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

Science.gov (United States)

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Affinity chromatography with pseudobiospecific ligands on high-performance supports for purification of proteins of biotechnological interest

Directory of Open Access Journals (Sweden)

N.B. Iannucci

2003-03-01

Full Text Available High-performance affinity matrices were obtained by attaching pseudobiospecific ligands to hollow-fibre membranes. The neutral protease contained in FlavourzymeTM was purified to homogeneity with Yellow 4R-HE affinity hollow-fibre membranes. Immobilisation of Red HE-3B allowed purification of a milk-clotting enzyme obtained by solid-state culture of Mucor bacilliformis. Copper immobilisation through iminodiacetic acid allowed fractionation of Biocon Bioconcentrated PlusTM to separate the pectinesterase-containing fraction. The productivity of the developed processes - 1900, 94 and 750 U/ml.min, respectively - was 10- to 15-fold higher than that achieved with the same ligands immobilised on agarose-based soft gels, mainly due to the shortening of the purification processes.

APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

Directory of Open Access Journals (Sweden)

О. V. Sviatenko

2014-04-01

Full Text Available Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding specificities and affinities of these proteins differ between species and antibody subclass. Protein А has high affinity to human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, IgG3, goat and sheep IgG2, dog, cat, guinea pig, rabbit IgG. Protein G binds strongly to human, mouse, cow, goat, sheep and rabbit IgG. Protein L has ability of strong binding to immunoglobulin kappa-chains of human, mouse, rat and pig. Expediency of application of affinity chromatography with usage of sorbents on the basis of immobilized proteins A, G, L are shown for isolation and purification of antibodies different classes. Previously mentioned method is used as an alternative to conventional methods of protein purification, such as ion-exchange, hydrophobic interactions, metal affinity chromatography, ethanol precipitation due to simplicity in usage, possibility of one-step purification process, obtaining of proteins high level purity, multiuse at maintenance of proper storage and usage conditions. Affinity sorbents on the basis of immobilized proteins A, G, L are used not only for antibodies purification, but also for extraction of different antibodies fractions from blood serum.

Affinity purification combined with mass spectrometry to identify herpes simplex virus protein-protein interactions.

Science.gov (United States)

Meckes, David G

2014-01-01

The identification and characterization of herpes simplex virus protein interaction complexes are fundamental to understanding the molecular mechanisms governing the replication and pathogenesis of the virus. Recent advances in affinity-based methods, mass spectrometry configurations, and bioinformatics tools have greatly increased the quantity and quality of protein-protein interaction datasets. In this chapter, detailed and reliable methods that can easily be implemented are presented for the identification of protein-protein interactions using cryogenic cell lysis, affinity purification, trypsin digestion, and mass spectrometry.

Rapid purification of circular DNA by triplex-mediated affinity capture

Science.gov (United States)

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.

Affinity column for purification of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor

International Nuclear Information System (INIS)

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

1987-01-01

The TXA 2 /PGH 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 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 2 /PGH 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

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

Science.gov (United States)

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

2014-02-01

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

Science.gov (United States)

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Science.gov (United States)

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

2016-11-11

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

DEFF Research Database (Denmark)

Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan

2010-01-01

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...

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

Science.gov (United States)

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

2017-01-01

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

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

Science.gov (United States)

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

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

Science.gov (United States)

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.

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Science.gov (United States)

Weber, Eva; Guth, Christina; Weiss, Ingrid M

2012-01-01

Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

Science.gov (United States)

Gardano, Laura; Holland, Linda; Oulton, Rena; Le Bihan, Thierry; Harrington, Lea

2012-01-01

Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. PMID:22187156

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

Science.gov (United States)

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

2005-10-05

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

Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

Directory of Open Access Journals (Sweden)

Christine Bee

Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein

International Nuclear Information System (INIS)

Rodriguez, K.; Talamantez, J.; Huang, W.; Reed, S.H.; Wang, Z.; Chen, L.; Feaver, W.J.; Friedberg, E.C.; Tomkinson, A.E.

1998-01-01

The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells

Aspartic acid incorporated monolithic columns for affinity glycoprotein purification.

Science.gov (United States)

Armutcu, Canan; Bereli, Nilay; Bayram, Engin; Uzun, Lokman; Say, Rıdvan; Denizli, Adil

2014-02-01

Novel aspartic acid incorporated monolithic columns were prepared to efficiently affinity purify immunoglobulin G (IgG) from human plasma. The monolithic columns were synthesised in a stainless steel HPLC column (20 cm × 5 mm id) by in situ bulk polymerisation of N-methacryloyl-L-aspartic acid (MAAsp), a polymerisable derivative of L-aspartic acid, and 2-hydroxyethyl methacrylate (HEMA). Monolithic columns [poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-aspartic acid) (PHEMAsp)] were characterised by swelling studies, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The monolithic columns were used for IgG adsorption/desorption from aqueous solutions and human plasma. The IgG adsorption depended on the buffer type, and the maximum IgG adsorption from aqueous solution in phosphate buffer was 0.085 mg/g at pH 6.0. The monolithic columns allowed for one-step IgG purification with a negligible capacity decrease after ten adsorption-desorption cycles. Copyright © 2013 Elsevier B.V. All rights reserved.

Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

Science.gov (United States)

Abdolalizadeh, Jalal; Majidi Zolbanin, Jafar; Nouri, Mohammad; Baradaran, Behzad; Movassaghpour, AliAkbar; Farajnia, Safar; Omidi, Yadollah

2013-01-01

Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods:In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications. PMID:24312807

Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

Directory of Open Access Journals (Sweden)

Safar Farajnia

2013-02-01

Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Directory of Open Access Journals (Sweden)

Eva Weber

Full Text Available Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3 (- as the first ionic interaction partner, but not necessarily for Ca(2+. The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins : a wash step with a low concentration of EDTA

NARCIS (Netherlands)

Westra, DF; Welling, GW; Koedijk, DGAM; Scheffer, AJ; The, TH; Welling-Wester, S

2001-01-01

Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.

Science.gov (United States)

Chen, Yong; Li, Yang; Liu, Peng; Sun, Qun; Liu, Zhu

2014-04-01

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

Directory of Open Access Journals (Sweden)

Jenkins Jeremy L

2001-10-01

Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

Science.gov (United States)

Jenkins, Jeremy L; Dean, Donald H

2001-01-01

Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

Science.gov (United States)

Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

2014-12-03

Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry.

Science.gov (United States)

Dubois, Marie-Line; Bastin, Charlotte; Lévesque, Dominique; Boisvert, François-Michel

2016-09-02

The extensive identification of protein-protein interactions under different conditions is an important challenge to understand the cellular functions of proteins. Here we use and compare different approaches including affinity purification and purification by proximity coupled to mass spectrometry to identify protein complexes. We explore the complete interactome of the minichromosome maintenance (MCM) complex by using both approaches for all of the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair, and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

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

Science.gov (United States)

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

1993-12-01

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

Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry.

Science.gov (United States)

Buhs, Sophia; Gerull, Helwe; Nollau, Peter

2017-01-01

Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.

Alternative affinity tools: more attractive than antibodies?

NARCIS (Netherlands)

Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.

2011-01-01

Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties.

Science.gov (United States)

Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet

2017-12-01

Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al 3+ , Cd 2+ and Mg 2+ , whereas Ca 2+ , Co 2+ and Ni 2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The K m and V max of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with K i value of 14.43μM, the IC 50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

Science.gov (United States)

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

Overview of the purification of recombinant proteins.

Science.gov (United States)

Wingfield, Paul T

2015-04-01

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 throughout Current Protocols in Protein Science. In the interim, there has been a shift toward 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, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

Science.gov (United States)

Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

2016-02-01

Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Overview of the recombinant proteins purification by affinity tags and ...

African Journals Online (AJOL)

From protein within isolation process which the same matter increases labor costs further and prevents application of these tags in industrial scale. Therefore proper replacement is emphasized for enzymatic removal of purification tags. Keywords: protein purification; recombinant proteins; self-cleavable tags; Intein tags; ...

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

Directory of Open Access Journals (Sweden)

Brian H. Carrick

2016-03-01

Full Text Available 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.

Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

Directory of Open Access Journals (Sweden)

Qiang Wang

Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

Czech Academy of Sciences Publication Activity Database

Tykvart, Jan; Šácha, Pavel; Bařinka, Cyril; Knedlík, Tomáš; Starková, Jana; Lubkowski, J.; Konvalinka, Jan

2012-01-01

Roč. 82, č. 1 (2012), s. 106-115 ISSN 1046-5928 R&D Projects: GA MŠk 1M0508; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : affinity purification * biotin acceptor peptide * recombinant protein expression * biotin -protein ligase (BirA) * co-localization * PSMA Subject RIV: CE - Biochemistry Impact factor: 1.429, year: 2012

Effects of gamma radiation immunogenicity of ribonucleoprotein (RNPs) of rabies virus and purification of anti-RNPs antibodies for diagnosis

International Nuclear Information System (INIS)

Costa, Ana Elena Boamorte da

2010-01-01

The World Health Organization recommends the direct immunofluorescence test for laboratory diagnosis and serological evaluation of rabies. To achieve this test, fluorescent anti-ribo nucleoproteins (RNPs) conjugates, produced from purified IgGs of RNP-immunized animals are employed. The aims of the present study were: investigate the effects of gamma radiation on the immunogenicity of RNPs, as well as to compare two chromatographic methodologies for the purification of anti-RNPs immunoglobulins. Sera from animals immunized with either native or irradiated RNPs were compared by direct immunofluorescence and immuno enzymatic assays. Our results indicate that the animals immunized with irradiated antigen requested a lower number of doses to reach high antibody titers. The immunofluorescence assays indicated that the conjugates produced with the anti-irradiated RNPs IgGs showed similar specificity to its anti-native counterpart, but with a higher definition of the virus inclusions. The purification methods were compared by Bradford and electrophoresis assays. According to the results, we concluded that the affinity-based process resulted in higher yields, lower execution time, and higher purity of the antibodies. (author)

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

Science.gov (United States)

Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

2015-07-01

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

Profiling of Parkin-binding partners using tandem affinity purification.

Directory of Open Access Journals (Sweden)

Alessandra Zanon

Full Text Available Parkinson's disease (PD is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2 are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells, we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

Profiling of Parkin-Binding Partners Using Tandem Affinity Purification

Science.gov (United States)

Blankenburg, Hagen; Doncheva, Nadezhda T.; Schwienbacher, Christine; Serafin, Alice; Alexa, Adrian; Weichenberger, Christian X.; Albrecht, Mario; Klein, Christine; Hicks, Andrew A.; Pramstaller, Peter P.

2013-01-01

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function. PMID:24244333

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

Science.gov (United States)

Salehi, Nasrin; Peng, Ching-An

2016-07-08

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. © 2016 American Institute of Chemical Engineers.

One-step FPLC-size-exclusion chromatography procedure for purification of rDMBT1 6 kb with increased biological activity

DEFF Research Database (Denmark)

Tuttolomondo, Martina; Hansen, Pernille Lund; Mollenhauer, Jan

2018-01-01

from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our...

Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of monospecific anti-p46 kDa/Myo1C antibodies for early diagnosis of multiple sclerosis patients

Czech Academy of Sciences Publication Activity Database

Horák, Daniel; Hlídková, Helena; Kit, Y.; Antonyuk, V.; Myronovsky, S.; Stoika, R.

2017-01-01

Roč. 37, č. 2 (2017), s. 1-10, č. článku BSR20160526. ISSN 0144-8463 R&D Projects: GA MŠk(CZ) LH14318 Institutional support: RVO:61389013 Keywords : poly(2-hydroxyethyl methacrylate) * magnetic microspheres * affinity purification Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 2.906, year: 2016

Affinity in electrophoresis.

Science.gov (United States)

Heegaard, Niels H H

2009-06-01

The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

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

Energy Technology Data Exchange (ETDEWEB)

James, W.M.; Emerick, M.C.; Agnew, W.S. (Yale Univ. School of medicine, New Haven, CT (USA))

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.

One-step purification of E. coli elongation factor Tu

DEFF Research Database (Denmark)

Knudsen, Charlotte Rohde; Clark, Brian F. C.; Degn, B

1993-01-01

The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one...

Strep-Tagged Protein Purification.

Science.gov (United States)

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). © 2015 Elsevier Inc. All rights reserved.

Biomimetic small peptide functionalized affinity monoliths for monoclonal antibody purification.

Science.gov (United States)

Wang, Xiangyu; Xia, Donghai; Han, Hai; Peng, Kun; Zhu, Peijie; Crommen, Jacques; Wang, Qiqin; Jiang, Zhengjin

2018-08-09

The rapid development of monoclonal antibodies (mAbs) in therapeutic and diagnostic applications has necessitated the advancement of mAbs purification technologies. In this study, a biomimetic small peptide ligand 3,5-di-tert-butyl-4-hydroxybenzoic acid-Arg-Arg-Gly (DAAG) functionalized monolith was fabricated through a metal ion chelation-based multi-step approach. The resulting monolith showed good chromatographic performance. Compared with the Ni 2+ based IMAC monolith, the DAAG functionalized monolith exhibited not only excellent specificity but also higher dynamic binding capacity (DBC). The 10% DBC and 50% DBC for hIgG reached as high values as 26.0 and 34.6 mg/mL, respectively, at a ligand density of 8.8 μmol/mL, due to the high porosity and accessibility of the monolithic matrix. Moreover, the stability of the DAAG functionalized monolith in successive breakthrough experiments indicates that it has a promising potential for long-term use in mAbs purification. Finally, the DAAG functionalized monolith was successfully applied to the purification of trastuzumab or human immunoglobulin G (hIgG) from biological samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Improved purification of native meningococcal porin PorB and studies on its structure/function.

Science.gov (United States)

Massari, Paola; King, Carol A; MacLeod, Heather; Wetzler, Lee M

2005-12-01

The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.

Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98

International Nuclear Information System (INIS)

Chauhan, Archana; Islam, Zeyaul; Jain, Rakesh Kumar; Karthikeyan, Subramanian

2009-01-01

Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported. Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively

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

Science.gov (United States)

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

2015-02-01

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

International Nuclear Information System (INIS)

Akduman, Begüm; Uygun, Murat; Uygun, Deniz Aktaş; Akgöl, Sinan; Denizli, Adil

2013-01-01

In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

Energy Technology Data Exchange (ETDEWEB)

Akduman, Begüm [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Uygun, Murat [Koçarlı Vocational and Training School, Adnan Menderes University, Aydın (Turkey); Uygun, Deniz Aktaş, E-mail: daktas@adu.edu.tr [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Akgöl, Sinan [Biochemistry Department, Ege University, İzmir (Turkey); Denizli, Adil [Chemistry Department, Hacettepe University, Ankara (Turkey)

2013-12-01

In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

DEFF Research Database (Denmark)

Domanski, Michal; Molloy, Kelly; Jiang, Hua

2012-01-01

An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

Transuranium element purification by liquid-liquid extraction

International Nuclear Information System (INIS)

Madic, C.; Koehly, G.

1976-01-01

In the transuranium element production, the liquid-liquid extraction purification is presented. The affinity of TBP and trilaurylammonium nitrate for these elements is given. Exemples of NP/Pu, Pu/Np, U/Pu, Am/Cm, Am and Cm/Ln separation are presented [fr

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

Science.gov (United States)

Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

2015-07-01

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Kelley, M.J.; Carman, G.M.

1987-01-01

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase was purified 2300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism

Homologous high-throughput expression and purification of highly conserved E coli proteins

Directory of Open Access Journals (Sweden)

Duchmann Rainer

2007-06-01

Full Text Available Abstract Background Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD. Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. Results As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP, N-utilizing substance A (NusA, or glutathione S-transferase (GST to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS. Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500

Purification of bovine thyroid-stimulating hormone by a monoclonal antibody

International Nuclear Information System (INIS)

Lock, A.J.; van Denderen, J.; Aarden, L.A.

1988-01-01

A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by [ 3 H]thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

Science.gov (United States)

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.

Semi-continuous protein fractionating using affinity cross-flow filtration

NARCIS (Netherlands)

Borneman, Zandrie; Zhang, W.; van den Boomgaard, Anthonie; Smolders, C.A.

2002-01-01

Protein purification by means of downstream processing is increasingly important. At the University of Twente a semi-continuous process is developed for the isolation of BSA out of crude protein mixtures. For this purpose an automated Affinity Cross-Flow Filtration, ACFF, process is developed. This

Expression, purification and characterization of the interferon ...

Indian Academy of Sciences (India)

2012-01-19

Jan 19, 2012 ... utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent ...

Identification of Protein Complexes from Tandem Affinity Purification/Mass Spectrometry Data via Biased Random Walk.

Science.gov (United States)

Cai, Bingjing; Wang, Haiying; Zheng, Huiru; Wang, Hui

2015-01-01

Systematic identification of protein complexes from protein-protein interaction networks (PPIs) is an important application of data mining in life science. Over the past decades, various new clustering techniques have been developed based on modelling PPIs as binary relations. Non-binary information of co-complex relations (prey/bait) in PPIs data derived from tandem affinity purification/mass spectrometry (TAP-MS) experiments has been unfairly disregarded. In this paper, we propose a Biased Random Walk based algorithm for detecting protein complexes from TAP-MS data, resulting in the random walk with restarting baits (RWRB). RWRB is developed based on Random walk with restart. The main contribution of RWRB is the incorporation of co-complex relations in TAP-MS PPI networks into the clustering process, by implementing a new restarting strategy during the process of random walk. Through experimentation on un-weighted and weighted TAP-MS data sets, we validated biological significance of our results by mapping them to manually curated complexes. Results showed that, by incorporating non-binary, co-membership information, significant improvement has been achieved in terms of both statistical measurements and biological relevance. Better accuracy demonstrates that the proposed method outperformed several state-of-the-art clustering algorithms for the detection of protein complexes in TAP-MS data.

