products by removing potential virus contamination. Virus removal by two affinity chromatography procedures, i.e. monoclonal antibody chromatography and metal chelate chromatography (immobilised metal ion affinity chromatography), used during the manufacture of the high purity factor VIII (Replenate®) and factor IX (Replenine®-VF), respectively, has been investigated. In addition, as these columns are recycled after use, the effectiveness of the sanitisation procedures for preventing possible cross-contamination, has also been investigated. Both chromatographic steps proved effective for eliminating a range of model enveloped and non-enveloped viruses by 4 to >6 and 5 to >8 log for the monoclonal and metal chelate columns, respectively. The effectiveness of the relatively mild column sanitisation conditions used, i.e. ethanol for factor IX and acetic acid for factor VIII, was confirmed using non-spiked column runs. The chemicals used contributed to virus elimination by inactivation and/or by physical removal of the virus. In summary, these studies demonstrate that potential virus contamination between chromatographic runs can be prevented when an effective column recycling and sanitisation procedure is included. PMID:24981392

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

    PubMed

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

    2001-10-30

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

  8. Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

    PubMed Central

    Sugden, E A; Brooks, B W; Young, N M; Watson, D C; Nielsen, K H; Corner, A H; Turcotte, C; Michaelides, A; Stewart, R B

    1991-01-01

    The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity. Images PMID:1761688

  9. Expression of cold-adapted β-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

    PubMed

    Kudou, Motonori; Okazaki, Fumiyoshi; Asai-Nakashima, Nanami; Ogino, Chiaki; Kondo, Akihiko

    2015-01-01

    Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes. PMID:25214227

  10. Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.

    PubMed

    Meininger, M; Stepath, M; Hennig, R; Cajic, S; Rapp, E; Rotering, H; Wolff, M W; Reichl, U

    2016-02-15

    Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO. PMID:26851523

  11. Purification and Properties of a Protein Which Binds Cytokinin-active 6-Substituted Purines 1

    PubMed Central

    Erion, Jack L.; Fox, J. Eugene

    1981-01-01

    A protein which binds 6-substituted purines of the cytokinin type with relatively high affinity has been extensively purified from wheat germ. Conventional chromatographic techniques, as well as an affinity matrix to which a cytokinin was covalently coupled, were used in the purification. The wheat germ cytokinin-binding protein (CBF-1) has four unlike subunits and an apparent molecular weight of 183,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CBF-1 is saturated at one cytokinin molecule per tetramer with a Kd for 6-benzylaminopurine of 5 × 10−7 molar. The protein exists both on the native wheat germ ribosome (1 molecule CBF-1 per 80S ribosome) and free in the cytosol with approximately three copies of the latter for each of the former. Data from affinity chromatography studies and cross-linking experiments strongly suggest that a specific binding site for CBF-1 occurs on the wheat germ ribosome. Images PMID:16661618

  12. Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis

    PubMed Central

    Veillard, Florian; Potempa, Barbara; Guo, Yonghua; Ksiazek, Miroslaw; Sztukowska, Maryta N.; Houston, John A.; Koneru, Lahari; Nguyen, Ky-Anh; Potempa, Jan

    2015-01-01

    Gingipain proteases are important virulence factors from the periodontal pathogen Porphyromonas gingivalis and are the target of many in vitro studies. Due to their close biochemical properties, purification of individual gingipains is difficult and requires multiple chromatographic steps. In this study, we demonstrate that insertion of a hexahistidine affinity tag upstream of a C-terminal outer membrane translocation signal in RgpB gingipain leads to the secretion of a soluble, mature form of RgpB bearing the affinity tag which can easily be purified by nickel-chelating affinity chromatography. The final product obtained in high yielding and high purity is biochemically indistinguishable from the native RgpB enzyme. PMID:25720118

  13. Purification of a putative brain somatostatin receptor

    SciTech Connect

    He, Haitao; Johnson, K.; Thermos, K.; Reisine, T. )

    1989-03-01

    The brain somatostatin receptor was purified by affinity chromatographic techniques. A protein of 60 kDa could be purified from rat brain. The protein was eluted from a (D-Trp{sup 8})SRIF affinity column with either sodium acetate (pH 5.5) or free (D-Trp{sup 8})SRIF. The binding of the protein to the affinity column was prevented by free (D-Trp{sup 8})SRIF or the stable SRIF analogue SMS 201-996 but not by the inactive somatostatin 28-(1-14). The purified receptor could be covalently labeled by the {sup 125}I-labeled SRIF analogue CGP 23996. Excess (D-Trp{sup 8})SRIF blocked the binding of {sup 125}I-labeled CGP 23996 to the purified receptor, but somatostatin 28-(1-14) did not affect the binding. A 60-kDa protein was also purified from the anterior pituitary cell line AtT-20, which has a high expression of SRIF receptors. In contrast, no 60-kDa protein could be purified from CHO cells, which have no detectable SRIF receptors. These findings present evidence for the purification of the SRIF receptor.

  14. Data for the identification of proteins and post-translational modifications of proteins associated to histones H3 and H4 in S. cerevisiae, using tandem affinity purification coupled with mass spectrometry.

    PubMed

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

    2016-03-01

    Tandem affinity purification method (TAP) allows the efficient purification of native protein complexes which incorporate a target protein fused with the TAP tag. Purified multiprotein complexes can then be subjected to diverse types of proteomic analyses. Here we describe the data acquired after applying the TAP strategy on histones H3 and H4 coupled with mass spectrometry to identify associated proteins and protein post-translational modifications in the budding yeast, Saccharomyces cerevisiae. The mass spectrometry dataset described here consists of 14 files generated from four different analyses in a 5600 Triple TOF (Sciex) by information-dependent acquisition (IDA) LC-MS/MS. The above files contain information about protein identification, protein relative abundance, and PTMs identification. The instrumental raw data from these files has been also uploaded to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PRIDE: PXD002671 and http://dx.doi.org/10.6019/PXD002671. These data are discussed and interpreted in http://dx.doi.org/10.1016/j.jprot.2016.01.004. Valero et al. (2016) [1]. PMID:26949727

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

    PubMed

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

    2003-04-01

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

  16. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    PubMed

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. PMID:26654254

  17. Production, purification and characterization of laccase from Pleurotus ostreatus grown on tomato pomace.

    PubMed

    Freixo, Maria do Rosário; Karmali, Amin; Arteiro, José Maria

    2012-01-01

    A strain of Pleurotus ostreatus was grown in tomato pomace as sole carbon source for production of laccase. The culture of P. ostreatus revealed a peak of laccase activity (147 U/L of fermentation broth) on the 4th day of culture with a specific activity of 2.8 U/mg protein. Differential chromatographic behaviour of laccase was investigated on affinity chromatographic matrices containing either urea, acetamide, ethanolamine or IDA as affinity ligands. Laccase exhibited retention on such affinity matrices and it was purified on a Sepharose 6B-BDGE-urea column with final enzyme recoveries of about 60%, specific activity of 6.0 and 18.0 U/mg protein and purification factors in the range of 14-46. It was also possible to demonstrate that metal-free laccase did not adsorb to Sepharose 6B-BDGE-urea column which suggests that adsorption of native laccase on this affinity matrix was apparently due to the specific interaction of carbonyl groups available on the matrix with the active site Cu (II) ions of laccase. The kinetic parameters (V(max), K(m), K(cat), and K(cat)/K(m)) of the purified enzyme for several substrates were determined as well as laccase stability and optimum pH and temperature of enzyme activity. This is the first report describing the production of laccase from P. ostreatus grown on tomato pomace and purification of this enzyme based on affinity matrix containing urea as affinity ligand. PMID:22806800

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

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

  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. PMID:6326622

  2. Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

    PubMed Central

    Matheson, Nicholas J.; Peden, Andrew A.; Lehner, Paul J.

    2014-01-01

    Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing. PMID:25360777

  3. Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols.

    PubMed

    Alba, Annia; Marcet, Ricardo; Otero, Oscar; Hernández, Hilda M; Figueredo, Mabel; Sarracent, Jorge

    2016-02-01

    Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties. PMID:26828226

  4. A new method of synthesizing biopolymeric affinity ligands.

    PubMed

    Chaga, G S; Guzman, R; Porath, J O

    1997-08-01

    (1) A new concept for producing soluble polymeric affinity ligands is proposed and exemplified. By solid-phase synthesis, an insoluble hydrophilic polymer is converted into an affinity gel. The gel is hydrolytically degraded to water-soluble affinity polymeric ligands which are recovered and purified. (2) A water-soluble biopolymeric metal-affinity carrier based on an iminodiacetic acid (IDA) derivative of dextran has been synthesized through the modification of Sephadex G-200 by IDA, followed by hydrolysis with dextranase and size-exclusion-chromatographic purification of the high-molecular-mass fragments. (3) The molecular size of the soluble products as a function of hydrolysis time with dextranase from Penicillium sp. was determined. The range of molecular size of the biopolymeric chelating ligand varies from around 200 Da to greater than 580 kDa. (4) The influence of three metal ions chelated with the Sephadex derivative on the hydrolysis rate and the molecular-size distribution of end products was studied. Eu3+ was found to improve the rate of solubilization. Ni2+ and Cu2+ decreased the hydrolysis rate, as compared with that of the metal-free IDA-Sephadex. (5) The method introduced here has the potential of being developed and applied as a general technology for synthesis of soluble multifunctional affinity ligands. Such ligands should be useful for liquid-phase extraction as well as for the synthesis of adsorbents with localized multiple binding sites. Other possible fields of applications are to be found in medicine, where they could be used for slow drug delivery or detoxification, and in analytical chemistry, where they could be used in various assays. PMID:9261997

  5. Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti(4+)-SPE enrichment for mass spectrometric analysis.

    PubMed

    Zhang, Ying; Peng, Ye; Bin, Zhichao; Wang, Huijie; Lu, Haojie

    2016-08-31

    N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti(4+)-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti(4+)-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified. PMID:27506354

  6. Preparation of core-shell structure Fe3 O4 @SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His-tag protein purification.

    PubMed

    Ni, Qian; Chen, Bing; Dong, Shaohua; Tian, Lei; Bai, Quan

    2016-04-01

    The core-shell structure Fe3 O4 /SiO2 magnetic microspheres were prepared by a sol-gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu(2+) , Ni(2+) and Zn(2+) , were chelated on the Fe3 O4 @SiO2 -IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni(2+) -chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3 O4 @SiO2 -IDA-Ni(2+) magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26268650

  7. Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.

    PubMed

    Meehan, M; Nowlan, P; Owen, P

    1998-04-01

    Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M(r) protein species (apparent M(r) 220,000 and 550,000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M(r) protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M(r) 54,597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M(r) estimation, fibrinogen binding and seroreactivity. PMID:9579073

  8. A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography.

    PubMed

    Qu, Jian-Bo; Huang, Yong-Dong; Jing, Guang-Lun; Liu, Jian-Guo; Zhou, Wei-Qing; Zhu, Hu; Lu, Jian-Ren

    2011-05-01

    Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251 cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography. PMID:21454141

  9. Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.

    PubMed

    Sriwanthana, B; Island, M D; Maneval, D; Mobley, H L

    1994-11-01

    Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations. PMID:7961442

  10. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

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

    PubMed

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

    2014-11-01

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

  12. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

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

  13. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

    PubMed

    Bigenzahn, Johannes W; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K; Scorzoni, Stefania; Vladimer, Gregory I; Müller, André C; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L; Superti-Furga, Giulio

    2016-03-01

    Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression. PMID:26933192

  14. Phosphoinositide-specific Phospholipase C β 1b (PI-PLCβ1b) Interactome: Affinity Purification-Mass Spectrometry Analysis of PI-PLCβ1b with Nuclear Protein*

    PubMed Central

    Piazzi, Manuela; Blalock, William L.; Bavelloni, Alberto; Faenza, Irene; D'Angelo, Antonietta; Maraldi, Nadir M.; Cocco, Lucio

    2013-01-01

    Two isoforms of inositide-dependent phospholipase C β1 (PI-PLCβ1) are generated by alternative splicing (PLCβ1a and PLCβ1b). Both isoforms are present within the nucleus, but in contrast to PLCβ1a, the vast majority of PLCβ1b is nuclear. In mouse erythroid leukemia cells, PI-PLCβ1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCβ1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCβ1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCβ1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule. PMID:23665500

  15. Preparation of λN-GST fusion protein for affinity immobilization of RNA.

    PubMed

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

    2012-01-01

    Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing conditions. However, the performance of affinity purification methods can vary tremendously depending on the RNA immobilization matrix. It was previously shown that RNA immobilization via an optimized λN-GST fusion protein bound to glutathione-Sepharose resin allows affinity purification of RNA with very high purity and yield. This Chapter outlines the experimental procedure employed to prepare the λN-GST fusion protein used for RNA immobilization in successful affinity purifications of RNA. It describes the details of protein expression and purification as well as routine quality control analyses. PMID:23065558

  16. Challenges and opportunities in the purification of recombinant tagged proteins.

    PubMed

    Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

    2014-01-01

    The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. PMID:24334194

  17. A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

    PubMed

    Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

    2016-01-01

    Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification). PMID:27279446

  18. Microminiature gas chromatographic column

    NASA Technical Reports Server (NTRS)

    Donaldson, R. W., Jr.

    1972-01-01

    Techniques commonly used for fabrication of integrated circuits are utilized to produce long capillary tubes for microminiature chromatographs. Method involves bonding of flat silicon plate to top of spirally grooved silicon chip to close groove and form capillary column.

  19. Liquid chromatographic extraction medium

    DOEpatents

    Horwitz, E.P.; Dietz, M.L.

    1994-09-13

    A method and apparatus are disclosed for extracting strontium and technetium values from biological, industrial and environmental sample solutions using a chromatographic column. An extractant medium for the column is prepared by generating a solution of a diluent containing a Crown ether and dispersing the solution on a resin substrate material. The sample solution is highly acidic and is introduced directed to the chromatographic column and strontium or technetium is eluted using deionized water. 1 fig.

  20. Liquid chromatographic extraction medium

    DOEpatents

    Horwitz, E. Philip; Dietz, Mark L.

    1994-01-01

    A method and apparatus for extracting strontium and technetium values from biological, industrial and environmental sample solutions using a chromatographic column is described. An extractant medium for the column is prepared by generating a solution of a diluent containing a Crown ether and dispersing the solution on a resin substrate material. The sample solution is highly acidic and is introduced directed to the chromatographic column and strontium or technetium is eluted using deionized water.

  1. Expression and purification of GST fusion proteins.

    PubMed

    Harper, S; Speicher, D W

    2001-05-01

    An increasingly common strategy for expressing proteins and large peptides in prokaryotic systems is to express the protein of interest connected to a "tag" that provides the basis for rapid high-affinity purification. This unit describes the expression and purification of fusion proteins containing the 26-kDa glutathione-S-transferase protein as well as methods for cleaving the affinity tag and repurifying the target protein. Advantages of this popular fusion protein system include high protein yields, high-affinity one-step protein purification of the fusion protein, existence of several alternative protease cleavage sites for removing the affinity tag when required, and ease of removal of the cleaved affinity tag. PMID:18429193

  2. Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands.

    PubMed

    Clune, A; Lee, K S; Ferenci, T

    1984-05-31

    Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein. PMID:6375667

  3. Chromatographic hydrogen isotope separation

    DOEpatents

    Aldridge, F.T.

    Intermetallic compounds with the CaCu/sub 5/ type of crystal structure, particularly LaNiCo/sub 4/ and CaNi/sub 5/, exhibit high separation factors and fast equilibrium times and therefore are useful for packing a chromatographic hydrogen isotope separation column. The addition of an inert metal to dilute the hydride improves performance of the column. A large scale multi-stage chromatographic separation process run as a secondary process off a hydrogen feedstream from an industrial plant which uses large volumes of hydrogen cn produce large quantities of heavy water at an effective cost for use in heavy water reactors.

  4. Chromatographic hydrogen isotope separation

    DOEpatents

    Aldridge, Frederick T.

    1981-01-01

    Intermetallic compounds with the CaCu.sub.5 type of crystal structure, particularly LaNiCo.sub.4 and CaNi.sub.5, exhibit high separation factors and fast equilibrium times and therefore are useful for packing a chromatographic hydrogen isotope separation colum. The addition of an inert metal to dilute the hydride improves performance of the column. A large scale mutli-stage chromatographic separation process run as a secondary process off a hydrogen feedstream from an industrial plant which uses large volumes of hydrogen can produce large quantities of heavy water at an effective cost for use in heavy water reactors.

  5. Purification of Lysozyme by Intrinsically Shielded Hydrogel Beads

    NASA Astrophysics Data System (ADS)

    Li, Cong; Zhang, R.; Wang, L.; Bowyer, A.; Eisenthal, R.; Shen, Yehua; Hubble, J.

    2013-07-01

    Macro-sized intrinsically shielded hydrogel beads have been prepared from BSA and CM-dextran grafted with CB using a technique based on freeze-thawing gelation method. The size of the beads lies in around 500 μm. Isothemal titration calorimetry (ITC) showed that the relative binding affinities of the lysozyme for CB, compared with BSA, at pH 3.0 was stronger than that at pH 7.4. They were employed for the affinity separation of lysozyme using chromatography column. Their adsorption capacity for lysozyme at pH 3.0 is higher than that at pH 9. In a binary mixture of lysozyme and ovalbumin, the beads showed very high selectivity toward lysozyme. Lysozyme of very high purity (> 93%) was obtained from a mixture of lysozyme and ovalbumin, and 85% from egg white solution. The results indicate that the macro-sized bead can be used for the separation, purification, and recovery of lysozyme in a chromatograph column.

  6. Californium purification and electrodeposition

    SciTech Connect

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of the feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.