Isolation and purification of wheat germ agglutinin and analysis of its properties

Science.gov (United States)

Wang, Han

2017-12-01

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography.

Directory of Open Access Journals (Sweden)

Indhu Kanakaraj

Full Text Available 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 Cu(2+-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 Cu(2+-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.

High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

Directory of Open Access Journals (Sweden)

Yi Ma

2016-01-01

Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

Science.gov (United States)

Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

2016-01-01

Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H 10 . The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

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

Science.gov (United States)

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

1983-01-01

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

The CRAPome: a contaminant repository for affinity purification–mass spectrometry data

NARCIS (Netherlands)

Mellacheruvu, D.; Wright, Z.; Couzens, A.L.; Low, T.Y.|info:eu-repo/dai/nl/411298437; Halim, V.A.|info:eu-repo/dai/nl/326157441; Heck, A.J.R.|info:eu-repo/dai/nl/105189332; Mohammed, S.|info:eu-repo/dai/nl/30483632X; Nesvizhskii, A.

2013-01-01

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background

Effects of gamma radiation immunogenicity of ribonucleoprotein (RNPs) of rabies virus and purification of anti-RNPs antibodies for diagnosis; Efeitos da radiacao gama na imunogenicidade das ribonucleoproteinas (RNPs) do virus da raiva e purificacao de anticorpos anti-RNPs para diagnostico

Energy Technology Data Exchange (ETDEWEB)

Costa, Ana Elena Boamorte da

2010-07-01

The World Health Organization recommends the direct immunofluorescence test for laboratory diagnosis and serological evaluation of rabies. To achieve this test, fluorescent anti-ribo nucleoproteins (RNPs) conjugates, produced from purified IgGs of RNP-immunized animals are employed. The aims of the present study were: investigate the effects of gamma radiation on the immunogenicity of RNPs, as well as to compare two chromatographic methodologies for the purification of anti-RNPs immunoglobulins. Sera from animals immunized with either native or irradiated RNPs were compared by direct immunofluorescence and immuno enzymatic assays. Our results indicate that the animals immunized with irradiated antigen requested a lower number of doses to reach high antibody titers. The immunofluorescence assays indicated that the conjugates produced with the anti-irradiated RNPs IgGs showed similar specificity to its anti-native counterpart, but with a higher definition of the virus inclusions. The purification methods were compared by Bradford and electrophoresis assays. According to the results, we concluded that the affinity-based process resulted in higher yields, lower execution time, and higher purity of the antibodies. (author)

Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth.

Science.gov (United States)

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.

Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

Science.gov (United States)

Peled, Michael; Tocheva, Anna S; Sandigursky, Sabina; Nayak, Shruti; Philips, Elliot A; Nichols, Kim E; Strazza, Marianne; Azoulay-Alfaguter, Inbar; Askenazi, Manor; Neel, Benjamin G; Pelzek, Adam J; Ueberheide, Beatrix; Mor, Adam

2018-01-16

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

Science.gov (United States)

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

Affinity purification and partial characterization of the zonulin/zonula occludens toxin (Zot) receptor from human brain.

Science.gov (United States)

Lu, R; Wang, W; Uzzau, S; Vigorito, R; Zielke, H R; Fasano, A

2000-01-01

The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood-brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of approximately 55 and approximately 45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55-kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14-kDa member of the S-100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB.

Bromelain: an overview of industrial application and purification strategies.

Science.gov (United States)

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.

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

Science.gov (United States)

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

2013-09-20

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

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

Science.gov (United States)

Li, Junhua; Zhang, Yang; Yang, Yanjun

2013-03-01

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

Directory of Open Access Journals (Sweden)

Jim F White

2010-09-01

Full Text Available Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1.To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein.Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

Native American medicine.

Science.gov (United States)

Cohen, K

1998-11-01

This article summarizes common principles, practices, and ethics of Native American healing, the traditional medicine of North America. Native American healing, spirituality, culture, and, in modern times, political, social, and economic concerns are closely intertwined. Intuition and spiritual awareness are a healer's most essential diagnostic tools. Therapeutic methods include prayer, music, ritual purification, herbalism, massage, ceremony, and personal innovations of individual healers. A community of friends, family, and helpers often participate in the healing intervention and help to alleviate the alienation caused by disease. A healthy patient has a healthy relationship with his or her community and, ultimately, with the greater community of nature known as "All Relations." The goal of Native American healing is to find wholeness, balance, harmony, beauty, and meaning. "Healing," making whole, is as important as curing disease; at times they are identical.

High-throughput purification of recombinant proteins using self-cleaving intein tags.

Science.gov (United States)

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

Synthesis of Ni-Zn ferrite nanoparticles in radiofrequency thermal plasma reactor and their use for purification of histidine-tagged proteins

International Nuclear Information System (INIS)

Feczko, Tivadar; Muskotal, Adel; Gal, Lorand; Szepvoelgyi, Janos; Sebestyen, Anett; Vonderviszt, Ferenc

2008-01-01

Superparamagnetic Ni-Zn ferrite nanoparticles were synthesized in radiofrequency thermal plasma reactor from aqueous solutions of Ni- and Zn-nitrates. The nanoparticles were studied for protein purification performance in both quantitative and qualitative terms. For comparison, experiments were also performed by Ni-charged affinity chromatography. It was proved that the Ni-Zn ferrite nanoparticles effectively purified histidine-tagged proteins with a maximum protein binding capacity of about 7% (w/w). Gel electrophoresis demonstrated better purification characteristics for magnetic nanoparticles than for affinity chromatography.

A novel affinity purification method to isolate peptide specific antibodies

DEFF Research Database (Denmark)

Karlsen, Alan E; Lernmark, A; Kofod, Hans

1990-01-01

Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

Structures of native and affinity-enhanced WT1 epitopes bound to HLA-A*0201: Implications for WT1-based cancer therapeutics

Energy Technology Data Exchange (ETDEWEB)

Borbulevych, Oleg Y.; Do, Priscilla; Baker, Brian M. (Notre)

2010-09-07

Presentation of peptides by class I or class II major histocompatibility complex (MHC) molecules is required for the initiation and propagation of a T cell-mediated immune response. Peptides from the Wilms Tumor 1 transcription factor (WT1), upregulated in many hematopoetic and solid tumors, can be recognized by T cells and numerous efforts are underway to engineer WT1-based cancer vaccines. Here we determined the structures of the class I MHC molecule HLA-A*0201 bound to the native 126-134 epitope of the WT1 peptide and a recently described variant (R1Y) with improved MHC binding. The R1Y variant, a potential vaccine candidate, alters the positions of MHC charged side chains near the peptide N-terminus and significantly reduces the peptide/MHC electrostatic surface potential. These alterations indicate that the R1Y variant is an imperfect mimic of the native WT1 peptide, and suggest caution in its use as a therapeutic vaccine. Stability measurements revealed how the R1Y substitution enhances MHC binding affinity, and together with the structures suggest a strategy for engineering WT1 variants with improved MHC binding that retain the structural features of the native peptide/MHC complex.

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Energy Technology Data Exchange (ETDEWEB)

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.

Lectins as carriers: preparation and purification of a concanavalin A-trypsin conjugate

International Nuclear Information System (INIS)

Shier, W.T.

1985-01-01

The scheme for the preparation and purification of Con A-trypsin demonstrates the presence of Con A in the conjugate by affinity purification and by hemagglutination. The presence of Con A was demonstrated in two additional ways. The conjugate was prepared using trypsin labeled with iodine 125 and unlabeled Con A. The resulting conjugate containing 42,800 cpm/mg protein was used to demonstrate prolonged retention of the conjugated trypsin in mouse footpads. The presence of trypsin was also demonstrated in the conjugate by affinity chromatography and by the esterase activity characteristic of trypsin with tosyl-L-arginine methyl ester as substrate. Con A-trypsin was also assayed for proteolytic activity with a macromolecular substrate azocasein. A comparison of proteolytic activities with these two substrates indicated that 50-80% of the proteolytic activity observed with the small substrate is retained with macromolecular substrates

Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

Science.gov (United States)

Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

2015-08-20

For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria.

Science.gov (United States)

Moreadith, R W; Lehninger, A L

1984-05-25

The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.

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

Directory of Open Access Journals (Sweden)

Isabelle Maxim

2010-04-01

Full Text Available Abstract 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-ribosylation. 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.

Probing SH2-domains using Inhibitor Affinity Purification (IAP).

Science.gov (United States)

Höfener, Michael; Heinzlmeir, Stephanie; Kuster, Bernhard; Sewald, Norbert

2014-01-01

Many human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds. In order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain. The results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors.

Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes One-step purification of Pfu DNA polymerase.