  7. Californium purification and electrodeposition

    DOE PAGESBeta

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of themore » feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.« less

  8. Protecting Gas Chromatographic Syringes

    NASA Astrophysics Data System (ADS)

    Ruekberg, Ben

    1995-12-01

    This article describes the construction of a device which protects gas chromatographic syringes. The device lessens the likelihood of syringes rolling off tables and breaking. If the syringe is dropped, the glass barrel is less apt to be struck and shattered.

  9. 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. PMID:24269760

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

    PubMed

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

    2015-03-01

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

  11. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

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

    PubMed

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

    2011-01-01

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

  13. Rational stabilization of the C-LytA affinity tag by protein engineering.

    PubMed

    Hernández-Rocamora, Víctor M; Maestro, Beatriz; Mollá-Morales, Almudena; Sanz, Jesús M

    2008-12-01

    The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization. PMID:18840883

  14. Chromatographic NMR in NMR solvents

    NASA Astrophysics Data System (ADS)

    Carrara, Caroline; Viel, Stéphane; Delaurent, Corinne; Ziarelli, Fabio; Excoffier, Grégory; Caldarelli, Stefano

    2008-10-01

    Recently, it was demonstrated that pseudo-chromatographic NMR experiments could be performed using typical chromatographic solids and solvents. This first setup yielded improved separation of the spectral components of the NMR spectra of mixtures using PFG self-diffusion measurements. The method (dubbed Chromatographic NMR) was successively shown to possess, in favorable cases, superior resolving power on non-functionalized silica, compared to its LC counterpart. To further investigate the applicability of the method, we studied here the feasibility of Chromatographic NMR in common deuterated solvents. Two examples are provided, using deuterated chloroform and water, for homologous compounds soluble in these solvents, namely aromatic molecules and alcohols, respectively.

  15. The Amicon Pro system--a centrifugal device capable of performing all steps in the protein purification workflow.

    PubMed

    Cappione, Amedeo; Mabuchi, Masaharu; Suhrawardy, Saosan; Briggs, David; Nadler, Timothy

    2013-01-01

    raditional protein purification is a long process with many steps utilizing multiple devices, often resulting in protein degradation and loss. The Amicon Pro device streamlines the affinity purification process by providing a single adaptable centrifugation unit capable of performing all steps in the affinity purification process. The device combines affinity-based spin column purification with downstream sample concentration and buffer exchange, eliminating the need for multiple sample transfers, thereby minimizing protein loss. The results presented in this work indicate that purification of His-tagged protein using the Amicon Pro device is faster, easier, and provides better yields than other traditional methods (eg. spin-column and slurry method). PMID:24364216

  16. Using Aspen to Teach Chromatographic Bioprocessing: A Case Study in Weak Partitioning Chromatography for Biotechnology Applications

    ERIC Educational Resources Information Center

    Evans, Steven T.; Huang, Xinqun; Cramer, Steven M.

    2010-01-01

    The commercial simulator Aspen Chromatography was employed to study and optimize an important new industrial separation process, weak partitioning chromatography. This case study on antibody purification was implemented in a chromatographic separations course. Parametric simulations were performed to investigate the effect of operating parameters…

  17. Microfabricated packed gas chromatographic column

    DOEpatents

    Kottenstette, Richard; Matzke, Carolyn M.; Frye-Mason, Gregory C.

    2003-12-16

    A new class of miniaturized gas chromatographic columns has been invented. These chromatographic columns are formed using conventional micromachining techniques, and allow packed columns having lengths on the order of a meter to be fabricated with a footprint on the order of a square centimeter.

  18. Use of a Phosphatidylinositol Phosphate Affinity Chromatography (PIP Chromatography) for the Isolation of Proteins Involved in Protein Quality Control and Proteostasis Mechanisms in Plants.

    PubMed

    Farmaki, T

    2016-01-01

    Protein functionality depends directly on its accurately defined three-dimensional organization, correct and efficient posttranslational modification, and transport. However, proteins are continuously under a hostile environment threatening with folding aberrations, aggregation, and mistargeting. Therefore, proteins must be constantly "followed up" by a tightly regulated homeostatic mechanism specifically known as proteostasis. To this end other proteins ensure this close surveillance including chaperones as well as structural and functional members of the proteolytic mechanisms, mainly the autophagy and the proteasome related. They accomplish their action via interactions not only with other proteins but also with lipids as well as cytoskeletal components. We describe a protocol based on an affinity chromatographic approach aiming at the isolation of phosphatidyl inositol phosphate binding proteins, a procedure which results into the enrichment and purification of several members of the proteostasis mechanism, e.g. autophagy and proteasome, among other components of the cell signaling pathways. PMID:27424758

  19. Microminiature gas chromatograph

    DOEpatents

    Yu, C.M.

    1996-12-10

    A microminiature gas chromatograph ({mu}GC) comprising a least one silicon wafer, a gas injector, a column, and a detector. The gas injector has a normally closed valve for introducing a mobile phase including a sample gas in a carrier gas. The valve is fully disposed in the silicon wafer(s). The column is a microcapillary in silicon crystal with a stationary phase and is mechanically connected to receive the mobile phase from the gas injector for the molecular separation of compounds in the sample gas. The detector is mechanically connected to the column for the analysis of the separated compounds of sample gas with electronic means, e.g., ion cell, field emitter and PIN diode. 7 figs.

  20. Microminiature gas chromatograph

    DOEpatents

    Yu, Conrad M.

    1996-01-01

    A microminiature gas chromatograph (.mu.GC) comprising a least one silicon wafer, a gas injector, a column, and a detector. The gas injector has a normally closed valve for introducing a mobile phase including a sample gas in a carrier gas. The valve is fully disposed in the silicon wafer(s). The column is a microcapillary in silicon crystal with a stationary phase and is mechanically connected to receive the mobile phase from the gas injector for the molecular separation of compounds in the sample gas. The detector is mechanically connected to the column for the analysis of the separated compounds of sample gas with electronic means, e.g., ion cell, field emitter and PIN diode.

  1. Chromatographic behaviors of proteins on cation-exchange column.

    PubMed

    Li, Rong; Chen, Guo-Liang; Zhao, Wen-Ming

    2004-12-01

    A weak cation-exchanger (XIDACE-WCX) has been synthesized by the indirect method. The chromatographic characteristics of the synthesized packing was studied in detail. The standard protein mixture and lysozyme from egg white were separated with the prepared chromatographic column. The chromatographic thermodynamics of proteins was studied in a wide temperature range. Thermodynamic parameters standard enthalpy change (deltaH0) and standard entropy change (deltaS0) and compensation temperature (beta) at protein denaturation were determined in the chromatographic system. By using obtained deltaS0, the conformational change of proteins was judged in the chromatographic process. The linear relationship between deltaH0 and deltaS0 can be used to identify the identity of the protein retention mechanism in the weak cation-exchange chromatography. The interaction between weak cation-exchanger and metal ions was investigated. Several metal chelate columns were prepared. The effects of introducing metal ion into the naked column on protein retention and the retention mechanism of proteins in the metal chalet affinity chromatography were discussed. PMID:15689030

  2. Hamiltonian purification

    SciTech Connect

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Pascazio, Saverio; Nakazato, Hiromichi; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-15

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians (h{sub 1}, …, h{sub m}) operating on a d-dimensional quantum system ℋ{sub d}, the problem consists in identifying a set of commuting Hamiltonians (H{sub 1}, …, H{sub m}) operating on a larger d{sub E}-dimensional system ℋ{sub d{sub E}} which embeds ℋ{sub d} as a proper subspace, such that h{sub j} = PH{sub j}P with P being the projection which allows one to recover ℋ{sub d} from ℋ{sub d{sub E}}. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.

  3. A novel chromatographic procedure for purification of bacterial plasmids.

    PubMed

    Bywater, M; Bywater, R; Hellman, L

    1983-07-01

    A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up. PMID:6312836

  4. Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase

    SciTech Connect

    Quirk, Stephen; Seley-Radtke, Katherine L.

    2006-04-01

    The human GTP fucose pyrophosphohydrolase protein has been crystallized via the hanging-drop technique over a reservoir of polyethylene glycol (MW 8000) and ethylene glycol. The orthorhombic crystals diffract to 2.8 Å resolution. The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 Å{sup 3} Da{sup −1} and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.

  5. Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.

    PubMed

    Sará-Páez, Martin; Contreras-Zentella, Martha; Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Gasca-Licea, Rolando; Mendoza-Hernández, Guillermo; Escamilla, José Edgardo; Reyes-Vivas, Horacio

    2015-02-01

    Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed. PMID:25576305

  6. Purification of monoclonal antibody against Ebola GP1 protein expressed in Nicotiana benthamiana

    PubMed Central

    Fulton, Andrew; Lai, Huafang; Chen, Qiang; Zhang, Chenming

    2016-01-01

    Monoclonal antibodies (mAbs) are one of the fastest growing drug molecules targeting the treatment of diseases ranging from arthritis, immune disorders, and infectious diseases to cancer. Due to its unique application principle, antibodies are commonly produced in large quantities. Plants, such as Nicotiana benthamiana, offer a unique production platform for bio-therapeutics due to their ability to produce large amounts of biomolecules in a relatively quick manner. However, purification of a target protein from plant is an arduous task due to the presence of toxic compounds in ground plant tissue and the large quantities of plant tissues to be processed. Here, a process was developed prior to the chromatographic purification of a mAb against Ebola GP1 protein expressed in N. benthamiana. The process includes a diafiltration step and a charged polyelectrolyte precipitation. The diafiltration step significantly improved the precipitation efficiency, reducing the usage of polyelectrolyte by more than 2000 fold while improving the native plant protein removed from 60% to 80%. The mAb can then be purified to near homogeneity judging from SDS-PAGE by either Protein A affinity chromatography or a tandem of hydrophobic interaction chromatography and a hydrophobic charge induction chromatography. The purified mAbs were shown to retain their binding specificity to irradiated Ebola virus. PMID:25746758

  7. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

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

  8. Process characterization for metal-affinity chromatography of an Fc fusion protein: a design-of-experiments approach.

    PubMed

    Shukla, A A; Sorge, L; Boldman, J; Waugh, S

    2001-10-01

    The utility of a design-of-experiments approach was investigated for process characterization of a metal-affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single-variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst-case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design-of-experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies. PMID:11592911

  9. Polyethyleneimine-grafted boronate affinity materials for selective enrichment of cis-diol-containing compounds.

    PubMed

    Xue, Yun; Shi, Wenjun; Zhu, Bangjie; Gu, Xue; Wang, Yan; Yan, Chao

    2015-08-01

    Polyethyleneimine (PEI)-grafted and 3-acrylamidophenylboronic acid (AAPBA)-functionalized SiO2 boronate affinity materials were synthesized for the selective enrichment of cis-diol-containing compounds. Characterization results of scanning electron microscopy, Fourier transform infrared spectroscopy, elemental analysis, zeta potential, and X-ray photoelectron spectroscopy indicated the successful fabrication of SiO2@PEI-AAPBA materials. Chromatographic separation of test mixtures reveals that SiO2@PEI-AAPBA has high selective enrichment ability for cis-diol-containing compounds. The binding pH between SiO2@PEI-AAPBA and catechol was found to be as low as pH 4.5, while that between SiO2@PEI-AAPBA and adenosine was only ~7.5. This difference might be attributed to the strong electrostatic repulsion between the solid phase and analytes at a low pH. Furthermore, a diphasic separation column was fabricated based on boronate affinity chromatography, C18-reversed-phase chromatography and applied in pressurized capillary electrochromatography (pCEC). Results showed that four polar nucleosides could be well captured by the boronate affinity chromatography (BAC) section and separated by reversed phase pCEC. Finally, SiO2@PEI600-AAPBA-based solid-phase extraction technology was applied to the purification of ribonucleosides in real urine samples, and results of UHPLC-MS/MS revealed that the intensities of the extracted ions (a neutral mass loss of m/z 132.04 Da) of the ribonucleosides were significantly enhanced after the enrichment. PMID:26048816

  10. Purification of specific loci for proteomic analysis

    PubMed Central

    Byrum, Stephanie D.; Taverna, Sean D.; Tackett, Alan J.

    2015-01-01

    Purification of small, native chromatin sections for proteomic identification of specifically bound proteins and histone posttranslational modifications is a powerful approach for studying mechanisms of chromosome metabolism. We detail a Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS) approach for affinity purification of ~1 kb sections of chromatin for targeted proteomic analysis. This approach utilizes quantitative, high resolution mass spectrometry to categorize proteins and histone posttranslational modifications co-enriching with the given chromatin section as either “specific” to the targeted chromatin or “non-specific” contamination. In this way, the ChAP-MS approach can help define and re-define mechanisms of chromatin-templated activities. PMID:25311124

  11. EVALUATION OF PORTABLE GAS CHROMATOGRAPHS

    EPA Science Inventory

    Limits of detection, linearity of responses, and stability of response factors and retention times for five commercially-available portable gas chromatographs (PGC) were determined during laboratory evaluation. he PGCs were also operated at the French Limited Superfund site near ...

  12. 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)

  13. [PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma.

    PubMed

    Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay; Elkak, Assem; Denizli, Adil

    2016-04-01

    The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PMID:26838913

  14. Pool Purification

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Caribbean Clear, Inc. used NASA's silver ion technology as a basis for its automatic pool purifier. System offers alternative approach to conventional purification chemicals. Caribbean Clear's principal markets are swimming pool owners who want to eliminate chlorine and bromine. Purifiers in Caribbean Clear System are same silver ions used in Apollo System to kill bacteria, plus copper ions to kill algae. They produce spa or pool water that exceeds EPA Standards for drinking water.

  15. Polonium purification

    SciTech Connect

    Baker, J.D.

    1996-09-01

    Three processes for the purification of {sup 210}Po from irradiated bismuth targets are described. Safety equipment includes shielded hotcells for the initial separation from other activation products, gloveboxes for handling the volatile and highly toxic materials, and provisions for ventilation. All chemical separations must be performed under vacuum or in inerted systems. Two of the processes require large amounts of electricity; the third requires vessels made from exotic materials.

  16. Aptamer-modified magnetic beads in affinity separation of proteins.

    PubMed

    Zhu, Guohong; Walter, Johanna-Gabriela

    2015-01-01

    Aptamers are valuable alternative ligands for affinity separations. Here, we describe the aptamer-based affinity separation of His-tagged proteins using an aptamer directed against the His-tag. The immobilization of the aptamer to magnetic beads is described as well as the aptamer-based purification and proper methods for the characterization of the process. Moreover, indications for the transfer of the process to other aptamers are given. PMID:25749947

  17. Strep-Tagged Protein Purification.

    PubMed

    Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

    2015-01-01

    The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). PMID:26096503

  18. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    PubMed

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA. PMID:23185899

  19. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

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

    1981-04-01

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

  20. Dual liquid and gas chromatograph system

    DOEpatents

    Gay, Don D.

    1985-01-01

    A chromatographic system that utilizes one detection system for gas chromatographic and micro-liquid chromatographic determinations. The detection system is a direct-current, atmospheric-pressure, helium plasma emission spectrometer. The detector utilizes a non-transparent plasma source unit which contains the plasma region and two side-arms which receive effluents from the micro-liquid chromatograph and the gas chromatograph. The dual nature of this chromatographic system offers: (1) extreme flexibility in the samples to be examined; (2) extremely low sensitivity; (3) element selectivity; (4) long-term stability; (5) direct correlation of data from the liquid and gas samples; (6) simpler operation than with individual liquid and gas chromatographs, each with different detection systems; and (7) cheaper than a commercial liquid chromatograph and a gas chromatograph.

  1. Dual liquid and gas chromatograph system

    DOEpatents

    Gay, D.D.

    A chromatographic system is described that utilizes one detection system for gas chromatographic and micro-liquid chromatographic determinations. The detection system is a direct-current, atmospheric-pressure, helium plasma emission spectrometer. The detector utilizes a nontransparent plasma source unit which contains the plasma region and two side-arms which receive effluents from the micro-liquid chromatograph and the gas chromatograph. The dual nature of this chromatographic system offers: (1) extreme flexibility in the samples to be examined; (2) extreme low sensitivity; (3) element selectivity; (4) long-term stability; (5) direct correlation of data from the liquid and gas samples; (6) simpler operation than with individual liquid and gas chromatographs, each with different detection systems; and (7) cheaper than a commercial liquid chromatograph and a gas chromatograph.

  2. Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif.

    PubMed

    Riahi, Nesrine; Cappadocia, Laurent; Henry, Olivier; Omichinski, James; De Crescenzo, Gregory

    2016-02-01

    A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides. PMID:26459292

  3. PURIFICATION PROCESS

    DOEpatents

    Wibbles, H.L.; Miller, E.I.

    1958-01-14

    This patent deals with the separation of uranium from molybdenum compounds, and in particular with their separation from ether solutions containing the molybdenum in the form of acids, such as silicomolybdic and phosphomolybdic acids. After the nitric acid leach of pitchblende, the molybdenum values present in the ore are found in the leach solution in the form of complex acids. The uranium bearing solution may be purified of this molybdenum content by comtacting it with activated charcoal. The purification is improved when the acidity of the solution is low ad agitation is also beneficial. The molybdenum may subsequently be recovered from the charcosl ad the charcoal reused.

  4. Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Silver ionization water purification technology was originally developed for Apollo spacecraft. It was later used to cleanse swimming pools and has now been applied to industrial cooling towers and process coolers. Sensible Technologies, Inc. has added two other technologies to the system, which occupies only six square feet. It is manufactured in three capacities, and larger models are custom built on request. The system eliminates scale, corrosion, algae, bacteria and debris, and because of the NASA technology, viruses and waterborne bacteria are also destroyed. Applications include a General Motors cooling tower, amusement parks, ice manufacture and a closed-loop process cooling system.

  5. Deconvolution of gas chromatographic data

    NASA Technical Reports Server (NTRS)

    Howard, S.; Rayborn, G. H.

    1980-01-01

    The use of deconvolution methods on gas chromatographic data to obtain an accurate determination of the relative amounts of each material present by mathematically separating the merged peaks is discussed. Data were obtained on a gas chromatograph with a flame ionization detector. Chromatograms of five xylenes with differing degrees of separation were generated by varying the column temperature at selected rates. The merged peaks were then successfully separated by deconvolution. The concept of function continuation in the frequency domain was introduced in striving to reach the theoretical limit of accuracy, but proved to be only partially successful.

  6. Recent development in chromatographic techniques

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromatographic techniques play a significant role in the determination of analytes in complex matrices, separating individual sample components prior to their detection. In the analysis of contaminants and chemical residues in foods, gas chromatography (GC) and liquid chromatography (LC) are two m...

  7. Ice-shell purification of ice-binding proteins.

    PubMed

    Marshall, Craig J; Basu, Koli; Davies, Peter L

    2016-06-01

    Ice-affinity purification is a simple and efficient method of purifying to homogeneity both natural and recombinant ice-binding proteins. The purification involves the incorporation of ice-binding proteins into slowly-growing ice and the exclusion of other proteins and solutes. In previous approaches, the ice was grown around a hollow brass finger through which coolant was circulated. We describe here an easily-constructed apparatus that employs ice affinity purification that not only shortens the time for purification from 1-2 days to 1-2 h, but also enhances yield and purity. In this apparatus, the surface area for the separation was increased by extracting the ice-binding proteins into an ice-shell formed inside a rotating round-bottom flask partially submerged in a sub-zero bath. In principle, any ice-binding compound can be recovered from liquid solution, and the method is readily scalable. PMID:27025155

  8. Development and application of high-performance affinity beads: toward chemical biology and drug discovery.

    PubMed

    Sakamoto, Satoshi; Kabe, Yasuaki; Hatakeyama, Mamoru; Yamaguchi, Yuki; Handa, Hiroshi

    2009-01-01

    In drug development research, the elucidation and understanding of the interactions between physiologically active substances and proteins that numerous genes produce is important. Currently, most commercially available drugs and physiologically active substances have been brought to market without knowledge of factors interacting with the drugs and the substances. Affinity purification is a useful and powerful technique employed to understand factors that are targeted by drugs and physiologically active substances. However, use of conventional matrices for affinity chromatography often causes a decrease in efficiency of affinity purification and, as a result, more practical matrices for affinity purification have been developed for application in drug discovery research. In this paper, we describe the development of high-performance affinity beads (SG beads and FG beads) that enable one-step affinity purification of drug targets and the elucidation of the mechanism of the action of the drugs. We also describe a chemical screening system using our affinity beads. We hope that utilization of the affinity beads will contribute to the progress of research in chemical biology. PMID:19243077

  9. Tagging recombinant proteins to enhance solubility and aid purification.

    PubMed

    Walls, Dermot; Loughran, Sinéad T

    2011-01-01

    Protein fusion technology has enormously facilitated the efficient production and purification of individual recombinant proteins. The use of genetically engineered affinity and solubility-enhancing polypeptide "tags" has increased greatly in recent years and there now exists a considerable repertoire of these that can be used to solve issues related to the expression, stability, solubility, folding, and purification of their fusion partner. In the case of large-scale proteomic studies, the development of purification procedures tailored to individual proteins is not practicable, and affinity tags have therefore become indispensable tools for structural and functional proteomic initiatives that involve the expression of many proteins in parallel. Here, the rationale and applications of a range of established and more recently developed solubility-enhancing and affinity tags are outlined. PMID:20978965

  10. Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.

    PubMed

    Scott, Daniel; Strachan, Jo; Krishna, Varun Gopala; Shaw, Barry; Tooth, David J; Searle, Mark S; Oldham, Neil J; Layfield, Rob

    2016-01-01

    Unanchored polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored polyubiquitin chains in purified fractions. PMID:27613037

  11. Quality improvements of cell membrane chromatographic column.

    PubMed

    Ding, Xuan; Chen, Xiaofei; Cao, Yan; Jia, Dan; Wang, Dongyao; Zhu, Zhenyu; Zhang, Juping; Hong, Zhanying; Chai, Yifeng

    2014-09-12

    Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using a silica stationary phase covered with specific cell membrane. However, its short life span and poor quality control was highlighted in a lot of research articles. In this study, special attention has been paid to the disruption, cell load and packing procedure in order to improve the quality of the CMC columns. Hereto, two newly established CMC models, HSC-T6/CMC and SMMC-7721/CMC have been developed and used in this research project. The optimization of the abovementioned parameters resulted in a better reproducibility of the retention time of the compound GFT (RSD<10%) and improved significantly the quality of the CMC columns. 3.5×10(7)cells were the optimal cell load for the preparation of the CMC columns, the disruption condition was optimized to 5 cycles (400W and 20s interval per cycle) by an ultrasonic processor reducing the total time of cell disruption to 1.5min and the packing flow rate was optimized by applying a linear gradient program. Additionally, 4% paraformaldehyde (PFA) was employed to improve the column quality and prolong the column life span. The results showed that the retention time was longer with PFA treated columns than the ones obtained with the control groups. PMID:25115453

  12. Production and purification of Clostridium botulinum type C and D neurotoxin.

    PubMed

    Gessler, F; Böhnel, H

    1999-07-01

    Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay. PMID:10397323

  13. 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. PMID:26606109

  14. 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…

  15. A membrane-based purification process for cell culture-derived influenza A virus.

    PubMed

    Weigel, Thomas; Solomaier, Thomas; Wehmeyer, Sebastian; Peuker, Alessa; Wolff, Michael W; Reichl, Udo

    2016-02-20

    A simple membrane-based purification process for cell culture-derived influenza virus was established that relies on only two chromatographic unit operations to achieve the contamination limits required according to regulatory authorities. After clarification and concentration, a pseudo-affinity membrane adsorber (sulfated cellulose, SCMA) was applied for virus capture. The subsequent polishing step consisted of a salt-tolerant anion exchange membrane adsorber (STMA) to bind residual DNA. For the presented process neither a buffer exchange step nor a nuclease step for further DNA digestion were required. As a starting point, a two-salt strategy (including a polyvalent ion) was employed to screen STMA conditions in a 96-well plate format. After optimization on chromatographic laboratory scale, the virus recovery was up to 97% with a residual DNA level below 0.82%. In addition, the STMA was characterized regarding its dynamic binding capacity and the impact of flow rate on yields and contamination levels. Overall, the total virus yield for influenza virus A/PR/8/34 (H1/N1) of this two-step membrane process was 75%, while the protein and the DNA contamination level could be reduced to 24% and at least 0.5%, respectively. With 19.8μg protein and 1.2ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived vaccines for human use. Overall, the presented downstream process might serve as a generic and economic platform technology for production of cell culture-derived viruses and viral vectors. PMID:26712479

  16. Pharmaceutical-grade pre-miR-29 purification using an agmatine monolithic support.

    PubMed

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

    2014-11-14

    MicroRNA-based therapeutic applications have fostered a growing interest in the development of microRNAs purification processes in order to obtain the final product with high purity degree, good quality and biologically active. The pre-miR-29 deficiency or overexpression has been associated to a number of clinically important diseases, and its therapeutic application can be considered. Monolithic columns emerged as a new class of chromatographic supports used in the plasmid DNA purification platforms, being an interesting alternative to the conventional particle-based columns. Thus, the current work describes, for the first time, a new affinity chromatography method that combines the high selectivity of agmatine ligands with the versatility of monoliths to specifically and efficiently purify pre-miR-29 from other small RNA species and Rhodovulum sulfidophilum impurities. The effect of different flow rates on pre-miR-29 separation was also evaluated. Moreover, breakthrough experiments were designed to study the effect of different RNA concentrations on the modified monolithic support binding capacity, being verified that the dynamic binding capacity for RNA molecules is dependent of the feed concentration. In order to achieve higher efficiency and selectivity, three different binding and elution strategies based on increased sodium chloride (1.75-3M) or arginine (100mM) and decreased ammonium sulfate (2.4-0M) stepwise gradients are described to purify pre-miR-29. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (97.33 and 94.88%, respectively) as well as purity (75.21 and 90.11%, respectively) were obtained. Moreover, the quality control analysis revealed that the level of impurities (proteins, endotoxins, sRNA) in the final pre-miR-29 sample was negligible. In fact, this new monolithic support arises as a powerful instrument on the microRNA purification to be used in further

  17. RAPID MEASUREMENTS OF NEPTUNIUM OXIDATION STATES USING CHROMATOGRAPHIC RESINS

    SciTech Connect

    Diprete, D; C Diprete, C; Mira Malek, M; Eddie Kyser, E

    2009-03-24

    The Savannah River Site's (SRS) H-Canyon facility uses ceric ammonium nitrate (CAN) to separate impure neptunium (Np) from a high sulfate feed stream. The material is processed using a two-pass solvent extraction purification which relies on CAN to oxidize neptunium to Np(VI) during the first pass prior to extraction. Spectrophotometric oxidation-state analyses normally used to validate successful oxidation to Np(VI) prior to extraction were compromised by this feed stream matrix. Therefore, a rapid chromatographic method to validate successful Np oxidation was developed using Eichrom Industries TRU and TEVA{reg_sign} resins. The method was validated and subsequently transferred to existing operations in the process analytical laboratories.

  18. A fast and easy strategy for protein purification using "teabags".

    PubMed

    Castaldo, M; Barlind, L; Mauritzson, F; Wan, P T; Snijder, H J

    2016-01-01

    Protein purification often involves affinity capture of proteins on stationary resin, alternatively proteins are captured on free flowing resin for subsequent separation from bulk fluid. Both methods require labour and time intensive separation of particulate matter from fluid. We present a method where affinity resin is contained within porous-walled containers, supporting clarification, product recovery, and concentration in a single step with minimal hands-on processing time, without significant investments in equipment. PMID:27356497

  19. Exhaust gas purification device

    SciTech Connect

    Fujiwara, H.; Hibi, T.; Sayo, S.; Sugiura, Y.; Ueda, K.

    1980-02-19

    The exhaust gas purification device includes an exhaust manifold , a purification cylinder connected with the exhaust manifold through a first honey-comb shaped catalyst, and a second honeycomb shaped catalyst positioned at the rear portion of the purification cylinder. Each catalyst is supported by steel wool rings including coarse and dense portions of steel wool. The purification device further includes a secondary air supplying arrangement.

  20. Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6'-O-fluorescein-atractyloside.

    PubMed Central

    Smith, Vernon R; Fearnley, Ian M; Walker, John E

    2003-01-01

    Atractyloside (ATR) is a high-affinity specific inhibitor of the mitochondrial ADP/ATP translocase (AAT). The binding of a fluorescent derivative, 6'- O -fluorescein-ATR (FATR), to mitochondria has been characterized. The binding constants obtained are in agreement with previously published values for ATR, demonstrating that FATR is a suitable probe of the AAT. AAT inhibited by FATR (FATR-AAT) was solubilized in dodecyl maltoside and purified by two separate ion-exchange chromatography steps at different pHs, which allowed FATR-AAT to be purified to homogeneity. The presence of the bound fluorescent probe enabled the inhibited AAT to be distinguished from the unliganded protein during chromatography, as they were markedly different in their chromatographic behaviour. The purified FATR-AAT was dimeric and in a single major conformation containing 1 mole FATR per mole of AAT dimer. In contrast, uninhibited AAT was monomeric and conformationally unstable. Use of the fluorescent ATR derivative in the development of the protocol enabled the stable dimeric AAT to be monitored directly and purified more effectively. The purification protocol was repeated using non-derivatized ATR, and highly pure AAT was obtained that was devoid of other members of the mitochondrial carrier family. PMID:14498831

  1. Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

    PubMed

    Yadav, Vikash Kumar; Chhikara, Nirmal; Gill, Kamaldeep; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2013-08-01

    The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research. PMID:23619703

  2. Vibrational Spectroscopy of Chromatographic Interfaces

    SciTech Connect

    Jeanne E. Pemberton

    2011-03-10

    Chromatographic separations play a central role in DOE-supported fundamental research related to energy, biological systems, the environment, and nuclear science. The overall portfolio of research activities in the Separations and Analysis Program within the DOE Office of Basic Energy Sciences includes support for activities designed to develop a molecular-level understanding of the chemical processes that underlie separations for both large-scale and analytical-scale purposes. The research effort funded by this grant award was a continuation of DOE-supported research to develop vibrational spectroscopic methods to characterize the interfacial details of separations processes at a molecular level.

  3. High-productivity membrane adsorbers: Polymer surface-modification studies for ion-exchange and affinity bioseparations

    NASA Astrophysics Data System (ADS)

    Chenette, Heather C. S.

    This dissertation centers on the surface-modification of macroporous membranes to make them selective adsorbers for different proteins, and the analysis of the performance of these membranes relative to existing technology. The common approach used in these studies, which is using membrane technology for chromatographic applications and using atom transfer radical polymerization (ATRP) as a surface modification technique, will be introduced and supported by a brief review in Chapter 1. The specific approaches to address the unique challenges and motivations of each study system are given in the introduction sections of the respective dissertation chapters. Chapter 2 describes my work to develop cation-exchange membranes. I discuss the polymer growth kinetics and characterization of the membrane surface. I also present an analysis of productivity, which measures the mass of protein that can bind to the stationary phase per volume of stationary phase adsorbing material per time. Surprisingly and despite its importance, this performance measure was not described in previous literature. Because of the significantly shorter residence time necessary for binding to occur, the productivity of these cation-exchange membrane adsorbers (300 mg/mL/min) is nearly two orders of magnitude higher than the productivity of a commercial resin product (4 mg/mL/min). My work studying membrane adsorbers for affinity separations was built on the productivity potential of this approach, as articulated in the conclusion of Chapter 2. Chapter 3 focuses on the chemical formulation work to incorporate glycoligands into the backbone of polymer tentacles grown from the surface of the same membrane stationary phase. Emphasis is given to characterizing and testing the working formulation for ligand incorporation, and details about how I arrived at this formulation are given in Appendix B. The plant protein, or lectin, Concanavalin A (conA) was used as the target protein. The carbohydrate affinity

  4. Para-aminobenzamidine linked regenerated cellulose membranes for plasminogen activator purification: Effect of spacer arm length and ligand density

    PubMed Central

    Fasoli, Ezio; Reyes, Yiaslin Ruiz; Guzman, Osiris Martinez; Rosado, Alexandra; Cruz, Vivian Rodriguez; Borges, Amaris; Martinez, Edmarie; Bansal, Vibha

    2013-01-01

    Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; ii) the affinity ligand (pABA) density on membrane surface (5–25 nmoles per cm2). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi- component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14- atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmole of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in monocomponent system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0 nmoles per cm2) in mono- and multi- component systems for 7- as well as 14- atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use. PMID:23703544

  5. Design of protease-resistant peptide ligands for the purification of antibodies from human plasma.

    PubMed

    Menegatti, Stefano; Bobay, Benjamin G; Ward, Kevin L; Islam, Tuhidul; Kish, William S; Naik, Amith D; Carbonell, Ruben G

    2016-05-01

    A strategy is presented for developing variants of peptide ligands with enhanced biochemical stability for the purification of antibodies from animal sera. Antibody-binding sequences HWRGWV, HYFKFD, and HFRRHL, previously discovered by our group, were modified with non-natural amino acids to gain resistance to proteolysis, while maintaining target affinity and selectivity. As trypsin and α-chymotrypsin were chosen as models of natural proteolytic enzymes, the basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) amino acids were replaced with non-natural analogs. Using the docking software HADDOCK, a virtual library of peptide variants was designed and screened in-silico against the known HWRGWV binding site on the pFc fragment of IgG. A pool of selected sequences with the highest predicted free energy of binding was synthesized on chromatographic resin, and the resulting adsorbents were tested for IgG binding and resistance to proteases. The ligand variants exhibited binding capacities and specificities comparable to the original sequences, yet with much higher proteolytic resistances. The sequences HWMetCitGWMetV and HFMetCitCitHL was used for purifying polyclonal IgG from IgG-rich fractions of human plasma, with yields and purity above 90%. Notably, due to electrical neutrality, the variant showed higher selectivity than the original sequence. Binding isotherms were also constructed, which confirmed the docking predictions. This method represents a general strategy for enhancing the biochemical stability as well as the affinity and selectivity of natural or synthetic peptide ligands for bioseparations. PMID:27072524

  6. Use of supercritical carbon dioxide fluid as a solvent for the purification of pet radiotracers

    SciTech Connect

    Ferrieri, R.A.; Fowler, J.S.; Wolf, A.P.

    1993-12-31

    We have identified superfluid chromatography (SFC) as a promising method which could offer advantages in radiotracer purification through rapid separation, as well as, improved recovery and purity of labeled product. Using SF CO{sub 2} as the mobile phase for chromatographic separation of labeled product would eliminate the need for solvent removal from product prior to delivery.

  7. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

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

  8. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    SciTech Connect

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  9. Purification of GST-Tagged Proteins.

    PubMed

    Schäfer, Frank; Seip, Nicole; Maertens, Barbara; Block, Helena; Kubicek, Jan

    2015-01-01

    This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure. GST-tagged proteins are often used to study protein-protein interactions, again making use of glutathione affinity in a procedure called a GST pull-down assay. The protocol is designed to process 200 ml of E. coli culture expressing intermediate to high amounts of a GST-tagged protein (~25 mg l(-1)). Depending on the expression rate or the available culture volume, the scale can be increased or decreased linearly. The protocol can also be used to purify GST-tagged proteins from other expression systems, such as insect or mammalian cells. Tips are provided to aid in modifying certain steps if proteins shall be recovered from alternative expression systems. PMID:26096507

  10. Evaluation of portable gas chromatographs

    SciTech Connect

    Berkley, R.E.; Miller, M.; Chang, J.C.; Oliver, K.; Fortune, C.