Science.gov (United States)

Melissis, S; Labrou, N E; Clonis, Y D

2006-07-28

The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (K(D)) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.

Membrane Affinity of Platensimycin and Its Dialkylamine Analogs

Directory of Open Access Journals (Sweden)

Ian Rowe

2015-08-01

Full Text Available Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes, signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation.

MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

Energy Technology Data Exchange (ETDEWEB)

Adams, Michael W. [University of Georgia; W. W. Adams, Michael

2014-01-07

Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

Purification of the functional plant membrane channel KAT1

International Nuclear Information System (INIS)

Hibi, Takao; Aoki, Shiho; Oda, Keisuke; Munemasa, Shintaro; Ozaki, Shunsuke; Shirai, Osamu; Murata, Yoshiyuki; Uozumi, Nobuyuki

2008-01-01

The inward-rectifying K + 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 + 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

High-throughput bioscreening system utilizing high-performance affinity magnetic carriers exhibiting minimal non-specific protein binding

International Nuclear Information System (INIS)

Hanyu, Naohiro; Nishio, Kosuke; Hatakeyama, Mamoru; Yasuno, Hiroshi; Tanaka, Toshiyuki; Tada, Masaru; Nakagawa, Takashi; Sandhu, Adarsh; Abe, Masanori; Handa, Hiroshi

2009-01-01

For affinity purification of drug target protein we have developed magnetic carriers, narrow in size distribution (184±9 nm), which exhibit minimal non-specific binding of unwanted proteins. The carriers were highly dispersed in aqueous solutions and highly resistant to organic solvents, which enabled immobilization of various hydrophobic chemicals as probes on the carrier surfaces. Utilizing the carriers we have automated the process of separation and purification of the target proteins that had been done by manual operation previously.

Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I

International Nuclear Information System (INIS)

Rehm, H.; Lazdunski, M.

1988-01-01

The binding protein for the K + -channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K d , 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO 4 /polyacrylamide gel revealed three bands of M r 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for 125 I-labeled mast cell degranulating peptide, another putative K + -channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

Science.gov (United States)

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.

Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column.

Science.gov (United States)

Wu, Zhihua; Li, Kun; Zhan, Shaode; Tong, Ping; Li, Xin; Yang, Anshu; Chen, Hongbing

2017-09-14

Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits' antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.

Native Mass Spectrometry in Fragment-Based Drug Discovery

Directory of Open Access Journals (Sweden)

Liliana Pedro

2016-07-01

Full Text Available The advent of native mass spectrometry (MS in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein–ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD. Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Native Mass Spectrometry in Fragment-Based Drug Discovery.

Science.gov (United States)

Pedro, Liliana; Quinn, Ronald J

2016-07-28

The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Purification and subunit structure of a putative K sup + -channel protein identified by its binding properties for dendrotoxin I

Energy Technology Data Exchange (ETDEWEB)

Rehm, H.; Lazdunski, M. (Centre National de la Recherche Scientifique, Nice (France))

1988-07-01

The binding protein for the K{sup +}-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K{sub d}, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO{sub 4}/polyacrylamide gel revealed three bands of M{sub r} 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for {sup 125}I-labeled mast cell degranulating peptide, another putative K{sup +}-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

Science.gov (United States)

El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

2000-02-15

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

Purification of a putative brain somatostatin receptor

International Nuclear Information System (INIS)

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

1989-01-01

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 8 ]SRIF affinity column with either sodium acetate (pH 5.5) or free [D-Trp 8 ]SRIF. The binding of the protein to the affinity column was prevented by free [D-Trp 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 125 I-labeled SRIF analogue CGP 23996. Excess [D-Trp 8 ]SRIF blocked the binding of 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

The affinity purification and characterization of ATP synthase complexes from mitochondria.

Science.gov (United States)

Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

2013-02-13

The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

Application of superparamagnetic microspheres for affinity adsorption and purification of glutathione

International Nuclear Information System (INIS)

Wang Qiang; Guan Yueping; Yang Mingzhu

2012-01-01

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. - Highlights: ► The magnetic microsphere with surface IDA–Cu groups was synthesized. ► The magnetic microspheres were applied for adsorption of GSH. ► The adsorption–desorption of glutathione was investigated. ► The maximum adsorption capacity of GSH was fitted at 42.4 mg/g.

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

Science.gov (United States)

Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

2002-01-01

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

Directory of Open Access Journals (Sweden)

Johan Nilvebrant

Full Text Available The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

Ligand-Induced Cross-Linking of Z-Elastin-like Polypeptide-Functionalized E2 Protein Nanoparticles for Enhanced Affinity Precipitation of Antibodies.

Science.gov (United States)

Swartz, Andrew R; Sun, Qing; Chen, Wilfred

2017-05-08

Affinity precipitation is an ideal alternative to chromatography for antibody purification because it combines the high selectivity of an affinity ligand with the operational benefits of precipitation. However, the widespread use of elastin-like polypeptide (ELP) capture scaffolds for antibody purification has been hindered by the high salt concentrations and temperatures necessary for efficient ELP aggregation. In this paper, we employed a tandem approach to enhance ELP aggregation by enlarging the dimension of the capturing scaffold and by creating IgG-triggered scaffold cross-linking. This was accomplished by covalently conjugating the Z-domain-ELP (Z-ELP) capturing scaffold to a 25 nm diameter E2 protein nanocage using Sortase A ligation. We demonstrated the isothermal recovery of IgG in the virtual absence of salt due to the significantly increased scaffold dimension and cross-linking from multivalent IgG-E2 interactions. Because IgG cross-linking is reversible at low pH, it may be feasible to achieve a high yielding IgG purification by isothermal phase separation using a simple pH trigger.

Effect of impurities and post-experimental purification in SAD phasing with serial femtosecond crystallography data.

Science.gov (United States)

Zhang, Tao; Gu, Yuanxin; Fan, Haifu

2016-06-01

In serial crystallography (SX) with either an X-ray free-electron laser (XFEL) or synchrotron radiation as the light source, huge numbers of micrometre-sized crystals are used in diffraction data collection. For a SAD experiment using a derivative with introduced heavy atoms, it is difficult to completely exclude crystals of the native protein from the sample. In this paper, simulations were performed to study how the inclusion of native crystals in the derivative sample could affect the result of SAD phasing and how the post-experimental purification proposed by Zhang et al. [(2015), Acta Cryst. D71, 2513-2518] could be used to remove the impurities. A gadolinium derivative of lysozyme and the corresponding native protein were used in the test. Serial femtosecond crystallography (SFX) diffraction snapshots were generated by CrystFEL. SHELXC/D, Phaser, DM, ARP/wARP and REFMAC were used for automatic structure solution. It is shown that a small amount of impurities (snapshots from native crystals) in the set of derivative snapshots can strongly affect the SAD phasing results. On the other hand, post-experimental purification can efficiently remove the impurities, leading to results similar to those from a pure sample.

Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

Directory of Open Access Journals (Sweden)

Eszter Szarka

2018-01-01

Full Text Available Background: In rheumatoid arthritis (RA, anti-citrullinated protein/peptide antibodies (ACPAs are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

Science.gov (United States)

Morrison, Kerrie A; Akram, Aneel; Mathews, Ashlyn; Khan, Zoeya A; Patel, Jaimin H; Zhou, Chumin; Hardy, David J; Moore-Kelly, Charles; Patel, Roshani; Odiba, Victor; Knowles, Tim J; Javed, Masood-Ul-Hassan; Chmel, Nikola P; Dafforn, Timothy R; Rothnie, Alice J

2016-12-01

The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein

DEFF Research Database (Denmark)

Balogh, Ria K.; Gyurcsik, Béla; Hunyadi-Gulyás, Éva

2016-01-01

. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes...... the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor...... any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure....

Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp paracasei CHCC 2115

DEFF Research Database (Denmark)

Thage, B.V.; Rattray, F.P.; Laustsen, M.W.

2004-01-01

Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. Methods and Results: The purification protocol consisted of anion exchange chromatography, affinity chromatography...... of other metal ions, thiol- and carbonyl-binding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively. Conclusions: The results suggest...

Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

Science.gov (United States)

Tejeda-Mansir, A; Montesinos, R M; Guzmán, R

2001-10-30

The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular

Investigation of molybdenum-crosslinker interfaces for affinity based electrochemical biosensing applications

Science.gov (United States)

Kamakoti, Vikramshankar; Shanmugam, Nandhinee Radha; Tanak, Ambalika Sanjeev; Jagannath, Badrinath; Prasad, Shalini

2018-04-01

Molybdenum (Mo) has been investigated for implementation as an electrode material for affinity based biosensing towards devloping flexibe electronic biosensors. Treatment of the native oxide of molybdenum was investigated through two surface treatment strategies namely thiol and carbodiimide crosslinking methods. The binding interaction between cross-linker molecules and Mo electrode surface has been characterized using Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and optical microscopy. The efficacy of treatment of Mo with its native oxide using carbodiimide cross linking methodology was established. The carbodiimide cross-linking chemistry was found to possess better surface coverage and binding affinity with Molybdenum electrode surface when compared to thiol cross-linking chemistry.Electrochemical characterization of Mo electrode using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltametry (CV) techniques was performed to evaluate the effect of ionic properties of solution buffer on the Mo electrode's performance. Affinity based biosensing of C-Reactive Protein (CRP) has been demonstrated on a flexible nanoporous polymeric substrate with detection threshold of 100 pg/ml in synthetic urine buffer medium. The biosensor has been evaluated to be developed as a dipstick based point of care device for detection of biomarkers in urine.

Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.

Science.gov (United States)

Scotter, Andrew J; Kuntz, Douglas A; Saul, Michelle; Graham, Laurie A; Davies, Peter L; Rose, David R

2006-06-01

We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.

Affinity partitioning of human antibodies in aqueous two-phase systems.

Science.gov (United States)

Rosa, P A J; Azevedo, A M; Ferreira, I F; de Vries, J; Korporaal, R; Verhoef, H J; Visser, T J; Aires-Barros, M R

2007-08-24

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.

Native human serum amyloid P component is a single pentamer

DEFF Research Database (Denmark)

Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH

1995-01-01

and ELISA, and SAP peak fractions were analysed by use of SDS-PAGE, Western blotting, and electron microscopy. The results indicate that native SAP circulates as a single pentamer, a part of which forms complexes with C4b-binding protein. The properties of SAP changed during purification as indicated...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Science.gov (United States)

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 ((NH4)2SO4). 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 which HIC media

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

Science.gov (United States)

Zang, Xiaonan; Zhang, Xuecheng; Mu, Xiaosheng; Liu, Bin

2013-03-01

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

Development of tantalum–zirconium alloy for hydrogen purification

Energy Technology Data Exchange (ETDEWEB)

Kumar, Sanjay, E-mail: sanjay.barc@gmail.com [Fusion Reactor Materials Section, MG, BARC, Mumbai 85 (India); IAMR, Hiroshima University, Higashihiroshima 739-8530 (Japan); Singh, Anamika [GSASM Hiroshima University, Higashihiroshima 739-8530 (Japan); Jain, Uttam; Dey, Gautam Kumar [Fusion Reactor Materials Section, MG, BARC, Mumbai 85 (India)

2016-11-01

Highlights: • Terminal solid solubility of Ta increases with Zr addition. • Increase in lattice parameters of Ta due to Zr addition may be the possible reason. • Enhance H solubility could also be explained on the change in e-DOS of Ta–Zr alloys. • Ta–Zr alloys could be possible combination for hydrogen purification membrane. - Abstract: Terminal solid solubility of hydrogen in Ta–Zr alloys has been studied in connection with the development of tantalum based metallic membrane for hydrogen/tritium purification. The alloys were prepared by vacuum arc melting technique and subsequently cold rolled to 0.2 mm thickness. The terminal solid solubility of hydrogen in these cold rolled samples was investigated in a modified Sieverts apparatus. The terminal solid solubility of hydrogen was marginally increased with zirconium content. The change in the lattices parameter of tantalum upon zirconium addition and the higher affinity of zirconium for hydrogen as compared to tantalum could be the possible reasons.

Modular microfluidics for point-of-care protein purifications.

Science.gov (United States)

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.

Silica-supported Macroporous Chitosan Bead for Affinity Purification of Trypsin Inhibitor

Institute of Scientific and Technical Information of China (English)

Feng Na XI; Jian Min WU; Ming Ming LUAN

2005-01-01

Macroporous cross-linking chitosan layer coated on silica gel (CTS-SiO2) was prepared by phase inversion and polyethylene glycol (PEG) molecular imprinting methods. Formation of macroporous surface was investigated by scanning electron microscopy (SEM) and BET analysis.The prepared bead was activated by reacting with 1,2-ethylene diglycidyl ether for introducing epoxy groups, and trypsin could be efficiently immobilized on the bead as a biospecific ligand.The bead bearing trypsin was employed to purify trypsin inhibitor (TIs) from egg white as affinity adsorbent.

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

Science.gov (United States)

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Purification of rat intestinal receptor for intrinsic factor-vitamin B12 complex

International Nuclear Information System (INIS)

Yamada, Shoji; Itaya, Harutaka; Nakazawa, Osamu; Fukuda, Morimichi.

1977-01-01

The intrinsic factor (IF) in a rat gastric mucosal extract was bound efficiently to vitamin B 12 -sepharose without significant change in its nature to produce IF-vitamin B 12 -sepharose. The purification of the intestinal receptor for the IF-vitamin B 12 complex was performed by the affinity chromatography using the IF-vitamin B 12 -sepharose as the affinity adsorbent. As a result of admixing the gastric mucosal extract sample with B 12 -sepharose while stirring for 4 hours, the adsorption was performed without any break through. Further, it was recognized that the B 12 -bound protein purified by the affinity chromatography using B 12 -sepharose was not much changed as compared with that before purification. Furthermore, it was recognized that IF-B 12 -sepharose was able to be made by binding IF with B 12 -sepharose which was made by coupling B 12 with the market-available AH-sepharose. The IF-B 12 -sepharose was washed with buffer solution, and then was loaded with the small intestine mucosal extract. Thereafter, the receptor was eluted by making di-valent cation inert with the buffer solution. After the removal of EDTA in the eluted solution by dialysis, the activity of the receptor was measured. 48.5% of the receptor activity loaded was recovered by the elution with EDTA. The specific activity of the receptor represented by the final amount of B 12 (pg)/the amount of protein (mg) in the purified substance was 335 folds of the original activity. (Iwakiri, K.)

Identification of Genes Enriched in GnRH Neurons by Translating Ribosome Affinity Purification and RNAseq in Mice.

Science.gov (United States)

Burger, Laura L; Vanacker, Charlotte; Phumsatitpong, Chayarndorn; Wagenmaker, Elizabeth R; Wang, Luhong; Olson, David P; Moenter, Suzanne M

2018-04-01

Gonadotropin-releasing hormone (GnRH) neurons are a nexus of fertility regulation. We used translating ribosome affinity purification coupled with RNA sequencing to examine messenger RNAs of GnRH neurons in adult intact and gonadectomized (GDX) male and female mice. GnRH neuron ribosomes were tagged with green fluorescent protein (GFP) and GFP-labeled polysomes isolated by immunoprecipitation, producing one RNA fraction enhanced for GnRH neuron transcripts and one RNA fraction depleted. Complementary DNA libraries were created from each fraction and 50-base, paired-end sequencing done and differential expression (enhanced fraction/depleted fraction) determined with a threshold of >1.5- or <0.66-fold (false discovery rate P ≤ 0.05). A core of ∼840 genes was differentially expressed in GnRH neurons in all treatments, including enrichment for Gnrh1 (∼40-fold), and genes critical for GnRH neuron and/or gonadotrope development. In contrast, non-neuronal transcripts were not enriched or were de-enriched. Several epithelial markers were also enriched, consistent with the olfactory epithelial origins of GnRH neurons. Interestingly, many synaptic transmission pathways were de-enriched, in accordance with relatively low innervation of GnRH neurons. The most striking difference between intact and GDX mice of both sexes was a marked downregulation of genes associated with oxidative phosphorylation and upregulation of glucose transporters in GnRH neurons from GDX mice. This may suggest that GnRH neurons switch to an alternate fuel to increase adenosine triphosphate production in the absence of negative feedback when GnRH release is elevated. Knowledge of the GnRH neuron translatome and its regulation can guide functional studies and can be extended to disease states, such as polycystic ovary syndrome.

Isolation and Purification of Jacalin from Artocarpus Heterophyllus Lam

Directory of Open Access Journals (Sweden)

M.R. Othman

2010-09-01

Full Text Available This paper presents investigation results of saturation conditions needed for purification of jacalin lectin from the extract seeds of Artocarpus heterophyllus by ammonium precipitation and affinity chromatography on Galactose-Affi gel Hz. Three different aspects of parameters encompassing the percentage of saturation of ammonium sulfate precipitation, the presence of ammonium sulfate on Lowry method and the suitable galactose concentration for optimum elution of the protein from Galactose-Affi gel Hz were investigated. With three different sets of fractional saturation of jacalin purification using ammonium sulfate precipitation, the maximum yield of 0.463 g/g was achieved at 0-90% saturation range in the absence of dialysis. Maximum yield of 0.425 g/g was obtained at 30-60% and 0-90% saturation range in the presence of dialysis. The result from this work also indicates that excessive quantity of NH4SO4 interferes with Lowry method for protein determination substantially. The 0-90% saturation range was found to be more potentially appropriate for large scale application than 30-60% saturation, since the former involves only 1 step NH4SO4 addition. From the affinity chromatography, elution of 0.2 M galactose (in 0.15 M NaCl from Galactose-Affi gel Hz produced the maximum peak profile and jacalin concentration. A reduction or increase in galactose concentration of more than 0.2 M did not increase concentration of purified jacalin purified using this method.

Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

Directory of Open Access Journals (Sweden)

Claire M. Gabe

2017-06-01

Full Text Available Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We

Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

DEFF Research Database (Denmark)

Andersen, P S; Geisler, C; Buus, S

2001-01-01

Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

A comprehensive review on biodiesel purification and upgrading

Directory of Open Access Journals (Sweden)

Hamed Bateni

2017-09-01

Full Text Available Serious environmental concerns regarding the use of fossil-based fuels have raised awareness regarding the necessity of alternative clean fuels and energy carriers. Biodiesel is considered a clean, biodegradable, and non-toxic diesel substitute produced via the transesterification of triglycerides with an alcohol in the presence of a proper catalyst. After initial separation of the by-product (glycerol, the crude biodiesel needs to be purified to meet the standard specifications prior to marketing. The presence of impurities in the biodiesel not only significantly affects its engine performance but also complicates its handling and storage. Therefore, biodiesel purification is an essential step prior to marketing. Biodiesel purification methods can be classified based on the nature of the process into equilibrium-based, affinity-based, membrane-based, reaction-based, and solid-liquid separation processes. The main adverse properties of biodiesel – namely moisture absorption, corrosiveness, and high viscosity – primarily arise from the presence of oxygen. To address these issues, several upgrading techniques have been proposed, among which catalytic (hydrodeoxygenation using conventional hydrotreating catalysts, supported metallic materials, and most recently transition metals in various forms appear promising. Nevertheless, catalyst deactivation (via coking and/or inadequacy of product yields necessitate further research. This paper provides a comprehensive overview on the techniques and methods used for biodiesel purification and upgrading.

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

Science.gov (United States)

Du, Ping; Sun, Shiqi; Dong, Jinjie; Zhi, Xiaoying; Chang, Yanyan; Teng, Zhidong; Guo, Huichen; Liu, Zaixin

2017-04-01

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification. Copyright © 2016. Published by Elsevier B.V.

Purification and characterization of mu-specific opioid receptor from rat brain

Energy Technology Data Exchange (ETDEWEB)

Hasegawa, J.; Cho, T.M.; Ge, B.L.; Loh, H.H.

1986-03-05

A mu-specific opioid receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, Ultrogel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by polyacrylamide gel electrophoresis, and was judged to be homogeneous by the following criteria: (1) a single band on the SDS gel; and (2) a specific opioid binding activity of 17,720 pmole/mg protein, close to the theoretical value. In addition, the 58,000 molecular weight value agrees closely with that determined by covalently labelling purified receptor with bromoacetyl-/sup 3/H-dihydromorphine or with /sup 125/I-beta-endorphin and dimethyl suberimidate. To their knowledge, this is the first complete purification of an opioid receptor that retains its ability to bind opiates.

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

Directory of Open Access Journals (Sweden)

Mora Xavier

2007-05-01

Full Text Available Abstract Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes in physiological salt (0.14 M NaCl at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl. The large differences in relative affinity of the H1 subtypes for

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

Science.gov (United States)

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain

DEFF Research Database (Denmark)

Krintel, Christian; Frydenvang, Karla; Ceravalls de Rabassa, Anna

2014-01-01

In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), L-Glu supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low affinity ligands because L-Glu is difficult to displace...... crystallized as a mixed dimer with L-Glu present in one subunit while neither L-Asp nor L-Glu were found in the other subunit. Thus, residual L-Glu is still present from the expression. On the other hand, only L-Asp was found at the binding site when using 50 mM or 250 mM L-Asp for crystallization. The binding...... mode observed for L-Asp at the GluA2 LBD is very similar to that described for L-Glu. Taken together, we have shown that L-Asp can be used instead of L-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low affinity ligands for lead...

Specificity and affinity quantification of protein-protein interactions.

Science.gov (United States)

Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

2013-05-01

Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

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

NARCIS (Netherlands)

J. Groen (Jan); N. Juntti; J.S. Teppema; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert); G.F. Rimmelzwaan (Guus)

1987-01-01

textabstractImmuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an

Isolation and purification of porcine LH for radioimmunoassay and radioreceptor assay

International Nuclear Information System (INIS)

Ziecik, A.; Goralska, M.; Krzymowski, T.; Pogorzelski, K.

1979-01-01

The procedure of isolation and purification of LH from porcine pituitary glands is described. From 1 kg of pituitary glands 150 mg of LH GPZ-1 preparation of high purity were obtained. Immunization of rabbits with the prepared hormone gave homogeneous antibodies against porcine LH with high affinity and low cross-reactions with FSH. Radioreceptor assay with the use of the prepared porcine LH demonstrated the high capacity of cell membrane receptors of the boar tests for binding this hormone. (author)

High quality protein microarray using in situ protein purification

Directory of Open Access Journals (Sweden)

Fleischmann Robert D

2009-08-01

Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

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

Science.gov (United States)

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. Copyright © 2014 John Wiley & Sons, Ltd.

Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

Science.gov (United States)

Horejsí, V; Tichá, M; Kocourek, J

1977-09-29

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

Influence of metallic surface states on electron affinity of epitaxial AlN films

Energy Technology Data Exchange (ETDEWEB)

Mishra, Monu; Krishna, Shibin; Aggarwal, Neha [Advanced Materials and Devices Division, CSIR-National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi110012 (India); Academy of Scientific and Innovative Research (AcSIR), CSIR-NPL Campus, Dr. K.S. Krishnan Marg, New Delhi 110012 (India); Gupta, Govind, E-mail: govind@nplindia.org [Advanced Materials and Devices Division, CSIR-National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi110012 (India); Academy of Scientific and Innovative Research (AcSIR), CSIR-NPL Campus, Dr. K.S. Krishnan Marg, New Delhi 110012 (India)

2017-06-15

The present article investigates surface metallic states induced alteration in the electron affinity of epitaxial AlN films. AlN films grown by plasma-assisted molecular beam epitaxy system with (30% and 16%) and without metallic aluminium on the surface were probed via photoemission spectroscopic measurements. An in-depth analysis exploring the influence of metallic aluminium and native oxide on the electronic structure of the films is performed. It was observed that the metallic states pinned the Fermi Level (FL) near valence band edge and lead to the reduction of electron affinity (EA). These metallic states initiated charge transfer and induced changes in surface and interface dipoles strength. Therefore, the EA of the films varied between 0.6–1.0 eV due to the variation in contribution of metallic states and native oxide. However, the surface barrier height (SBH) increased (4.2–3.5 eV) adversely due to the availability of donor-like surface states in metallic aluminium rich films.

Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

Directory of Open Access Journals (Sweden)

TIJANA SAVIC

2006-02-01

Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

Large-Scale Purification and Characterization of Barley Limit Dextrinase, a Member of the α-Amylase Structural Family

DEFF Research Database (Denmark)

Kristensen, Michael; Planchot, Véronique; Abe, Jun-ichi

1998-01-01

Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol...... consists of precipitation from the extract at 20-70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on b-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mM b-cyclodextrin and contains...

Recombinant spider silk genetically functionalized with affinity domains.

Science.gov (United States)

Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

2014-05-12

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Science.gov (United States)

Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

2000-01-01

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

Dye-Affinity Nanofibrous Membrane for Adsorption of Lysozyme: Preparation and Performance Evaluation

Directory of Open Access Journals (Sweden)

Steven Sheng-Shih Wang

2018-01-01

Full Text Available Polyacrylonitrile (PAN nanofibrous membrane was prepared by an electrospinning technique. After heat treatment and alkaline hydrolysis, the weak ion exchange membrane was grafted with chitosan molecule and then covalently immobilized with a Cibacron Blue F3GA (CB. Fibre diameter, porosity and pore size of the membrane and immobilized dye density were characterized. Furthermore, the membrane was applied to evaluate the binding performance of lysozyme under various operating parameters (pH, chitosan mass per volume ratio, dye concentration, ionic strength and temperature in batch mode. The experimental results were directly applied to purify lysozyme from chicken egg white by membrane chromatography. The results showed that the capture efficiency, recovery yield and purification factor were 90 and 87 %, and 47-fold, respectively, in a single step. The binding capacity remained consistent after five repeated cycles of adsorption-desorption operations. This work demonstrates that the dye-affinity nanofibrous membrane holds great potential for purification of lysozyme from real feedstock.