    1993-01-01

    Limits of detection, linearity of responses, and stability of response factors and retention times for five commercially-available portable gas chromatographs (PGC) were determined during laboratory evaluation. The PGCs were also operated at the French Limited Superfund site near Houston, TX during startup of bioremediation. Concentrations of volatile organic compounds (VOC) at the site were slightly above ambient background levels. Concurrent collocated grab samples were collected periodically in canisters and analyzed by Method TO-14 using a mass-selective detector. Canister data were taken to indicate correct concentrations and were used to assess the accuracy of PGC data. Durability, reliability, and complexity of operation of PGCs were also evaluated. The principal goal of the study was to determine the best way to use each instrument as a monitor for airborne VOCs.

  11. An alternate high yielding purification method for Clitoria ternatea lectin.

    PubMed

    Naeem, Aabgeena; Ahmad, Ejaz; Khan, Rizwan Hasan

    2007-10-01

    In our previous publication we had reported the purification and characterization of Clitoria ternatea agglutinin from its seeds on fetuin CL agarose affinity column, designated CTA [A. Naeem, S. Haque, R.H. Khan. Protein J., 2007]. Since CTA binds beta-d-galactosides, this lectin can be used as valuable tool for glycobiology studies in biomedical and cancer research. So an attempt was made for a high yielding alternative purification method employing the use of asialofetuin CL agarose column for the above-mentioned lectin, designated CTL. The fetuin affinity purified agglutinin was found similar to asialofetuin affinity purified lectin in SDS pattern, HPLC and N-terminal sequence. The content of lectin was found to be 30mg/30g dry weight of pulse. The yield was 2.8% as compared to 0.3% obtained on fetuin column. The number of tryptophan and tyrosine estimated was four and six per subunit. PMID:17590430

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

    PubMed

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

    2008-03-28

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

  13. DNA purification by triplex-affinity capture and affinity capture electrophoresis

    DOEpatents

    Cantor, C.R.; Ito, Takashi; Smith, C.L.

    1996-01-09

    The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel. 6 figs.

  14. DNA purification by triplex-affinity capture and affinity capture electrophoresis

    DOEpatents

    Cantor, Charles R.; Ito, Takashi; Smith, Cassandra L.

    1996-01-01

    The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

  15. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  16. Chromatographic resolution of altered forms of protein kinase C

    SciTech Connect

    Ashendel, C.L.; Minor, P.L.; Baudoin, P.A.; Carlos, M.

    1987-05-01

    Rapid chromatographic resolution of protein kinase C (PKC) in extracts of rat brain on DEAE-cellulose yielded two major peaks of activity. These fractions bound phorbol esters with identical affinity and specificity and had similar ratios of PKC to phorbol ester-binding activities. Chicken egg yolk antibodies raised to PKC in the first fraction reacted with 74 to 76 kilodalton peptides in the second fraction. Chromatography of each fraction on hydroxylapatite yielded similar distributions of three PKC isozymes. Rechromatography of the DEAE-cellulose fractions on DEAE-cellulose confirmed that these forms of PKC were not rapidly interconvertible. Results of experiments in which extracts or fractions were incubated with MgATP and phosphatase inhibitors were consistent with elution of dephospho-PKC in the first fraction while the second fraction contained phospho-PKC. If confirmed, this suggests that a substantial fraction of PKC in rat and mouse tissues exists in the phosphorylated form.

  17. Derivatization and gas chromatographic-mass spectrometric detection of anabolic steroid residues isolated from edible muscle tissues.

    PubMed

    Daeseleire, E; De Guesquière, A; Van Peteghem, C

    1991-01-01

    A method was developed for the detection of anabolic steroid residues in edible muscle tissues. After enzymic digestion of the tissue and purification on disposable C18 solid-phase extraction columns, the extract was injected onto a C18 reversed-phase high-performance liquid chromatographic column. Three fractions or windows were collected, each containing specific analytes. After evaporation to dryness, the residues were subjected to a derivatization procedure which yielded suitable derivatives. After gas chromatographic-mass spectrometric analysis, both gas chromatographic retention data and mass spectral data were used for the detection and identification of nortestosterone, testosterone, estradiol, ethinylestradiol, trenbolone, methyltestosterone, chlormadinone acetate, medroxyprogesterone acetate and megestrol acetate. PMID:2026730

  18. Detergent-Free Membrane Protein Purification.

    PubMed

    Rothnie, Alice J

    2016-01-01

    Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment. PMID:27485341

  19. Expression and purification of rhIL-10-RGD from Escherichia coli as a potential wound healing agent.

    PubMed

    Yang, Fangfang; Wan, Yi; Liu, Jiaqi; Yang, Xuekang; Wang, Hongtao; Tao, Ke; Han, Juntao; Shi, Jihong; Hu, Dahai

    2016-08-01

    Various protocols for recombinant Interleukin-10 (IL-10) purification in wound healing have been reported previously. However, the therapeutic effect was not obvious. Thus, it is of great importance to find new and effective approaches for therapy. In this study, we propose that IL-10 and Arginine-Glycine-Aspartic (RGD) peptide would be a valuable therapeutic for wound healing. To explore a high-efficiency and cost-effective approach for the production of IL-10 and RGD peptide with bioactivity, a synthetic gene was cloned into a recombinant pTWIN1 vector. As a consequence, rhIL-10-RGD and the pH-induced self-cleavable Ssp DnaB mini-intein as a fusion protein was highly expressed by IPTG induction in Escherichia coli Rosetta without extra residues in a bioreactor. After Ni affinity chromatographic purification, rhIL-10-RGD was released by the Ssp DnaB intein-mediated self-cleavage that is triggered by pH shift. SDS-PAGE and silver staining showed a major band with an estimated molecular mass of 19.3kDa. Cell proliferation assay confirmed its potent proliferation activity on MC/9 murine mast cells. In conclusion, we report a novel strategy to produce rhIL-10-RGD mediated by the pH-induced self-cleavable Ssp DnaB mini-intein, and show that rhIL-10-RGD could play an effective role in wound healing of BALB/c mice. PMID:27241829

  20. Purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity.

    PubMed

    Luth, Eric S; Bartels, Tim; Dettmer, Ulf; Kim, Nora C; Selkoe, Dennis J

    2015-01-20

    Despite two decades of research, the structure-function relationships of endogenous, physiological forms of α-synuclein (αSyn) are not well understood. Most in vitro studies of this Parkinson's disease-related protein have focused on recombinant αSyn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that αSyn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant αSyn, physiological tetramers purified from human erythrocytes have substantial α-helical content and resist pathological aggregation into β-sheet rich fibers. Here, we report the first method to fully purify soluble αSyn from the most relevant source, human brain. We describe protocols that purify αSyn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant αSyn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue β-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived αSyn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying αSyn to homogeneity destabilizes native, α-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification. PMID:25490121

  1. Rapid Purification of a New P-I Class Metalloproteinase from Bothrops moojeni Venom with Antiplatelet Activity

    PubMed Central

    de Queiroz, Mayara R.; Mamede, Carla C. Neves; Fonseca, Kelly C.; de Morais, Nadia C. G.; de Sousa, Bruna B.; Santos-Filho, Norival A.; Beletti, Marcelo E.; Arantes, Eliane C.; Stanziola, Leonilda; de Oliveira, Fábio

    2014-01-01

    The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMPα-II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMPα-II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMPα-II cleaves Aα-chain of fibrinogen followed by Bβ-chain, and does not show any effect on the γ-chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30–50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMPα-II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMPα-II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMPα-II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders. PMID:24982866

  2. High-yield expression and purification of the Hsp90-associated p23, FKBP52, HOP and SGTα proteins.

    PubMed

    Hildenbrand, Zacariah L; Molugu, Sudheer K; Paul, Atanu; Avila, Gustavo A; Herrera, Nadia; Xiao, Chuan; Cox, Marc B; Bernal, Ricardo A

    2010-10-15

    Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of hormone-dependent pathologies such as cancer. An important role for Hsp90 is to facilitate the stable binding of steroid hormones to their respective receptors enabling the ligand-based signal to be carried to the nucleus and ultimately resulting in the up-regulation of gene expression. Along with Hsp90, this dynamic and transient process also involves the recruitment of additional proteins and co-chaperones that add further stability to the mature receptor-chaperone complex. In the work presented here, we describe four new protocols for the bacterial over-expression and column chromatographic purification of the human p23, FKBP52, HOP and SGTα proteins. Each of these proteins plays a distinct role in the steroid hormone receptor regulatory cycle. Affinity, ion-exchange and size-exclusion techniques were used to produce target yields greater than 50mg/L of cultured media, with each purified sample reaching near absolute sample homogeneity. These results reveal a reliable system for the production of p23, FKBP52, HOP and SGTα substrate proteins for use in the investigation of the Hsp90-associated protein interactions of the steroid hormone receptor cycle. PMID:20829124

  3. An open tubular ion chromatograph.

    PubMed

    Yang, Bingcheng; Zhang, Min; Kanyanee, Tinakorn; Stamos, Brian N; Dasgupta, Purnendu K

    2014-12-01

    We describe an open tubular ion chromatograph (OTIC) that uses anion exchange latex coated 5 μm radius silica and 9.8 μm radius poly(methyl methacrylate) tubes and automated time/pressure based hydrodynamic injection for pL-nL scale injections. It is routinely possible to generate 50,000 plates or more (up to 150,000 plates/m, columns between 0.3 and 0.8 m have been used), and as such, fast separations are possible, comparable to or in some cases better than the current practice of IC. With an optimized admittance detector, nonsuppressed detection permits LODs of submicromolar to double digit micromolar for a variety of analytes. However, large volume injections are possible and can significantly improve on this. A variety of eluents, the use of organic modifiers, and variations of eluent pH can be used to tailor a given separation. The approach is discussed in the context of extraterrestrial exploration, especially Mars, where the existence of large amounts of perchlorate in the soil needs to be confirmed. These columns can survive drying and freezing, and small footprint, low power consumption, and simplicity make OTIC a good candidate for such a mission. PMID:25394230

  4. Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part II. Impact on chromatographic separations.

    PubMed

    Hubbuch, Jürgen; Linden, Thomas; Knieps, Esther; Thömmes, Jörg; Kula, Maria-Regina

    2003-12-22

    The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent. PMID:14735979

  5. 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

  6. Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): a production process developed for a lead candidate recombinant hookworm vaccine antigen.

    PubMed

    Goud, Gaddam Narsa; Deumic, Vehid; Gupta, Richi; Brelsford, Jill; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan L; Tsao, Eric I; Hotez, Peter J; Bottazzi, Maria Elena

    2012-06-01

    The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing. PMID:22503665

  7. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

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

    2015-07-01

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

  8. A Quantitative Gas Chromatographic Ethanol Determination.

    ERIC Educational Resources Information Center

    Leary, James J.

    1983-01-01

    Describes a gas chromatographic experiment for the quantitative determination of volume percent ethanol in water ethanol solutions. Background information, procedures, and typical results are included. Accuracy and precision of results are both on the order of two percent. (JN)

  9. Gas Chromatographic Determination of Enrivonmentally Significant Pesticides.

    ERIC Educational Resources Information Center

    Rudzinski, Walter E.; Beu, Steve

    1982-01-01

    A chromatographic procedure for analyzing organophosphorus pesticides (such as PCB's, nitrosamines, and phthalate esters) in orange juice is described, including a summary of the method, instrumentation, methodology, results/discussion, and calculations. (JN)

  10. A Versatile, Automatic Chromatographic Column Packing Device

    ERIC Educational Resources Information Center

    Barry, Eugene F.; And Others

    1977-01-01

    Describes an inexpensive apparatus for packing liquid and gas chromatographic columns of high efficiency. Consists of stainless steel support struts, an Automat Getriebmotor, and an associated three-pulley system capable of 10, 30, and 300 rpm. (MLH)

  11. Gas chromatograph injection port protective device

    NASA Technical Reports Server (NTRS)

    Robertson, M. D.; Welz, E. A.

    1969-01-01

    To prevent samples containing foreign matter from poisoning the gas chromatographic columns, a pre-filter insertion is placed in the injection port. The packing becomes a variable reactant, for example, acids are removed by using an alkaline liquid.

  12. 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

  13. Purification and identification of endogenous polySUMO conjugates.

    PubMed

    Bruderer, Roland; Tatham, Michael H; Plechanovova, Anna; Matic, Ivan; Garg, Amit K; Hay, Ronald T

    2011-02-01

    The small ubiquitin-like modifier (SUMO) can undergo self-modification to form polymeric chains that have been implicated in cellular processes such as meiosis, genome maintenance and stress response. Investigations into the biological role of polymeric chains have been hampered by the absence of a protocol for the purification of proteins linked to SUMO chains. In this paper, we describe a rapid affinity purification procedure for the isolation of endogenous polySUMO-modified species that generates highly purified material suitable for individual protein studies and proteomic analysis. We use this approach to identify more than 300 putative polySUMO conjugates from cultured eukaryotic cells. PMID:21252943

  14. Nanofluidic Size-Exclusion Chromatograph

    NASA Technical Reports Server (NTRS)

    Feldman, Sabrina; Svehla, Danielle; Grunthaner, Frank; Feldman, Jason; Shakkottai, P.

    2004-01-01

    Efforts are under way to develop a nanofluidic size-exclusion chromatograph (SEC), which would be a compact, robust, lightweight instrument for separating molecules of interest according to their sizes and measuring their relative abundances in small samples. About as large as a deck of playing cards, the nanofluidic SEC would serve, in effect, as a laboratory on a chip that would perform the functions of a much larger, conventional, bench-top SEC and ancillary equipment, while consuming much less power and much smaller quantities of reagent and sample materials. Its compactness and low power demand would render it attractive for field applications in which, typically, it would be used to identify and quantitate a broad range of polar and nonpolar organic compounds in soil, ice, and water samples. Size-exclusion chromatography is a special case of high-performance liquid chromatography. In a conventional SEC, a sample plug is driven by pressure along a column packed with silica or polymer beads that contain uniform nanopores. The interstices between, and the pores in, the beads collectively constitute a size-exclusion network. Molecules follow different paths through the size-exclusion network, such that characteristic elution times can be related to sizes of molecules: basically, smaller molecules reach the downstream end of the column after the larger ones do because the smaller ones enter minor pores and stay there for a while, whereas the larger ones do not enter the pores. The volume accessible to molecules gradually diminishes as their size increases. All molecules bigger than a pore size elute together. For most substances, the elution times and sizes of molecules can be correlated directly with molecular weights. Hence, by measuring the flux of molecules arriving at the downstream end as a function of time, one can obtain a liquid mass spectrum for the molecules present in a sample over a broad range of molecular weights.

  15. Purification of Oat and Rye Phytochrome 1

    PubMed Central

    Rice, Harbert V.; Briggs, Winslow R.; Jackson-White, Cecil J.

    1973-01-01

    A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A280 nm/A665 nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants. Images PMID:16658440

  16. The Borexino purification system

    NASA Astrophysics Data System (ADS)

    Benziger, Jay

    2014-05-01

    Purification of 278 tons of liquid scintillator and 889 tons of buffer shielding for the Borexino solar neutrino detector is performed with a system of combined distillation, water extraction, gas stripping and filtration. The purification system removed K, U and Th by distillation of the pseudocumene solvent and the PPO fluor. Noble gases, Rn, Kr and Ar were removed by gas stripping. Distillation was also employed to remove optical impurities and reduce the attenuation of scintillation light. The success of the purification system has facilitated the first time real time detection of low energy solar neutrinos.

  17. A process for the separation and purification of yttrium-90 for medical applications

    DOEpatents

    Horwitz, P.E.; Dietz, M.L.

    1993-01-01

    An extraction chromatographic method for the preparation of {sup 90}Y of high chemical and radiochemical purity is disclosed. After an initial purification of a {sup 90}Sr stock solution and a suitable period of {sup 90}Y ingrowth, the solution is passed through a series of strontium-selective chromatographic columns, each of which lowers the {sup 90}Sr content of the mixture by a factor of about 10{sup 3}. The {sup 90}Y remaining is freed from any residual {sup 90}Sr, from its {sup 90}Zr daughter, and from any remaining impurities by passing the sample through a final column designed to selectively retain yttrium.

  18. Process for the separation and purification of yttrium-90 for medical applications

    DOEpatents

    Horwitz, Philip E.; Dietz, Mark L.

    1994-01-01

    An extraction chromatographic method for the preparation of .sup.90 Y of high chemical and radiochemical purity is disclosed. After an initial purification of a .sup.90 Sr stock solution and a suitable period of .sup.90 Y ingrowth, the solution is passed through a series of strontium-selective chromatographic columns, each of which lowers the .sup.90 Sr content of the mixture by a factor of about 10.sup.3. The .sup.90 Y remaining is freed from any residual .sup.90 Sr, from its .sup.90 Zr daughter, and from any remaining impurities by passing the sample through a final column designed to selectively retain yttrium.

  19. Process for the separation and purification of yttrium-90 for medical applications

    DOEpatents

    Horwitz, P.E.; Dietz, M.L.

    1994-11-29

    An extraction chromatographic method for the preparation of [sup 90]Y of high chemical and radiochemical purity is disclosed. After an initial purification of a [sup 90]Sr stock solution and a suitable period of [sup 90]Y ingrowth, the solution is passed through a series of strontium-selective chromatographic columns, each of which lowers the [sup 90]Sr content of the mixture by a factor of about 10[sup 3]. The [sup 90]Y remaining is freed from any residual [sup 90]Sr, from its [sup 90]Zr daughter, and from any remaining impurities by passing the sample through a final column designed to selectively retain yttrium. 5 figures.

  20. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

    PubMed Central

    Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

    2012-01-01

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

  1. Local Affinity Release.

    PubMed

    Delplace, Vianney; Obermeyer, Jaclyn; Shoichet, Molly S

    2016-07-26

    The use of hydrogels for therapeutic delivery is a burgeoning area of investigation. These water-swollen polymer matrices are ideal platforms for localized drug delivery that can be further combined with specific ligands or nanotechnologies to advance the controlled release of small-molecule drugs and proteins. Due to the advantage of hydrophobic, electrostatic, or specific extracellular matrix interactions, affinity-based strategies can overcome burst release and challenges associated with encapsulation. Future studies will provide innovative binding tools, truly stimuli-responsive systems, and original combinations of emerging technologies to control the release of therapeutics spatially and temporally. Local drug delivery can be achieved by directly injecting a therapeutic to its site of action and is advantageous because off-target effects associated with systemic delivery can be minimized. For prolonged benefit, a vehicle that provides sustained drug release is required. Hydrogels are versatile platforms for localized drug release, owing to the large library of biocompatible building blocks from which they can be formed. Injectable hydrogel formulations that gel quickly in situ and provide sustained release of therapeutics are particularly advantageous to minimize invasiveness. The incorporation of polymers, ligands or nanoparticles that have an affinity for the therapeutic of interest improve control over the release of small-molecule drugs and proteins from hydrogels, enabling spatial and temporal control over the delivery. Such affinity-based strategies can overcome drug burst release and challenges associated with protein instability, allowing more effective therapeutic molecule delivery for a range of applications from therapeutic contact lenses to ischemic tissue regeneration. PMID:27403513

  2. Bromelain: an overview of industrial application and purification strategies.

    PubMed

    Arshad, Zatul Iffah Mohd; Amid, Azura; Yusof, Faridah; Jaswir, Irwandi; Ahmad, Kausar; Loke, Show Pau

    2014-09-01

    This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future. PMID:24965557

  3. Novel lipase purification methods - a review of the latest developments.

    PubMed

    Tan, Chung Hong; Show, Pau Loke; Ooi, Chien Wei; Ng, Eng-Poh; Lan, John Chi-Wei; Ling, Tau Chuan

    2015-01-01

    Microbial lipases are popular biocatalysts due to their ability to catalyse diverse reactions such as hydrolysis, esterification, and acidolysis. Lipases function efficiently on various substrates in aqueous and non-aqueous media. Lipases are chemo-, regio-, and enantio-specific, and are useful in various industries, including those manufacturing food, detergents, and pharmaceuticals. A large number of lipases from fungal and bacterial sources have been isolated and purified to homogeneity. This success is attributed to the development of both conventional and novel purification techniques. This review highlights the use of these techniques in lipase purification, including conventional techniques such as: (i) ammonium sulphate fractionation; (ii) ion-exchange; (iii) gel filtration and affinity chromatography; as well as novel techniques such as (iv) reverse micellar system; (v) membrane processes; (vi) immunopurification; (vi) aqueous two-phase system; and (vii) aqueous two-phase floatation. A summary of the purification schemes for various bacterial and fungal lipases are also provided. PMID:25273633

  4. Petasis-Ugi ligands: New affinity tools for the enrichment of phosphorylated peptides.

    PubMed

    Batalha, Íris L; Roque, Ana C A

    2016-09-15

    Affinity chromatography is a widespread technique for the enrichment and isolation of biologics, which relies on the selective and reversible interaction between affinity ligands and target molecules. Small synthetic affinity ligands are valuable alternatives due to their robustness, low cost and fast ligand development. This work reports, for the first time, the use of a sequential Petasis-Ugi multicomponent reaction to generate rationally designed solid-phase combinatorial libraries of small synthetic ligands, which can be screened for the selection of new affinity adsorbents towards biological targets. As a proof of concept, the Petasis-Ugi reaction was here employed in the discovery of affinity ligands suitable for phosphopeptide enrichment. A combinatorial library of 84 ligands was designed, synthesized on a chromatographic solid support and screened in situ for the specific binding of phosphopeptides binding human BRCA1C-terminal domains. The success of the reaction on the chromatographic matrix was confirmed by both inductively coupled plasma atomic emission spectroscopy and fluorescence microscopy. Three lead ligands were identified due to their superior performance in terms of binding capacity and selectivity towards the phosphorylated moiety on peptides, which showed the feasibility of the Petasis-Ugi reaction for affinity ligand development. PMID:27469904

  5. Chromatographic separation of germanium and arsenic for the production of high purity (77)As.

    PubMed

    Gott, Matthew D; DeGraffenreid, Anthony J; Feng, Yutian; Phipps, Michael D; Wycoff, Donald E; Embree, Mary F; Cutler, Cathy S; Ketring, Alan R; Jurisson, Silvia S

    2016-04-01

    A simple column chromatographic method was developed to isolate (77)As (94±6% (EtOH/HCl); 74±11 (MeOH)) from germanium for potential use in radioimmunotherapy. The separation of arsenic from germanium was based on their relative affinities for different chromatographic materials in aqueous and organic environments. Using an organic or mixed mobile phase, germanium was selectively retained on a silica gel column as germanate, while arsenic was eluted from the column as arsenate. Subsequently, enriched (76)Ge (98±2) was recovered for reuse by elution with aqueous solution (neutral to basic). Greater than 98% radiolabeling yield of a (77)As-trithiol was observed from methanol separated [(77)As]arsenate [17]. PMID:26947162

  6. Inexpensive, serotype-independent protocol for native and bioengineered recombinant adeno-associated virus purification

    PubMed Central

    Arden, Erik; Metzger, Joseph M.

    2016-01-01

    Recombinant adeno-associated virus (AAV) is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes and includes bioengineered viruses. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimization to isolate purified AAV. These methods may also limit crude viral lysate processing volume resulting in a significant loss of viral titer. To circumvent these issues, we have developed an AAV purification protocol independent of toxic compounds, supernatant volume and capsid moiety. This purification method standardizes virus purification across native serotype and bioengineered mosaic capsids. PMID:27294171

  7. Refined separation of combined Fe–Hf from rock matrices for isotope analyses using AG-MP-1M and Ln-Spec chromatographic extraction resins

    PubMed Central

    Cheng, Ting; Nebel, Oliver; Sossi, Paolo A.; Chen, Fukun

    2014-01-01

    A combined procedure for separating Fe and Hf from a single rock digestion is presented. In a two-stage chromatographic extraction process, a purified Fe fraction is first quantitatively separated from the rock matrix using AG-MP-1M resin in HCl. Hafnium is subsequently isolated using a modified version of a commonly applied method using Eichrom LN-Spec resin. Our combined method includes:•Purification of Fe from the rock matrix using HCl, ready for mass spectrometric analysis.•Direct loading of the matrix onto the resin that is used for Hf purification.•Collection of a Fe-free Hf fraction. PMID:26150946

  8. Portable gas chromatograph-mass spectrometer

    DOEpatents

    Andresen, Brian D.; Eckels, Joel D.; Kimmons, James F.; Myers, David W.

    1996-01-01

    A gas chromatograph-mass spectrometer (GC-MS) for use as a field portable organic chemical analysis instrument. The GC-MS is designed to be contained in a standard size suitcase, weighs less than 70 pounds, and requires less than 600 watts of electrical power at peak power (all systems on). The GC-MS includes: a conduction heated, forced air cooled small bore capillary gas chromatograph, a small injector assembly, a self-contained ion/sorption pump vacuum system, a hydrogen supply, a dual computer system used to control the hardware and acquire spectrum data, and operational software used to control the pumping system and the gas chromatograph. This instrument incorporates a modified commercial quadrupole mass spectrometer to achieve the instrument sensitivity and mass resolution characteristic of laboratory bench top units.

  9. Portable gas chromatograph-mass spectrometer

    DOEpatents

    Andresen, B.D.; Eckels, J.D.; Kimmons, J.F.; Myers, D.W.

    1996-06-11

    A gas chromatograph-mass spectrometer (GC-MS) is described for use as a field portable organic chemical analysis instrument. The GC-MS is designed to be contained in a standard size suitcase, weighs less than 70 pounds, and requires less than 600 watts of electrical power at peak power (all systems on). The GC-MS includes: a conduction heated, forced air cooled small bore capillary gas chromatograph, a small injector assembly, a self-contained ion/sorption pump vacuum system, a hydrogen supply, a dual computer system used to control the hardware and acquire spectrum data, and operational software used to control the pumping system and the gas chromatograph. This instrument incorporates a modified commercial quadrupole mass spectrometer to achieve the instrument sensitivity and mass resolution characteristic of laboratory bench top units. 4 figs.

  10. Portable gas chromatograph-mass spectrometer

    SciTech Connect

    Andresen, B.D.; Eckels, J.D.; Kimmins, J.F.; Myers, D.W.

    1994-12-31

    A gas chromatograph-mass spectrometer (GC-MS) for use as a field portable organic chemical analysis instrument. The GC-MS is designed to be contained in a standard size suitcase, weighs less than 70 pounds, and requires less than 600 watts of electrical power at peak power (all systems on). The GC-MS includes: a conduction heated, forced air cooled small bore capillary gas chromatograph, a small injector assembly, a self-contained ion/sorption pump vacuum system, a hydrogen supply, a dual computer system used to control the hardware and acquire spectrum data, and operational software used to control the pumping system and the gas chromatograph. This instrument incorporates a modified commercial quadrupole mass spectrometer to achieve the instrument sensitivity and mass resolution characteristic of laboratory bench top units.

  11. Bioengineering of bacteria to assemble custom-made polyester affinity resins.

    PubMed

    Hay, Iain D; Du, Jinping; Burr, Natalie; Rehm, Bernd H A

    2015-01-01

    Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

  12. Purification of a rat neurotensin receptor expressed in Escherichia coli.

    PubMed Central

    Tucker, J; Grisshammer, R

    1996-01-01

    A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress. PMID:8760379

  13. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  14. Morphometric affinities of gigantopithecus.

    PubMed

    Gelvin, B R

    1980-11-01

    Multivariate analyses, supplemented by univariate statistical methods, of measurements from mandibular tooth crown dimensions and the mandible of Gigantopithecus blacki, G. bilaspurensis, Plio-Plelstocene hominids, Homo erectus, and seven Neogene ape species from the genera Proconsul, Sivapithecus, Ouranopithecus, and Dryopithecus were used to assess the morphometric affinities of Gigantopithecus. The results show that Gigantopithecus displays affinities to Ouranopithecus and to the hominids, particularly the Plio-Plelstocene hominids, rather than to the apes. Ouranopithecus demonstrated dental resemblances to G. bilaspurensis and the Plio-Pleistocene hominids but mandibular similarities to the apes. Results of analyses of tooth and mandibular shape indices, combined with multivariate distance and temporal relationships, suggest that Ouranopithecus is a more likely candidate for Gigantopithecus ancestry than is Silvapithecus indicus. Shape and allometric differences between G. bilaspurensis and the robust australopithecines weaken the argument for an ancestral-descendant relationship between these groups. The results support the hypothesis that Gigantopithecus is an extinct side branch of the Hominidae. PMID:7468790

  15. Ion chromatographic determination of sulfur in fuels

    NASA Technical Reports Server (NTRS)

    Mizisin, C. S.; Kuivinen, D. E.; Otterson, D. A.

    1978-01-01

    The sulfur content of fuels was determined using an ion chromatograph to measure the sulfate produced by a modified Parr bomb oxidation. Standard Reference Materials from the National Bureau of Standards, of approximately 0.2 + or - 0.004% sulfur, were analyzed resulting in a standard deviation no greater than 0.008. The ion chromatographic method can be applied to conventional fuels as well as shale-oil derived fuels. Other acid forming elements, such as fluorine, chlorine and nitrogen could be determined at the same time, provided that these elements have reached a suitable ionic state during the oxidation of the fuel.

  16. Analysis of chromatograph systems using orthogonal collocation

    NASA Technical Reports Server (NTRS)

    Woodrow, P. T.

    1974-01-01

    Research is generating fundamental engineering design techniques and concepts for the chromatographic separator of a chemical analysis system for an unmanned, Martian roving vehicle. A chromatograph model is developed which incorporates previously neglected transport mechanisms. The numerical technique of orthogonal collocation is studied. To establish the utility of the method, three models of increasing complexity are considered, the latter two being limiting cases of the derived model: (1) a simple, diffusion-convection model; (2) a rate of adsorption limited, inter-intraparticle model; and (3) an inter-intraparticle model with negligible mass transfer resistance.

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

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

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

  18. Monoclonal antibody purification with hydroxyapatite.

    PubMed

    Gagnon, Pete

    2009-06-01

    Hydroxyapatite (HA) has been used for IgG purification since its introduction in the 1950s. Applications expanded to include IgA and IgM in the 1980s, along with elucidation of its primary binding mechanisms and the development of ceramic HA media. With the advent of recombinant monoclonal antibodies, HA was demonstrated to be effective for removal of antibody aggregates, as well as host cell proteins and leached protein A. HA's inherent abilities have been enhanced by the development of elution strategies that permit differential control of its primary binding mechanisms: calcium metal affinity and phosphoryl cation exchange. These strategies support reduction of antibody aggregate content from greater than 60% to less than 0.1%, in conjunction with enhanced removal of DNA, endotoxin, and virus. HA also has a history of discriminating various immunological constructs on the basis of differences in their variable regions, or discriminating Fab fragments from Fc contaminants in papain digests of purified monoclonal IgG. Continuing development of novel elution strategies, alternative forms of HA, and application of robotic high throughput screening systems promise to expand HA's utility in the field. PMID:19491046

  19. Analysis of Free Fractions for Chiral Drugs Using Ultrafast Extraction and Multi-Dimensional High-Performance Affinity Chromatography†

    PubMed Central

    Zheng, Xiwei; Yoo, Michelle J.; Hage, David S.

    2013-01-01

    A multi-dimensional chromatographic approach was developed to measure the free fractions of drug enantiomers in samples that also contained a binding protein or serum. This method, which combined ultrafast affinity extraction with a chiral stationary phase, was demonstrated using the drug warfarin and the protein human serum albumin. PMID:23979112

  20. Succinonitrile Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The Succinonitrile (SCN) Purification Facility provides succinonitrile and succinonitrile alloys to several NRA selected investigations for flight and ground research at various levels of purity. The purification process employed includes both distillation and zone refining. Once the appropriate purification process is completed, samples are characterized to determine the liquidus and/or solidus temperature, which is then related to sample purity. The lab has various methods for measuring these temperatures with accuracies in the milliKelvin to tenths of milliKelvin range. The ultra-pure SCN produced in our facility is indistinguishable from the standard material provided by NIST to well within the stated +/- 1.5mK of the NIST triple point cells. In addition to delivering material to various investigations, our current activities include process improvement, characterization of impurities and triple point cell design and development. The purification process is being evaluated for each of the four vendors to determine the efficacy of each purification step. We are also collecting samples of the remainder from distillation and zone refining for analysis of the constituent impurities. The large triple point cells developed will contain SCN with a melting point of 58.0642 C +/- 1.5mK for use as a calibration standard for Standard Platinum Resistance Thermometers (SPRTs).

  1. A fast and easy strategy for protein purification using “teabags”

    PubMed Central

    Castaldo, M.; Barlind, L.; Mauritzson, F.; Wan, P. T.; Snijder, H. J.

    2016-01-01

    Protein purification often involves affinity capture of proteins on stationary resin, alternatively proteins are captured on free flowing resin for subsequent separation from bulk fluid. Both methods require labour and time intensive separation of particulate matter from fluid. We present a method where affinity resin is contained within porous-walled containers, supporting clarification, product recovery, and concentration in a single step with minimal hands-on processing time, without significant investments in equipment. PMID:27356497

  2. Expression and purification of recombinant antibody formats and antibody fusion proteins.

    PubMed

    Siegemund, Martin; Richter, Fabian; Seifert, Oliver; Unverdorben, Felix; Kontermann, Roland E

    2014-01-01

    In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains. PMID:24515473

  3. Purification of genuine multipartite entanglement

    SciTech Connect

    Huber, Marcus; Plesch, Martin

    2011-06-15

    In tasks where multipartite entanglement plays a central role, state purification is, due to inevitable noise, a crucial part of the procedure. We consider a scenario exploiting the multipartite entanglement in a straightforward multipartite purification algorithm and compare it to bipartite purification procedures combined with state teleportation. While complete purification requires an infinite amount of input states in both cases, we show that for an imperfect output fidelity the multipartite procedure exhibits a major advantage in terms of input states used.

  4. 40 CFR 1065.267 - Gas chromatograph.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 33 2011-07-01 2011-07-01 false Gas chromatograph. 1065.267 Section 1065.267 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS ENGINE-TESTING PROCEDURES Measurement Instruments Hydrocarbon Measurements § 1065.267 Gas...

  5. 40 CFR 1065.267 - Gas chromatograph.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Gas chromatograph. 1065.267 Section 1065.267 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS ENGINE-TESTING PROCEDURES Measurement Instruments Hydrocarbon Measurements § 1065.267 Gas...

  6. Nano-fabricated size exclusion chromatograph

    NASA Technical Reports Server (NTRS)

    Svehla, D.; Feldman, S.; Feldman, J.; Grunthaner, F.; Shakkottai, P.; dle Castillo, L.; White, V.