Chromatographic techniques used in the laboratory scale fractionation and purification of plasma

International Nuclear Information System (INIS)

Siti Najila Mohd Janib; Wan Hamirul Bahrin Wan Kamal; Shaharuddin Mohd

2004-01-01

Chromatography is a powerful technique used in the separation as well as purification of proteins for use as biopharmaceuticals or medicines. Scientists use many different chromatographic techniques in biotechnology as they bring a molecule from its initial identification stage to the stage of it becoming a marketed product. The most commonly used of these techniques is liquid chromatography (1,C). This technique can be used to separate the target molecule from undesired contaminants, as well as to analyse the final product for the requisite purity as established by governmental regulatory groups such as the FDA. Some examples of LC techniques include: ion exchange (IEC), hydrophobic interaction (HIC), gel filtration (GF), affinity (AC) and reverse phase (RPC) chromatography. These techniques are very versatile and can be used at any stage of the purification process i.e. capture, intermediate purification phase and polishing. The choice of a particular technique is dependent upon the nature of the target protein as well as its intended final use. This paper describes the preliminary work done on the chromatographic purification of factor VIII (FVIII), factor IX (FIX), albumin and IgG from plasma. Results, in particular, in the isolation of albumin and IgG using IEC, have been promising. Preparation and production of cryoprecipitate to yield FVIII and FIX have also been successful. (Author)

Large-scale functional purification of recombinant HIV-1 capsid.

Directory of Open Access Journals (Sweden)

Magdeleine Hung

Full Text Available During human immunodeficiency virus type-1 (HIV-1 virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.

Isolation, one-step affinity purification, and characterization of a polyextremotolerant laccase from the halophilic bacterium Aquisalibacillus elongatus and its application in the delignification of sugar beet pulp.

Science.gov (United States)

Rezaei, Shahla; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali

2017-04-01

The aim of the present work was to study the ability of a halophilic bacterial laccase to efficient delignification in extreme conditions. Here, a highly stable extracellular laccase showing ligninolytic activity from halophilic Aquisalibacillus elongatus is described. The laccase production was strongly influenced by NaCl and CuSO 4 and under optimal conditions reached 4.8UmL -1 . The monomeric enzyme of 75kDa was purified by a synthetic affinity column with 68.2% yield and 99.8-fold purification. The enzyme showed some valuable features viz. stability against a wide range of organic solvents, salts, metals, inhibitors, and surfactants and specificity to a wide spectrum of substrates diverse in structure and redox potential. It retained more than 50% of the original activity at 25-75°C and pH 5.0-10.0. Furthermore, the enzyme was found to be effective in the delignification of sugar beet pulp in an ionic liquid that makes it useful for industrial applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system.

Science.gov (United States)

de Albuquerque Wanderley, Maria Carolina; Wanderley Duarte Neto, José Manoel; Campos Albuquerque, Wendell Wagner; de Araújo Viana Marques, Daniela; de Albuquerque Lima, Carolina; da Cruz Silvério, Sara Isabel; de Lima Filho, José Luiz; Couto Teixeira, José António; Porto, Ana Lúcia Figueiredo

2017-05-01

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method. Copyright © 2017 Elsevier Inc. All rights reserved.

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.

Science.gov (United States)

Ogata, Makoto; Kameshima, Yumiko; Hattori, Takeshi; Michishita, Kousuke; Suzuki, Tomohiro; Kawagishi, Hirokazu; Totani, Kazuhide; Hiratake, Jun; Usui, Taichi

2010-12-10

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters. Copyright © 2010 Elsevier Ltd. All rights reserved.

Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor.

NARCIS (Netherlands)

Ratnala, V.R.; Swarts, H.G.P.; Oostrum, J. van; Leurs, R.; Groot, H.J.M. de; Bakker, R.; Grip, W.J. de

2004-01-01

This report describes an efficient strategy for amplified functional purification of the human H1 receptor after heterologous expression in Sf9 cells. The cDNA encoding a C-terminally histidine-tagged (10xHis) human histamine H1 receptor was used to generate recombinant baculovirus in a Spodoptera

Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.

Science.gov (United States)

Ahn, Mi Young; Hahn, Bum-Soo; Ryu, Kang Sun; Hwang, Jae Sam; Kim, Yeong Shik

2005-07-01

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

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

Science.gov (United States)

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

2017-01-01

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

Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC

Czech Academy of Sciences Publication Activity Database

Sadílková, Lenka; Osička, Radim; Šulc, Miroslav; Linhartová, Irena; Novák, Petr; Šebo, Peter

2008-01-01

Roč. 17, č. 10 (2008), s. 1834-1843 ISSN 0961-8368 R&D Projects: GA AV ČR KAN200520702; GA ČR GA310/06/0720 Institutional research plan: CEZ:AV0Z50200510 Keywords : asp-pro bond * frpc * purification Subject RIV: EE - Microbiology, Virology Impact factor: 3.115, year: 2008

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

Directory of Open Access Journals (Sweden)

Ram Sarup Singh

Full Text Available Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis.Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay.Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae.This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

Science.gov (United States)

Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The

Purification and Autoactivation Method for Recombinant Coagulation Factor VII.

Science.gov (United States)

Granovski, Vladimir; Freitas, Marcela C C; Abreu-Neto, Mario Soares; Covas, Dimas T

2018-01-01

Recombinant coagulation factor VII is a very important and complex protein employed for treatment of hemophiliac patients (hemophilia A/B) who develop inhibitors antibodies to conventional treatments (FVIII and FIX). The rFVII is a glycosylated molecule and circulates in plasma as zymogen of 50 kDa. When activated the molecule is cleaved to 20-30 kDa and has a half-life of about 3 h, needing to be processed fast and efficiently until freeze-drying. Here, we describe a very simple and fast purification sequence for rFVII using affinity FVII Select resin and a dialysis system that can be easily scaled up.

A new mannose-specific lectin from daylily (Hemerocallis fulva L. rhizome: purification and properties

Directory of Open Access Journals (Sweden)

V. O. Antonyuk

2013-04-01

Full Text Available A new lectin was purified from the daylily (Hemerocallis fulva L. with the yield of approximately 10 mg per kg of fresh plant rhizome. The purification procedure was based on application of the affinity chromathography on the column with yeast mannan and the ion-exchange chromatography on the column with DEAE-Toyopearl. The lectin possessed low affinity for α-methyl-D-mannopyranoside, D-fructose, D-turanose and 2-acetamido-D-galactopyranose and hight affinity for the yeast mannan. The lectin bound with greatly less affinity for the mannose-containig glycoproteins, such as ovoalbumin, ovomucoid and horseradish peroxidase. According to the results of electrophoresis in 20% DSNa-PAGE, the lectin consists of subunits of 12 kDa molecular weight. According to the results of gel-chromatography on the Toyopearl HW-55, the lectin’s molecular weight is 48 kDa. It agglutinated rabbit erythrocytes very well, while rat and guinea-pig erythrocytes were agglutinated worse, and human erythrocytes were not agglutinated at all. Lectin’s dialysis against 1% EDTA or heating to 60 ºC for 60 min did not stop its hemagglutinating activity.

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

Science.gov (United States)

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

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

Science.gov (United States)

Li, Xu; Gao, Min; Choi, Jong Min; Kim, Beom-Jun; Zhou, Mao-Tian; Chen, Zhen; Jain, Antrix N; Jung, Sung Yun; Yuan, Jingsong; Wang, Wenqi; Wang, Yi; Chen, Junjie

2017-04-01

Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined c lustered, r egularly i nterspaced s hort p alindromic r epeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

OpenAIRE

Boopathy, R; Balasubramanian, A S

1986-01-01

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel elec...

Effects of monitor examination intervals on resource affinity by Reticulitermes spp. (Isoptera: Rhinotermitidae)

Science.gov (United States)

Thomas Shelton; Terence Wagner; Patrick Gerard

2011-01-01

Monthly visits to 2 field sites in southern Mississippi were made to determine the influence of monitor examination on feeding site affinity by southeastern native subterranean termites (Reticulitermes spp.). Wooden board monitors were examined once every 30, 60, or 90 days. Presence of live termites and damage to the boards (both binary parameters) were collected for...

Simultaneous isolation of mRNA and native protein from minute samples of cells

DEFF Research Database (Denmark)

Petersen, Tonny Studsgaard; Andersen, Claus Yding

2014-01-01

Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

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

Directory of Open Access Journals (Sweden)

Hideyuki Arimitsu

Full Text Available 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.

Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

International Nuclear Information System (INIS)

Beck, J.T.; Ullman, B.

1989-01-01

An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 μM concentration of either activated [ 3 H]folate or activated [ 3 H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms

Purification of biomaterials by phase partitioning

Science.gov (United States)

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.

Affine and quasi-affine frames for rational dilations

DEFF Research Database (Denmark)

Bownik, Marcin; Lemvig, Jakob

2011-01-01

In this paper we extend the investigation of quasi-affine systems, which were originally introduced by Ron and Shen [J. Funct. Anal. 148 (1997), 408-447] for integer, expansive dilations, to the class of rational, expansive dilations. We show that an affine system is a frame if, and only if......, the corresponding family of quasi-affine systems are frames with uniform frame bounds. We also prove a similar equivalence result between pairs of dual affine frames and dual quasi-affine frames. Finally, we uncover some fundamental differences between the integer and rational settings by exhibiting an example...