    2002-01-01

    This poster describes the development of a nano-fabricated size exclusion chromatograph (nSEC) based on the principle that molecules traveling through a microcolumn containing nano-fabricated features will have characteristics elution times that directly correlate to molecular weight.

  7. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  8. Comparative oxygen affinity of fish and mammalian myoglobins.

    PubMed

    Nichols, J W; Weber, L J

    1989-01-01

    Myoglobins from rat, coho salmon (Oncorhynchus kisutch), buffalo sculpin (Enophrys bison) hearts, and yellowfin tuna (Thunnus albacares) red skeletal muscle were partially purified and their O2 binding affinities determined. Commercially prepared sperm whale myoglobin was employed as an internal standard. Tested at 20 degrees C, myoglobins from salmon and sculpin bound O2 with lower affinity than myoglobins from the rat or sperm whale. Oxygen binding studies at 12 degrees C and 37 degrees C suggest that this difference is adaptive, permitting myoglobins from cold-adapted fish to function at physiologically relevant temperatures. Taken together, purification and O2 binding data obtained in this study reveal a previously unrecognized diversity of myoglobin structure and function. PMID:2760286

  9. High Throughput Identification, Purification and Structural Characterization of Water Soluble Protein Complexes in Desulfovibrio vulgaris

    SciTech Connect

    Dong,, Ming; Han, Bong-Gyoon; Liu, Hui-Hai; Malik, J.; Geller, Jil; Yang, Li; Choi, M.; Chandonia, John-Marc; Arbelaez, Pablo; Sterling, H. J.; Typke, Dieter; Shatsky, Max; Brenner, Steve; Fisher, Susan; Williams, Evan; Szakal, Evelin; Allen, S.; Hall, S. C.; Hazen, Terry; Witkowska, H. E.; Jin, Jiming; Glaeser, Robert; Biggin, Mark

    2010-05-17

    Our scheme for the tagless purification of water soluble complexes. 10 g of protein from a crude bacterial extract is first fractionated by ammonium sulfate precipitation and then by a series of chromatographic steps: anion exchange (IEX), hydrophobic interaction (HIC), and finally size exclusion (Gel Filtration). Fractions from the last chromatography step are trypsin digested and peptides labeled with iTRAQ reagents to allow multiplexing and quantitation during mass spectrometric analysis. Elution profiles of identified proteins are then subjected to clustering analysis.

  10. A Small-Scale Low-Cost Gas Chromatograph

    ERIC Educational Resources Information Center

    Gros, Natasa; Vrtacnik, Margareta

    2005-01-01

    The design and application of a small-scale portable gas chromatograph for learning of the basic concepts of chromatography is described. The apparatus consists of two basic separable units, which includes a chromatographic unit and an electronic unit.

  11. Method to fabricate silicon chromatographic column comprising fluid ports

    DOEpatents

    Manginell, Ronald P.; Frye-Mason, Gregory C.; Heller, Edwin J.; Adkins, Douglas R.

    2004-03-02

    A new method for fabricating a silicon chromatographic column comprising through-substrate fluid ports has been developed. This new method enables the fabrication of multi-layer interconnected stacks of silicon chromatographic columns.

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

    PubMed Central

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

    2011-01-01

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

  13. Bringing the excitement and motivation of research to students; Using inquiry and research-based learning in a year-long biochemistry laboratory: Part I-guided inquiry-purification and characterization of a fusion protein: Histidine tag, malate dehydrogenase, and green fluorescent protein.

    PubMed

    Knutson, Kristopher; Smith, Jennifer; Wallert, Mark A; Provost, Joseph J

    2010-09-01

    A successful laboratory experience provides the foundation for student success, creating active participation in the learning process. Here, we describe a new approach that emphasizes research, inquiry and problem solving in a year-long biochemistry experience. The first semester centers on the purification, characterization, and analysis of a novel fusion protein within a guided research experience. Throughout the semester, students gradually acquire skills as they are allowed to work independently. A fusion protein, malate dehydrogenase-green fluorescent protein with a histidine affinity tag (MGH), is used throughout the semester. The fusion protein allows for a high throughput analysis and is stable for duration of the semester. Students start with the purification and analysis of the plasmid DNA and end with an enzymatic analysis of MGH. As students take ownership of their experiments and choose two different chromatographic resins, they make many choices throughout the semester. Skills, motivation, confidence levels, and attitudes were assessed before and after the semester. Students achieved high levels of critical biochemical laboratory skills and critical thinking while increasing their confidence and motivation for working in a biochemical research setting. PMID:21567851

  14. Water purification in Borexino

    SciTech Connect

    Giammarchi, M.; Balata, M.; Ioannucci, L.; Nisi, S.; Goretti, A.; Ianni, A.; Miramonti, L.

    2013-08-08

    Astroparticle Physics and Underground experiments searching for rare nuclear events, need high purity materials to act as detectors or detector shielding. Water has the advantage of being cheap, dense and easily available. Most of all, water can be purified to the goal of obatining a high level of radiopurity. Water Purification can be achieved by means of a combination of processes, including filtration, reverse osmosis, deionization and gas stripping. The Water Purification System for the Borexino experiment, will be described together with its main performances.

  15. Purification of triolein for use in semipermeable membrane devices (SPMDs)

    USGS Publications Warehouse

    Lebo, J.A.; Almeida, F.V.; Cranor, W.L.; Petty, J.D.; Huckins, J.N.; Rastall, A.; Alvarez, D.A.; Mogensen, B.B.; Johnson, B.T.

    2004-01-01

    Analyses of triolein-containing semipermeable membrane devices (SPMDs) have sometimes been impeded by interferences caused by impurities endemic to triolein that codialyze with the analytes. Oleic acid and methyl oleate have been the most troublesome of these impurities because of their relatively high concentrations in triolein and because significant residues of both can persist even after size exclusion chromatographic (SEC) fractionation. These residues have also been blamed for false-positive signals during bioindicator testing of SPMD dialysates. To prevent these problems, a simple, cost-effective procedure was developed for purifying triolein destined for use in SPMDs: the bulk triolein is repeatedly (6x) partitioned against methanol. Tests of the procedure show that 14C-oleic acid is completely removed from the triolein. After SEC fractionation, dialysates of standard-size SPMDs made with the purified triolein contain less than 5 ??g of methyl oleate as compared to sometimes more than 500 ??g for dialysates (also after SEC) of SPMDs made with unpurified triolein. Gas chromatographic analyses with flame ionization and electron capture detection show that the purification treatment also greatly reduces the number and size of peaks caused by unidentified contaminants in the triolein. Microtox basic assay of dialysates of SPMDs shows that those made with the purified triolein have lower acute toxicities than dialysates of SPMDs made with unpurified triolein. Yeast estrogen screen (YES) testing of SPMDs fabricated with unpurified and purified triolein demonstrates that the purification process removes all background estrogenic activity. Published by Elsevier Ltd.

  16. Purification and properties of cytosolic malic enzyme from human skeletal muscle.

    PubMed

    Taroni, F; Di Donato, S

    1988-01-01

    1. An NADP+-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for L-malate and NADP+ were 0.246 mM and 5.8 microM, and 0.304 mM and 5.8 microM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 microM and 22.2 mM, respectively. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. The following physical parameters were established: s0(20.w) = 10.48, Stokes' radius = 5.61 nm, pI = 5.72 Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f/fo = 1.33 by combining the chromatographic data with the sedimentation measurements. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme. PMID:3169368

  17. Purification of the neurotensin receptor from bovine brain

    SciTech Connect

    Mills, A.; Demoliou-Mason, C.D.; Barnard, E.A.

    1988-01-05

    The neurotensin receptor protein, solubilized with digitonin/asolectin from bovine cerebral cortex membranes, was purified to apparent homogeneity by affinity chromatography using immobilized neurotensin. The product exhibits saturable and specific binding of (3,11-tyrosyl-3,5-/sup 3/H) neurotensin with an apparent affinity (K/sub d/ = 5.5 nM) comparable to that measured in intact membranes and crude soluble extracts. The affinity-purified material, after reduction with 100 mM dithiothreitol, in denaturing gel electrophoresis showed a single polypeptide of M/sub r/ 72,000. Under nonreducing conditions the apparent M/sub r/, however, was 50,000, suggesting the presence of intramolecular disulfide bonds. The purified neurotensin receptor was judged to be homogenous, in that (i) only a single polypeptide was detectable; and (ii) the overall purification was 30,000-50,000-fold, giving a specific neurotensin-binding activity close to the theoretical maximum.

  18. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

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

  19. Qualitative assessment of IC50 values of inhibitors of the neuronal nicotinic acetylcholine receptor using a single chromatographic experiment and multivariate cluster analysis.

    PubMed

    Jozwiak, Krzysztof; Moaddel, Ruin; Yamaguchi, Rika; Ravichandran, Sarangan; Collins, Jack R; Wainer, Irving W

    2005-05-01

    It has been widely demonstrated that affinity chromatography can be used to derive binding affinities, and that these affinities can be correlated to data obtained using standard techniques such as membrane binding, ultrafiltration and equilibrium dialysis. The purpose of this study is to evaluate the use of immobilized nicotinic acetylcholine receptor stationary phase in chromatographic experiments to assess the functional activity of series of noncompetitive inhibitors (NCIs) as reflected in their IC50 values. Chromatographically determined retention values and computer generated molecular descriptors were obtained for 29 compounds and the data were analyzed by cluster analysis. The approach qualitatively ranked the test compounds as efficient NCIs (low IC50 values) or poor NCIs (high IC50 values). The data obtained with the 29 compounds used in this study demonstrate that the experimental approach had been able to place 25 of these compounds in the correct IC(50) clusters. To our knowledge, this is the first relationship established between chromatographic retention and IC50 for membrane-bound receptors. These results suggest that the chromatographic approach may be useful in development of lead drug candidates including the determination of off-target binding. PMID:15797535

  20. Chromatographic fingerprint analysis of Pycnogenol dietary supplements.

    PubMed

    Chen, Pei; Song, Fenhong; Lin, Long-Ze

    2009-01-01

    The bark of maritime pine (Pinus pinaster Aiton) has been widely used as a remedy for various degenerative diseases. A standard high-performance liquid chromatographic (HPLC) procedure for Pycnogenol analysis is a method specified in the United States Pharmacopeia (USP) monograph, which requires measurement of peak areas and identification of four components of the extract: caffeic acid, catechin, ferulic acid, and taxifolin. In this study, a fingerprint analysis using an HPLC method based on the USP monograph has been developed to provide additional qualitative information for the analysis of Pycnogenol-containing dietary supplements (PDS). Twelve commercially available PDS samples were purchased and analyzed along with a standard Pycnogenol extract. Their chromatographic fingerprints were analyzed using principal component analysis. The results showed that two of the samples were not consistent with the standard reference Pycnogenol extract. One contained other active ingredients in addition to Pycnogenol, and the other may have resulted from a quality control issue in manufacturing. PMID:19485224

  1. A nonlinear model for gas chromatograph systems

    NASA Technical Reports Server (NTRS)

    Feinberg, M. P.

    1975-01-01

    Fundamental engineering design techniques and concepts were studied for the optimization of a gas chromatograph-mass spectrometer chemical analysis system suitable for use on an unmanned, Martian roving vehicle. Previously developed mathematical models of the gas chromatograph are found to be inadequate for predicting peak heights and spreading for some experimental conditions and chemical systems. A modification to the existing equilibrium adsorption model is required; the Langmuir isotherm replaces the linear isotherm. The numerical technique of Crank-Nicolson was studied for use with the linear isotherm to determine the utility of the method. Modifications are made to the method eliminate unnecessary calculations which result in an overall reduction of the computation time of about 42 percent. The Langmuir isotherm is considered which takes into account the composition-dependent effects on the thermodynamic parameter, mRo.

  2. Micro-column plasma emission liquid chromatograph

    DOEpatents

    Gay, Don D.

    1984-01-01

    In a direct current plasma emission spectrometer for use in combination with a micro-column liquid chromatograph, an improved plasma source unit. The plasma source unit includes a quartz capillary tube having an inlet means, outlet off gas means and a pair of spaced electrodes defining a plasma region in the tube. The inlet means is connected to and adapted to receive eluant of the liquid chromatograph along with a stream of plasma-forming gas. There is an opening through the wall of the capillary tube penetrating into the plasma region. A soft glass capillary light pipe is disposed at the opening, is connected to the spectrometer, and is adapted to transmit light passing from the plasma region to the spectrometer. There is also a source of electromotive force connected to the electrodes sufficient to initiate and sustain a plasma in the plasma region of the tube.

  3. Flow in a metal hydride chromatographic column

    SciTech Connect

    Nichols, G.S.

    1990-01-01

    The flow of hydrogen isotopes in a metal hydride chromatographic column is calculated by a one-dimensional finite difference method. The Ergun equation is used to define the gas flow; and equilibrium pressure isotherms are used to define the column holdup. Solid phase loadings are shown to move as a wave front on absorption, but remain more uniform on desorption. 3 refs., 4 figs.

  4. Nano-fabricated size exclusion chromatograph

    NASA Technical Reports Server (NTRS)

    Svehla, D.; Feldman, S.; Feldman, J.; Grunthaner, F.; Shakkottai, P.; Castillo, L. del; White, V.

    2002-01-01

    This paper describes the development of a nano-fabricated size exclusion chromatograph (nSEC) based on the principle that molecules traveling through amicrocolumn containing nano-fabricated features will have characteristic elution times that directly correlate to molecular weight. Compared to conventional size exclusion chromatography, the nSEC offers greater control over the size exclusion process; mass fabrication; integration of the separation column with associated valves, pumps, and detectors; and dramatic reductions in instrument mass and power requirements.

  5. Purification of biomaterials by phase partitioning

    NASA Technical Reports Server (NTRS)

    Harris, J. M.

    1984-01-01

    A technique which is particularly suited to microgravity environments and which is potentially more powerful than electrophoresis is phase partitioning. Phase partitioning is purification by partitioning between the two immiscible aqueous layers formed by solution of the polymers poly(ethylene glycol) and dextran in water. This technique proved to be very useful for separations in one-g but is limited for cells because the cells are more dense than the phase solutions thus tend to sediment to the bottom of the container before reaching equilibrium with the preferred phase. There are three phases to work in this area: synthesis of new polymers for affinity phase partitioning; development of automated apparatus for ground-based separations; and design of apparatus for performing simple phase partitioning space experiments, including examination of mechanisms for separating phases in the absence of gravity.

  6. Purification and characterization of the Oligosaccharyl transferase

    SciTech Connect

    Kapoor, T.M.

    1990-11-01

    Oligosaccharyl transferase was characterized to be a glycoprotein with at least one saccharide unit that had a D-manno or D- glucopyranose configuration with unmodified hydroxy groups at C-3, C-4 and C-6, using a Concanavalin A affinity column. This afforded a 100 fold increase in the transferase purity in the solubilized microsomal sample and also removed over 90% of the microsomal proteins (the cytosolic ones being removed before solubilization). The detergent, N,N-Dimethyldodecylamine N-oxide (LDAO) was used for solubilization and it yielded a system compatible with the assay and the purification steps. An efficient method for detergent extraction without dilution of sample or protein precipitation was also developed.

  7. Investigation of purification process stresses on erythropoietin peptide mapping profile

    PubMed Central

    Sepahi, Mina; Kaghazian, Hooman; Hadadian, Shahin; Norouzian, Dariush

    2015-01-01

    Background: Full compliance of recombinant protein peptide mapping chromatogram with the standard reference material, is one of the most basic quality control tests of biopharmaceuticals. Changing a single amino acid substitution or side chain diversity for a given peptide changes protein hydrophobicity and causes peak shape or retention time alteration in a peptide mapping assay. In this work, the effect of different stresses during the recombinant erythropoietin (EPO) purification process, including pH 4, pH 5, and room temperature were checked on product peptide mapping results. Materials and Methods: Cell culture harvest was purified under stress by different chromatographic techniques consisting of gel filtration, anionic ion exchange, concentration by ultrafiltration, and high resolution size exclusion chromatography. To induce more pH stresses, the purified EPO was exposed to pH stress 4 and 5 by exchanging buffer by a 10 KDa dialysis sac overnight. The effects of temperature and partial deglycosylation (acid hydrolysis) on purified EPO were also studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping analysis. Removal of sialic acid by mild hydrolysis was performed by exposure to two molar acetic acid at 80°C for 3 h. Results: No significant effect was observed between intact and stressed erythropoietin peptide mapping profiles and SDS-PAGE results. To validate the sensibility of the technique, erythropoietin was partially acid hydrolyzed and significant changes in the chromatographic peptide map of the intact form and a reduction on its molecular weight were detected, which indicates some partial deglycosylation. Conclusions: Purification process does not alter the peptide mapping profile and purification process stresses are not the cause of peptide mapping noncompliance. PMID:26261816

  8. Native Purification and Analysis of Long RNAs

    PubMed Central

    Chillón, Isabel; Marcia, Marco; Legiewicz, Michal; Liu, Fei; Somarowthu, Srinivas; Pyle, Anna Marie

    2015-01-01

    The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation–renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2′-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing. PMID:26068736

  9. A semi-automated method for purification of milligram quantities of proteins on the QIAcube

    PubMed Central

    McGraw, J; Tatipelli, VK; Traore, MC; Feyijinmi, O; Eangoor, P; Lane, S; Stollar, EJ

    2015-01-01

    A growing number of studies require the purification of multiple proteins simultaneously and the development of simple economical high-throughput purification methods is essential. We have tested the purification of two related proteins in a variety of conditions to benchmark the semi-automated affinity chromatography method for the QIAcube that we have developed. We find that this new QIAcube method can successfully purify milligram quantities of proteins with minimal user involvement and performs as well as methods based on gravity. The method could easily be adapted to other chromatography resins and should prove to be a versatile method for optimizing protein expression or purification conditions for multiple proteins while obtaining sufficient amounts for subsequent biochemical analyses. PMID:24508590

  10. Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds.

    PubMed

    Roy; Sardar; Gupta

    2000-07-01

    Two polysaccharides, alginate and chitosan, showed unusual affinity and bound alpha-amylase (from various sources) and Aspergillus niger cellulase, respectively. The beads prepared from these polymers were successfully used for the purification of the respective enzymes by fluidized bed affinity chromatography. alpha-amylase from wheat germ could be purified by 58-fold with about 90% recovery of activity. Aspergillus niger cellulase, on the other hand, was purified by 30-fold with 80% recovery of enzyme activity. Both purified preparations show single band on SDS-PAGE. PMID:10862902

  11. Purification of large plasmids with methacrylate monolithic columns.

    PubMed

    Krajnc, Nika Lendero; Smrekar, Franci; Cerne, Jasmina; Raspor, Peter; Modic, Martina; Krgovic, Danijela; Strancar, Ales; Podgornik, Ales

    2009-08-01

    The rapid evolution of gene therapy and DNA vaccines results in an increasing interest in producing large quantities of pharmaceutical grade plasmid DNA. Most current clinical trials involve plasmids of 10 kb or smaller in size, however, future requirements for multigene vectors including extensive control regions may require the production of larger plasmids, e. g., 20 kb and bigger. The objective of this study was to examine certain process conditions for purification of large plasmids with the size of up to 93 kb. Since there is a lack of knowledge about production and purification of bigger plasmid DNA, cell lysis and storage conditions were investigated. The impact of chromatographic system and methacrylate monolithic column on the degradation of plasmid molecules under nonbinding conditions at different flow rates was studied. Furthermore, capacity measurements varying salt concentration in loading buffer were performed and the capacities up to 13 mg of plasmid per mL of the monolithic column were obtained. The capacity flow independence in the range from 130 to 370 cm/h was observed. Using high resolution monolithic column the separation of linear and supercoiled isoforms of large plasmids was obtained. Last but not least, since the baseline separation of RNA and pDNA was achieved, the one step purification on larger CIM DEAE 8 mL tube monolithic column was performed and the fractions were analyzed by CIM analytical monolithic columns. PMID:19598166

  12. Water Purification Systems

    NASA Technical Reports Server (NTRS)

    1992-01-01

    A water purification/recycling system developed by Photo-Catalytics, Inc. (PCI) for NASA is commercially available. The system cleanses and recycles water, using a "photo-catalysis" process in which light or radiant energy sparks a chemical reaction. Chemically stable semiconductor powders are added to organically polluted water. The powder absorbs ultraviolet light, and pollutants are oxidized and converted to carbon dioxide. Potential markets for the system include research and pharmaceutical manufacturing applications, as well as microchip manufacture and wastewater cleansing.

  13. Probabilistic theories with purification

    SciTech Connect

    Chiribella, Giulio; D'Ariano, Giacomo Mauro; Perinotti, Paolo

    2010-06-15

    We investigate general probabilistic theories in which every mixed state has a purification, unique up to reversible channels on the purifying system. We show that the purification principle is equivalent to the existence of a reversible realization of every physical process, that is, to the fact that every physical process can be regarded as arising from a reversible interaction of the system with an environment, which is eventually discarded. From the purification principle we also construct an isomorphism between transformations and bipartite states that possesses all structural properties of the Choi-Jamiolkowski isomorphism in quantum theory. Such an isomorphism allows one to prove most of the basic features of quantum theory, like, e.g., existence of pure bipartite states giving perfect correlations in independent experiments, no information without disturbance, no joint discrimination of all pure states, no cloning, teleportation, no programming, no bit commitment, complementarity between correctable channels and deletion channels, characterization of entanglement-breaking channels as measure-and-prepare channels, and others, without resorting to the mathematical framework of Hilbert spaces.

  14. High-pressure liquid chromatographic gradient mixer

    DOEpatents

    Daughton, C.G.; Sakaji, R.H.

    1982-09-08

    A gradient mixer effects the continuous mixing of any two miscible solvents without excessive decay or dispersion of the resultant isocratic effluent or of a linear or exponential gradient. The two solvents are fed under low or high pressure by means of two high performance liquid chromatographic pumps. The mixer comprises a series of ultra-low dead volume stainless steel tubes and low dead volume chambers. The two solvent streams impinge head-on at high fluxes. This initial nonhomogeneous mixture is then passed through a chamber packed with spirally-wound wires which cause turbulent mixing thereby homogenizing the mixture with minimum band-broadening.

  15. High pressure liquid chromatographic gradient mixer

    DOEpatents

    Daughton, Christian G.; Sakaji, Richard H.

    1985-01-01

    A gradient mixer which effects the continuous mixing of any two miscible solvents without excessive decay or dispersion of the resultant isocratic effluent or of a linear or exponential gradient. The two solvents are fed under low or high pressure by means of two high performance liquid chromatographic pumps. The mixer comprises a series of ultra-low dead volume stainless steel tubes and low dead volume chambers. The two solvent streams impinge head-on at high fluxes. This initial nonhomogeneous mixture is then passed through a chamber packed with spirally-wound wires which cause turbulent mixing thereby homogenizing the mixture with minimum "band-broadening".

  16. Hand-held multiple system gas chromatograph

    DOEpatents

    Yu, Conrad M.

    2001-01-01

    A multiple parallel hand-held gas chromatograph (GC) system which includes several independent GCs. Each independent GC has its own injector, separation column, detector and oven and the GCs are mounted in a light weight hand-held assembly. Each GC operates independently and simultaneously. Because of different coatings in different separation columns, different retention times for the same gas will be measured. Thus, for a GC system with multiple parallel GCs, the system can measure, in a short period, different retention times and provide a cross-reference in the determination of the measured gas and to become a two-dimensional system for direct field use.

  17. Development of Gas Chromatographic Mass Spectrometry.

    PubMed

    Hites, Ronald A

    2016-07-19

    Gas chromatographic mass spectrometry is now widely used for the quantitation and identification of organic compounds in almost any imaginable sample. These applications include the measurement of chlorinated dioxins in soil samples, the identification of illicit drugs in human blood, and the quantitation of accelerants in arson investigations, to name just a few. How did GC/MS get so popular? It turns out that it required parallel developments in mass spectrometry, gas chromatography, and computing and that no one person "invented" the technique. This Perspective traces this history from the 1950s until today. PMID:27384908

  18. Purification of the functional plant membrane channel KAT1

    SciTech Connect

    Hibi, Takao Aoki, Shiho; Oda, Keisuke; Munemasa, Shintaro; Ozaki, Shunsuke; Shirai, Osamu; Murata, Yoshiyuki; Uozumi, Nobuyuki

    2008-09-26

    The inward-rectifying K{sup +} channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K{sup +} channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a 'test set' of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.

  19. Crystal structures of fusion proteins with large-affinity tags.

    PubMed

    Smyth, Douglas R; Mrozkiewicz, Marek K; McGrath, William J; Listwan, Pawel; Kobe, Bostjan

    2003-07-01

    The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest. PMID:12824478

  20. A novel and effective chromatographic approach to the separation of isoflavone derivatives from Pueraria lobata.

    PubMed

    Fu, Jiang; Jing, Wenguang; Wang, Weihao; Chen, Sha; Zhang, Jun; Liu, An

    2015-01-01

    A novel and effective chromatographic approach to the separation and purification of isoflavone compounds from Pueraria lobata is described. The method is based on flash chromatography (FC), coupled to preparative high performance liquid chromatography (prep-HPLC) via a six-way valve. The FC step comprised tandem reversed phase columns, pre-packed with MCI gel (Mitsubishi Chemical Corp., Tokyo, Japan) and C18 (Fuji Silysia Chemical Ltd, Osaka, Japan) resin, respectively, and was designed to separate a crude Pueraria lobata extract into several preliminary fractions. Fractions containing the target compounds were then directly injected via the six-way valve into prep-HPLC columns, without further treatment, for final isolation and purification. Nine isoflavonoids were successfully isolated, three through an online mode and the other six through an offline mode. The purities of all compounds exceeded 95.0%, as determined by HPLC with an UV-vis photodiode array detector. The convenience, low solvent consumption, and time-saving advantages of this method offer an attractive and promising approach to the isolation of natural products. PMID:25751785

  1. 32P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase.

    PubMed

    Reddy, M V; Bleicher, W T; Blackburn, G R

    1991-04-01

    Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive 32P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO4). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO4-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO4 selectively forms cis-Tg adducts. With OsO4-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO4-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2025496

  2. sup 32 P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase

    SciTech Connect

    Reddy, M.V.; Bleicher, W.T.; Blackburn, G.R. )

    1991-04-01

    Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive {sup 32}P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO{sub 4}). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO{sub 4}-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO{sub 4} selectively forms cis-Tg adducts. With OsO{sub 4}-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO{sub 4}-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.

  3. High performance hand-held gas chromatograph

    SciTech Connect

    Yu, C.M.

    1998-04-28

    The Microtechnology Center of Lawrence Livermore National Laboratory has developed a high performance hand-held, real time detection gas chromatograph (HHGC) by Micro-Electro-Mechanical-System (MEMS) technology. The total weight of this hand-held gas chromatograph is about five lbs., with a physical size of 8{close_quotes} x 5{close_quotes} x 3{close_quotes} including carrier gas and battery. It consumes about 12 watts of electrical power with a response time on the order of one to two minutes. This HHGC has an average effective theoretical plate of about 40k. Presently, its sensitivity is limited by its thermal sensitive detector at PPM. Like a conventional G.C., this HHGC consists mainly of three major components: (1) the sample injector, (2) the column, and (3) the detector with related electronics. The present HHGC injector is a modified version of the conventional injector. Its separation column is fabricated completely on silicon wafers by means of MEMS technology. This separation column has a circular cross section with a diameter of 100 pm. The detector developed for this hand-held GC is a thermal conductivity detector fabricated on a silicon nitride window by MEMS technology. A normal Wheatstone bridge is used. The signal is fed into a PC and displayed through LabView software.

  4. Versatile gas/particle ion chromatograph.

    PubMed

    Ullah, S M Rahmat; Takeuchi, Masaki; Dasgupta, Purnendu K

    2006-02-01

    A new, compact gas/particle ion chromatograph has been developed for measuring ionic constituents in PM2.5 (particulate matter of aerodynamic diameter < or = 2.5 microm) and water-soluble ionogenic gases. The instrument has separate sampling channels for gases and particles. In one, a membrane denuder collects soluble gases for preconcentration and analysis. In the other, a cyclone removes larger particles, a membrane denuder removes soluble gases, and a continuously wetted hydrophilic filter collects particles. A single, multiport, syringe pump handles liquid transport, and one conductivity detector measures anions and ammonium for both channels. Electrodialytically generated gradient hydroxide eluent permits 20 min chromatographic runs. Gas/particle samples are each collected for 40 min, butthe sampling intervals are staggered by 20 min. Liquid samples from the gas denuder and particle collector are aspirated and preconcentrated on sequential cation and anion concentrators and transferred respectively to an ammonia transfer device and an anion separation column. The flow configuration results in an ammonium peak before anion peaks in the chromatogram. The system measures ammonia, organic acids (such as acetic, formic, and oxalic acids), HCl, HONO, SO2, HNO3, and the corresponding ions in the aerosol phase. Low ng/m3 to sub-ng/m3 limits of detection (LODs) are attained for most common gases and particulate constituents, the LODs for gaseous SO2 to NH3 range, for example, from sub parts per trillion by volume (sub-pptv) to approximately 5 pptv. PMID:16509343

  5. Estimating optimal time for fast chromatographic separations.

    PubMed

    Welch, Christopher J; Regalado, Erik L

    2014-09-01

    The term t(min cc) provides a ready estimate of the shortest time that can be obtained by "column cutting" for baseline resolution of two components showing excess chromatographic resolution. While actual column cutting is impractical, the t(min cc) value is shown to be closely related to the minimum separation time obtainable by adjusting other parameters such as flow rate, mobile phase composition, and temperature, affording scientists interested in the development of fast chromatographic separations a convenient tool for estimating the minimum separation time that can be obtained by modifying a given method development screening result. Furthermore, the relationship between t(min cc) and the minimum separation time obtainable by adjusting other parameters is shown to be dependent on the speed of the screening method, with aggressive screening gradients affording t(min cc) estimates that match the actual minimum separation time, and "lazy" screening gradients affording t(min cc) values that overestimate minimum separation time. Consequently, the analysis of the relationship between t(min cc) and actual minimum separation time may be a useful tool for determining the "fitness" of method development screening methods. PMID:24995384

  6. Process for purification of silicon

    NASA Technical Reports Server (NTRS)

    Rath, H. J.; Sirtl, E.; Pfeiffer, W.

    1981-01-01

    The purification of metallurgically pure silicon having a silicon content of more than 95% by weight is accomplished by leaching with an acidic solution which substantially does not attack silicon. A mechanical treatment leading to continuous particle size reduction of the granulated silicon to be purified is combined with the chemical purification step.

  7. Oxygen Sag and Stream Purification.

    ERIC Educational Resources Information Center

    Neal, Larry; Herwig, Roy

    1978-01-01

    Presents a literature review of water quality related to oxygen sag and stream purification, covering publications of 1976-77. This review includes: (1) self-purification models; (2) oxygen demand; and (3) reaeration and oxygen transfer. A list of 60 references is also presented. (HM)

  8. DNA PURIFICATION BY POLYCARBONATE FILTERS

    EPA Science Inventory

    Organic solvent free procedure s are described for the purification of mammalian DNA from rat liver, kidney, spleen, lung and brain. he basis of the purification procedures are the use of the detergent sodium dodecyl sulfate (SDS) and inert polycarbonate filters with 2 um pores w...

  9. Chromatographic biopanning for the selection of peptides with high specificity to Pb2+ from phage displayed peptide library.

    PubMed

    Nian, Rui; Kim, Duck Sang; Nguyen, Thuong; Tan, Lihan; Kim, Chan-Wha; Yoo, Ik-Keun; Choe, Woo-Seok

    2010-09-17

    Toxic heavy metal pollution is a global problem occurring in air, soil as well as water. There is a need for a more cost effective, renewable remediation technique, but most importantly, for a recovery method that is selective for one specific metal of concern. Phage display technology has been used as a powerful tool in the discovery of peptides capable of exhibiting specific affinity to various metals or metal ions. However, traditional phage display is mainly conducted in batch mode, resulting in only one equilibrium state hence low-efficiency selection. It is also unable to monitor the selection process in real time mode. In this study, phage display technique was incorporated with chromatography procedure with the use of a monolithic column, facilitating multiple phage-binding equilibrium states and online monitoring of the selection process in search of affinity peptides to Pb2+. In total, 17 candidate peptides were found and their specificity toward Pb2+ was further investigated with bead-based enzyme immunoassay (EIA). A highly specific Pb2+ binding peptide ThrAsnThrLeuSerAsnAsn (TNTLSNN) was obtained. Based on our knowledge, this is the first report on a new chromatographic biopanning method coupled with monolithic column for the selection of metal ion specific binding peptides. It is expected that this monolith-based chromatographic biopanning will provide a promising approach for a high throughput screening of affinity peptides cognitive of a wide range of target species. PMID:20709321

  10. An automated multidimensional preparative gas chromatographic system for isolation and enrichment of trace amounts of xenon from ambient air.

    PubMed

    Larson, Tuula; Östman, Conny; Colmsjö, Anders

    2011-04-01

    The monitoring of radioactive xenon isotopes is one of the principal methods for the detection of nuclear explosions in order to identify clandestine nuclear testing. In this work, a miniaturized, multiple-oven, six-column, preparative gas chromatograph was constructed in order to isolate trace quantities of radioactive xenon isotopes from ambient air, utilizing nitrogen as the carrier gas. The multidimensional chromatograph comprised preparative stainless steel columns packed with molecular sieves, activated carbon, and synthetic carbon adsorbents (e.g., Anasorb®-747 and Carbosphere®). A combination of purification techniques--ambient adsorption, thermal desorption, back-flushing, thermal focusing, and heart cutting--was selectively optimized to produce a well-defined xenon peak that facilitated reproducible heart cutting and accurate quantification. The chromatographic purification of a sample requires approximately 4 h and provides complete separation of xenon from potentially interfering components (such as water vapor, methane, carbon dioxide, and radon) with recovery and accuracy close to 100%. The preparative enrichment process isolates and concentrates a highly purified xenon gas fraction that is suitable for subsequent ultra-low-level γ-, ß/γ-spectroscopic or high-resolution mass spectrometric measurement (e.g., to monitor the gaseous fission products of nuclear explosions at remote locations). The Xenon Processing Unit is a free-standing, relatively lightweight, and transportable system that can be interfaced to a variety of sampling and detection systems. It has a relatively inexpensive, rugged, and compact modular (19-inch rack) design that provides easy access to all parts for maintenance and has a low power requirement. PMID:21347675

  11. Water Purification Product

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Ecomaster, an affiliate of BioServe Space Technologies, this PentaPure technology has been used to purify water for our nation's Space Shuttle missions since 1981. WTC-Ecomaster of Mirneapolis, Minnesota manufactures water purification systems under the brand name PentaPure (TM). BioServe researcher Dr. George Marchin, of Kansas State University, first demonstrated the superiority of this technology and licensed it to WTC. Marchin continues to perform microgravity research in the development of new technologies for the benefit of life on Earth.

  12. Air/Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    After 18 years of research into air/water pollution at Stennis Space Center, Dr. B. C. Wolverton formed his own company, Wolverton Environmental Services, Inc., to provide technology and consultation in air and water treatment. Common houseplants are used to absorb potentially harmful materials from bathrooms and kitchens. The plants are fertilized, air is purified, and wastewater is converted to clean water. More than 100 U.S. communities have adopted Wolverton's earlier water hyacinth and artificial marsh applications. Catfish farmers are currently evaluating the artificial marsh technology as a purification system.

  13. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  14. Preparative two-dimensional liquid chromatography/mass spectrometry for the purification of complex pharmaceutical samples.

    PubMed

    Zhang, Yinong; Zeng, Lu; Pham, Catherine; Xu, Rongda

    2014-01-10

    A new preparative two-dimensional liquid chromatography/mass spectrometry system (2D LC-LC/MS) has been designed and implemented to enhance capability and resolving power for the separation and purification of pharmaceutical samples. The system was constructed by modifications of a conventional preparative LC/MS instrument with the addition of a set of switching valves and a sample loop, as well as interfacing a custom software program with MassLynx. The system integrates two chromatographic separations from the first and second dimensions into a single automated run to perform the purification of a target compound from a complex mixture without intermediate steps of sample preparation. The chromatography in the first dimension, operated in the heart-cutting mode, separates the target compound from the impurities by mass-triggered fractionation based on its molecular weight. This purified fraction from the first dimension is stored in the sample loop, and then gets transferred to the second column by using at-column dilution. A control software program, coined Prep 2D LCMS, was designed to integrate with MassLynx to retrieve data acquisition status. All of the chromatographic hardware components used in this preparative 2D LC-LC/MS system are from the original open access preparative LC/MS system, which has high level of robustness and affords easy and user-friendly operation. The new system is very versatile and capable of collecting multiple fractions with different masses under various purification modes as configured in the methods, such as conventional one-dimensional (1D) purification and/or 2D purification. This new preparative 2D LC-LC/MS system is therefore the ideal tool for medicinal chemistry lab in drug discovery environment. PMID:24309715

  15. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII. PMID:23244324

  16. Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

    SciTech Connect

    Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

    2012-04-01

    Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

  17. Modular microfluidics for point-of-care protein purifications.

    PubMed

    Millet, L J; Lucheon, J D; Standaert, R F; Retterer, S T; Doktycz, M J

    2015-04-21

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules. PMID:25740172

  18. Modular microfluidics for point-of-care protein purifications

    SciTech Connect

    Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; Retterer, S. T.; Doktycz, M. J.

    2015-01-01

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

  19. FIELD COMPARISON OF PORTABLE GAS CHROMATOGRAPHS WITH METHOD TO-14

    EPA Science Inventory

    A field-deployable prototype fast gas chromatograph (FGC) and two commercially-available portable gas chromatographs (PGC) were evaluated by measuring organic vapors in ambient air at a field monitoring site in metropolitan San Juan, Puerto Rico. he data were compared with simult...

  20. Gas-Chromatographic Determination Of Water In Freon PCA

    NASA Technical Reports Server (NTRS)

    Melton, Donald M.

    1994-01-01

    Gas-chromatographic apparatus measures small concentrations of water in specimens of Freon PCA. Testing by use of apparatus faster and provides greater protection against accidental contamination of specimens by water in testing environment. Automated for unattended operation. Also used to measure water contents of materials, other than Freon PCA. Innovation extended to development of purgeable sampling accessory for gas chromatographs.

  1. Gas Chromatographic Detectors for Exobiology Flight Experiments

    NASA Technical Reports Server (NTRS)

    Kojiro, Daniel R.; Humphry, Donald E.; Takeuchi, Nori; Chang, Sherwood (Technical Monitor)

    1997-01-01

    Exobiology flight experiments require highly sensitive instrumentation for in situ chemical analysis of the volatile chemical species that occur in the atmospheres and surfaces of various bodies within the solar system. The complex mixtures encountered place a heavy burden on the analytical instrumentation to detect and identify all species present. Future missions to Mars', comets, or planetary moons such as Europa, will perform experiments with complex analyses. In addition, instrumentation for such missions must perform under severely restricted conditions with limited resources. To meet these analytical requirements, improved methods and highly sensitive yet smaller instruments must continually be developed with increasingly greater capabilities. We describe here efforts to achieve this objective, for past and future missions, through the development of new or the improvement of existing sensitive, miniaturized gas chromatographic detectors.

  2. Interface for liquid chromatograph-mass spectrometer

    DOEpatents

    Andresen, Brian D.; Fought, Eric R.

    1989-01-01

    A moving belt interface for real-time, high-performance liquid chromatograph (HPLC)/mass spectrometer (MS) analysis which strips away the HPLC solvent as it emerges from the end of the HPLC column and leaves a residue suitable for mass-spectral analysis. The interface includes a portable, stand-alone apparatus having a plural stage vacuum station, a continuous ribbon or belt, a drive train magnetically coupled to an external drive motor, a calibrated HPLC delivery system, a heated probe tip and means located adjacent the probe tip for direct ionization of the residue on the belt. The interface is also capable of being readily adapted to fit any mass spectrometer.

  3. Interface for liquid chromatograph-mass spectrometer

    DOEpatents

    Andresen, B.D.; Fought, E.R.

    1989-09-19

    A moving belt interface is described for real-time, high-performance liquid chromatograph (HPLC)/mass spectrometer (MS) analysis which strips away the HPLC solvent as it emerges from the end of the HPLC column and leaves a residue suitable for mass-spectral analysis. The interface includes a portable, stand-alone apparatus having a plural stage vacuum station, a continuous ribbon or belt, a drive train magnetically coupled to an external drive motor, a calibrated HPLC delivery system, a heated probe tip and means located adjacent the probe tip for direct ionization of the residue on the belt. The interface is also capable of being readily adapted to fit any mass spectrometer. 8 figs.

  4. Multiscale peak alignment for chromatographic datasets.

    PubMed

    Zhang, Zhi-Min; Liang, Yi-Zeng; Lu, Hong-Mei; Tan, Bin-Bin; Xu, Xiao-Na; Ferro, Miguel

    2012-02-01

    Chromatography has been extensively applied in many fields, such as metabolomics and quality control of herbal medicines. Preprocessing, especially peak alignment, is a time-consuming task prior to the extraction of useful information from the datasets by chemometrics and statistics. To accurately and rapidly align shift peaks among one-dimensional chromatograms, multiscale peak alignment (MSPA) is presented in this research. Peaks of each chromatogram were detected based on continuous wavelet transform (CWT) and aligned against a reference chromatogram from large to small scale gradually, and the aligning procedure is accelerated by fast Fourier transform cross correlation. The presented method was compared with two widely used alignment methods on chromatographic dataset, which demonstrates that MSPA can preserve the shapes of peaks and has an excellent speed during alignment. Furthermore, MSPA method is robust and not sensitive to noise and baseline. MSPA was implemented and is available at http://code.google.com/p/mspa. PMID:22222564

  5. Detection system for a gas chromatograph

    DOEpatents

    Hayes, John M.; Small, Gerald J.

    1984-01-01

    A method and apparatus are described for the quantitative analysis of vaporizable compounds, and in particular of polycyclic aromatic hydrocarbons which may be induced to fluoresce. The sample to be analyzed is injected into a gas chromatography column and is eluted through a narrow orifice into a vacuum chamber. The free expansion of the eluted sample into the vacuum chamber creates a supersonic molecular beam in which the sample molecules are cooled to the extent that the excited vibrational and rotational levels are substantially depopulated. The cooled molecules, when induced to fluoresce by laser excitation, give greatly simplified spectra suitable for analytical purposes. The laser induced fluorimetry provides great selectivity, and the gas chromatograph provides quantitative transfer of the sample to the molecular beam.

  6. Fast gas chromatographic separation of biodiesel.

    SciTech Connect

    Pauls, R. E.

    2011-05-01

    A high-speed gas chromatographic method has been developed to determine the FAME distribution of B100 biodiesel. The capillary column used in this work has dimensions of 20 m x 0.100 mm and is coated with a polyethylene glycol film. Analysis times are typically on the order of 4-5 min depending upon the composition of the B100. The application of this method to a variety of vegetable and animal derived B100 is demonstrated. Quantitative results obtained with this method were in close agreement with those obtained by a more conventional approach on a 100 m column. The method, coupled with solid-phase extraction, was also found suitable to determine the B100 content of biodiesel-diesel blends.

  7. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  8. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  9. High-capacity pressurized continuous chromatograph

    SciTech Connect

    Begovich, J.M.; Byers, C.H.; Sisson, W.G.

    1983-01-01

    Multicomponent liquid chromatographic separations have been achieved by using a slowly rotating annular bed of sorbent material. The feed material is continuously introduced at a stationary point at the top of the bed, and eluent is allowed to flow everywhere else around the annulus. The rotation of the sorbent bed causes the separated components to appear as helical bands, each of which has a characteristic, stationary exit point; hence the separation process is truly continuous. The concept has been developed primarily on a 279-mm-diam by 0.6-m-long device with a 12.7-mm-wide annulus. The effects of annulus width and diameter have been studied using the same device with annulus widths up to 114.3 mm. With this largest width, approximately 96% of the area available within the outer cylinder is devoted to the rotating sorbent bed. Further annulus-width studies have been pursued on units with 89- and 445-mm diameters. These geometric extensions to the basic concept allow extremely large capacity increases with minimal loss in separation and no increase in chromatograph diameter. The effects associated with increased feed concentration have also been studied. In this effort as well as in the annulus-width program, the separation of copper, nickel, and cobalt components from a carbonate solution was studied in detail. The nickel and cobalt components are found in the leach liquor of the Caron process for recovering nickel and cobalt from laterite ores. Nominally 50-..mu..m-diam Dowex 50W-X8 cation exchange resin was used as the bed material. The nickel concentration of the feed was varied tenfold, from 136.1 to approximately 1400 meq/L. The combined effects of the bed loading and annulus width were studied and compared with nonlinear theory. 17 references, 9 figures, 1 table.

  10. High-capacity pressurized continuous chromatograph

    SciTech Connect

    Begovich, J.M.; Byers, C.H.; Sisson, W.G.

    1983-01-01

    Multicomponent liquid chromatographic separations have been achieved by using a slowly rotating annular bed of sorbent material. The feed material is continuously introduced at a stationary point at the top of the bed, and eluent is allowed to flow everywhere else around the annulus. The rotation of the sorbent bed causes the separated components to appear as helical bands, each of which has a characteristic, stationary exit point; hence the separation process is truly continuous. The concept has been developed primarily on a 279-mm-diam by 0.6m-long device with a 12.7-mm-wide annulus. The effects of annulus width and diameter have been studied using the same device with annulus widths up to 114.3 mm. With this largest width, approximately 96% of the area available within the outer cylinder is devoted to the rotating sorbent bed. Further annulus-width studies have been pursued on units with 89- and 445-mm diameters. These geometric extensions to the basic concept allow extremely large capacity increases with minimal loss in separation and no increase in chromatograph diameter. The effects associated with increased feed concentration have also been studied. In this effort as well as in the annulus-width program, the separation of copper, nickel, and cobalt components from a carbonate solution was studied in detail. The nickel and cobalt components are found in the leach liquor of the Caron process for recovering nickel and cobalt from laterite ores. Nominally 50-..mu..m-diam Dowex 50W-X8 cation exchange resin was used as the bed material. The nickel concentration of the feed was varied tenfold, from 136.1 to approximately 1400 meq/L. The combined effects of the bed loading and annulus width were studied and compared with nonlinear theory. 9 figures, 1 table.

  11. High-capacity pressurized continuous chromatograph

    SciTech Connect

    Begovich, J.M.; Byers, C.H.; Sisson, W.G.

    1983-01-01

    Multicomponent liquid chromatographic separations have been achieved by using a slowly rotating annular bed of sorbent material. The feed material is continuously introduced at a stationary point at the top of the bed, and eluent is allowed to flow everwhere else around the annulus. The rotation of the sorbent bed causes the separation components to appear as helical bands, each of which has a characteristic, stationary exit point; hence the separation process is truly continuous. The concept has been developed primarily on a 279-mm-diam by 0.6-m-long device with a 12.7-mm-wide annulus. The effects of annulus width and diameter have been studied using the same device with annulus widths up to 114.3 mm. With this largest width, approximately 96% of the area available within the outer cylinder is devoted to the rotating sorbent bed. Further annulus-width studies have been pursued on units with 89- and 445-mm diameters. These geometric extensions to the basic concept allow extremely large capacity increases with minimal loss in separation and no increase in chromatograph diameter. The effects associated with increased feed concentration have also been studied. In this effort as well as in the annulus-width program, the separation of copper, nickel, and cobalt components from a carbonate solution was studied in detail. The nickel and cobalt components are found in the leach liquor of the Caron process for recovering nickel and cobalt from laterite ores. Nominally 50-..mu..m0-diam Dowex 50W-X8 cation exchange resin was used as the bed material. The nickel concentration of the feed was varied tenfold, from 136.1 to approximately 1400 meq/L. The combined effects of the bed loading and annulus width were studied and compared with nonlinear theory.

  12. FTIR gas chromatographic analysis of perfumes

    NASA Astrophysics Data System (ADS)

    Diederich, H.; Stout, Phillip J.; Hill, Stephen L.; Krishnan, K.

    1992-03-01

    Perfumes, natural or synthetic, are complex mixtures consisting of numerous components. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) techniques have been extensively utilized for the analysis of perfumes and essential oils. A limited number of perfume samples have also been analyzed by FT-IR gas chromatographic (GC-FTIR) techniques. Most of the latter studies have been performed using the conventional light pipe (LP) based GC-FTIR systems. In recent years, cold-trapping (in a matrix or neat) GC-FTIR systems have become available. The cold-trapping systems are capable of sub-nanogram sensitivities. In this paper, comparison data between the LP and the neat cold-trapping GC- FTIR systems is presented. The neat cold-trapping interface is known as Tracer. The results of GC-FTIR analysis of some commercial perfumes is also presented. For comparison of LP and Tracer GC-FTIR systems, a reference (synthetic) mixture containing 16 major and numerous minor constituents was used. The components of the mixture are the compounds commonly encountered in commercial perfumes. The GC-FTIR spectra of the reference mixture was obtained under identical chromatographic conditions from an LP and a Tracer system. A comparison of the two sets of data thus generated do indeed show the enhanced sensitivity level of the Tracer system. The comparison also shows that some of the major components detected by the Tracer system were absent from the LP data. Closer examination reveals that these compounds undergo thermal decomposition on contact with the hot gold surface that is part of the LP system. GC-FTIR data were obtained for three commercial perfume samples. The major components of these samples could easily be identified by spectra search against a digitized spectral library created using the Tracer data from the reference mixture.

  13. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

    PubMed

    Dolgosheina, Elena V; Jeng, Sunny C Y; Panchapakesan, Shanker Shyam S; Cojocaru, Razvan; Chen, Patrick S K; Wilson, Peter D; Hawkins, Nancy; Wiggins, Paul A; Unrau, Peter J

    2014-10-17

    Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes. PMID:25101481

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  16. Affinity separation in magnetically stabilized fluidized beds: synthesis and performance of packing materials

    SciTech Connect

    Lochmueller, C.H.; Wigman, L.S.

    1987-11-01

    A magnetically stabilized fluidized-bed separator designed to test the use of pellicular, ferromagnetic affinity chromatography packing materials has been developed. A wire wound solenoid was used to produce the magnetic field. The ferromagnetic packing material is comprised of a magnetite-containing, polyurethane gel coated onto polystyrene beads. The gel contains free carboxyl groups. These were carbodiimide-coupled to soy trypsin inhibitor and the material used for trypsin purification. Narrow-band affinity chromatography was carried out in packed-bed, fluidized-bed, and magnetically stabilized, fluidized-bed separators. Pressure drop, capacity, dilution, and peak asymmetry were evaluated for each type of separator. The three types provide comparable efficiency but the fluidized separators exhibit a much lower pressure drop. As might be expected, fluidized-bed separators perform well for affinity chromatography (large k') but poorly for size exclusion chromatography.

  17. Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

  18. Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity.

    PubMed

    Arendt, R M; Greenblatt, D J; Liebisch, D C; Luu, M D; Paul, S M

    1987-01-01

    Factors influencing brain uptake of benzodiazepine derivatives were evaluated in adult Sprague Dawley rats (n = 8-10 per drug). Animals received single intraperitoneal doses of alprazolam, triazolam, lorazepam, flunitrazepam, diazepam, midazolam, desmethyldiazepam, or clobazam. Concentrations of each drug (and metabolites) in whole brain and serum 1 h after dosage were determined by gas chromatography. Serum free fraction was measured by equilibrium dialysis. In vitro binding affinity (apparent Ki) of each compound was estimated based on displacement of tritiated flunitrazepam in washed membrane preparations from rat cerebral cortex. Lipid solubility of each benzodiazepine was estimated using the reverse-phase liquid chromatographic (HPLC) retention index at physiologic pH. There was no significant relation between brain:total serum concentration ratio and either HPLC retention (r = 0.18) or binding Ki (r = -0.34). Correction of uptake ratios for free as opposed to total serum concentration yielded a highly significant correlation with HPLC retention (r = 0.78, P less than 0.005). However, even the corrected ratio was not correlated with binding Ki (r = -0.22). Thus a benzodiazepine's capacity to diffuse from systemic blood into brain tissue is much more closely associated with the physicochemical property of lipid solubility than with specific affinity. Unbound rather than total serum or plasma concentration most accurately reflects the quantity of drug available for diffusion. PMID:2888155

  19. Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth.

    PubMed

    Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou

    2006-09-01

    An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods. PMID:16736086

  20. Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.

    PubMed

    Lyons, Russell E; Elvin, Christopher M; Taylor, Karin; Lekieffre, Nicolas; Ramshaw, John A M

    2012-12-01

    Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. PMID:22627880