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

Science.gov (United States)

Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

2018-02-23

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression, purification and crystallization of an atypical class C acid phosphatase from Mycoplasma bovis

International Nuclear Information System (INIS)

Singh, Harkewal; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

2011-01-01

Methods for the expression, purification and crystallization of the class C acid phosphatase from M. bovis are reported. This enzyme is atypical in that it is nearly 20 kDa larger than other known class C acid phosphatases. Class C acid phosphatases (CCAPs) are 25–30 kDa bacterial surface proteins that are thought to function as broad-specificity 5′,3′-nucleotidases. Analysis of the newly published complete genome sequence of Mycoplasma bovis PG45 revealed a putative CCAP with a molecular weight of 49.9 kDa. The expression, purification and crystallization of this new family member are described here. Standard purification procedures involving immobilized metal-ion affinity chromatography and ion-exchange chromatography yielded highly pure and crystallizable protein. Crystals were grown in sitting drops at room temperature in the presence of PEG 3350 and HEPES buffer pH 7.5 and diffracted to 2.3 Å resolution. Analysis of diffraction data suggested a primitive monoclinic space group, with unit-cell parameters a = 78, b = 101, c = 180 Å, β = 92°. The asymmetric unit is predicted to contain six molecules, which are likely to be arranged as three dimers

Isolation, purification, and partial characterization of a membrane-bound Cl-/HCO3--activated ATPase complex from rat brain with sensitivity to GABAAergic ligands.

Science.gov (United States)

Menzikov, Sergey A

2017-02-07

This study describes the isolation and purification of a protein complex with [Formula: see text]-ATPase activity and sensitivity to GABA A ergic ligands from rat brain plasma membranes. The ATPase complex was enriched using size-exclusion, affinity, and ion-exchange chromatography. The fractions obtained at each purification step were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed four subunits with molecular mass ∼48, 52, 56, and 59 kDa; these were retained at all stages of the purification process. Autoradiography revealed that the ∼52 and 56 kDa subunits could bind [ 3 H]muscimol. The [Formula: see text]-ATPase activity of this enriched protein complex was regulated by GABA A ergic ligands but was not sensitive to blockers of the NKCC or KCC cotransporters.

Expression, purification and auto-activation of cathepsin E from insect cells.

Science.gov (United States)

Železnik, Tajana Z; Puizdar, Vida; Dolenc, Iztok

2015-01-01

Cathepsin E is an aspartic protease that belongs to the pepsin family. This protease is similar to cathepsin D but differs in its tissue distribution and cell localization. Elevated levels of this enzyme are linked to several tumors, including devastating pancreatic ductal adenocarcinoma. In this manuscript, we present a new protocol for the high-yield purification of recombinant human cathepsin E in the baculovirus expression system. The recombinant protein was produced by the Sf9 insect cell line and secreted into the medium in the form of an inactive zymogen. Procathepsin E was purified using ion-exchange and size exclusion chromatographies followed by pepstatin- and heparin-affinity chromatography steps. The zymogen was activated at an acidic pH, resulting in a high yield of the activated intermediate of cathepsin E. The enzymatic activity, stability, and molecular weight corresponded to those of cathepsin E. The new purification procedure will promote further studies of this enzyme to improve the understanding of its structure-function relationship and consequently enable the development of better therapeutic approaches.

An Ecosystem-Service Approach to Evaluate the Role of Non-Native Species in Urbanized Wetlands

Directory of Open Access Journals (Sweden)

Rita S. W. Yam

2015-04-01

Full Text Available Natural wetlands have been increasingly transformed into urbanized ecosystems commonly colonized by stress-tolerant non-native species. Although non-native species present numerous threats to natural ecosystems, some could provide important benefits to urbanized ecosystems. This study investigated the extent of colonization by non-native fish and bird species of three urbanized wetlands in subtropical Taiwan. Using literature data the role of each non-native species in the urbanized wetland was evaluated by their effect (benefits/damages on ecosystem services (ES based on their ecological traits. Our sites were seriously colonized by non-native fishes (39%–100%, but <3% by non-native birds. Although most non-native species could damage ES regulation (disease control and wastewater purification, some could be beneficial to the urbanized wetland ES. Our results indicated the importance of non-native fishes in supporting ES by serving as food source to fish-eating waterbirds (native, and migratory species due to their high abundance, particularly for Oreochromis spp. However, all non-native birds are regarded as “harmful” species causing important ecosystem disservices, and thus eradication of these bird-invaders from urban wetlands would be needed. This simple framework for role evaluation of non-native species represents a holistic and transferable approach to facilitate decision making on management priority of non-native species in urbanized wetlands.

Multimodal charge-induction chromatography for antibody purification.

Science.gov (United States)

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

2016-01-15

Hydrophobic charge-induction chromatography (HCIC) has advantages of high capacity, salt-tolerance and convenient pH-controlled elution. However, the binding specificity might be improved with multimodal molecular interactions. New ligand W-ABI that combining tryptophan and 5-amino-benzimidazole was designed with the concept of mutimodal charge-induction chromatography (MCIC). The indole and benzimidazole groups of the ligand could provide orientated mutimodal binding to target IgG under neutral pH, while the imidazole groups could induce the electrostatic repulsion forces for efficient elution under acidic pH. W-ABI ligand was coupled successfully onto agarose gel, and IgG adsorption behaviors were investigated. High affinity to IgG was found with the saturated adsorption capacity of 70.4 mg/ml at pH 7, and the flow rate of mobile phase showed little impact on the dynamic binding capacity. In addition, efficient elution could be achieved at mild acidic pH with high recovery. Two separation cases (IgG separation from albumin containing feedstock and monoclonal antibody purification from cell culture supernatant) were verified with high purity and recovery. In general, MCIC with the specially-designed ligand is an expanding of HCIC with improved adsorption selectivity, which would be a potential alternative to Protein A-based capture for the cost-effective purification of antibodies. Copyright © 2015 Elsevier B.V. All rights reserved.

Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

Science.gov (United States)

Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

2017-10-01

Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l -1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

Science.gov (United States)

Pohl, Thomas; Uhlmann, Mareike; Kaufenstein, Miriam; Friedrich, Thorsten

2007-09-18

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.

The universal statistical distributions of the affinity, equilibrium constants, kinetics and specificity in biomolecular recognition.

Directory of Open Access Journals (Sweden)

Xiliang Zheng

2015-04-01

Full Text Available We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity, the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics.

Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii.

Science.gov (United States)

Stressler, Timo; Tanzer, Coralie; Ewert, Jacob; Claaßen, Wolfgang; Fischer, Lutz

2017-03-01

The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His 6 -tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His 6 -tag harboring PepA; the K M value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

The development of a purification procedure for saxitoxin-induced protein.

Science.gov (United States)

Smith, D S; Kitts, D D; Fenske, B; Owen, T G; Shyng, S

1995-02-01

A simple economical procedure for purifying saxitoxin-induced protein (SIP) from crude extracts of the small shore crab, Hemigrapsus oregenesis, was developed. (NH4)2SO4 precipitation, chymotrypsin digestion, heat treatment, gel filtration and ion-exchange-chromatography procedures were evaluated in purifying SIP. An enzyme immunoassay was used to determine the SIP yield and relative purity at each step of three procedures, thus permitting an assessment of the conditions required for maximum recovery. Response surface analysis was used in an attempt to determine the optimum temperature and exposure time for the heat treatment. A 20 min incubation at 65 degrees C was confirmed by electrophoretic analysis to be the best combination of time and temperature for achieving both an acceptable yield and purity of SIP. SIP in desalted concentrate was shown to be resistant to chymotrypsin proteolysis; however, this enzyme had deleterious effects on SIP purification at later stages of the procedure. The omission of the chymotrypsin digestion, and the inclusion of gel-filtration chromatography in the final clean-up step, resulted in the purification of SIP comparable with that achieved with affinity chromatography.

A new polyethyleneglycol-derivatized hemoglobin derivative with decreased oxygen affinity and limited toxicity.

Science.gov (United States)

Zolog, Oana; Mot, Augustin; Deac, Florina; Roman, Alina; Fischer-Fodor, Eva; Silaghi-Dumitrescu, Radu

2011-01-01

A new protocol is described for derivatization of hemoglobin with polyethyleneglycol (PEG) via reaction of the unmodified native hemoglobin with an activated amine-reacting polyethylene glycol derivative which, unlike protocols previously described, leads to formation of a peptide bond between hemoglobin and PEG. Dioxygen binding and peroxide reactivities of the derivatized hemoglobin are examined, and found to be within reasonable limits, with the particular observation that, unlike with a few other derivatization protocols, the dioxygen affinity is slightly lower than that of native Hb. In cell culture tests (human umbilical vein epithelial cells, HUVEC), the derivatization protocol induces no toxic effect. These results show promise towards applicability for production of hemoglobin-based blood substitutes.

Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host.

Science.gov (United States)

Noda, Shuhei; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

2015-01-13

Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host. In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in E. coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of S. lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by E. coli. The amount of Sav using S. lividans was about 9 times higher than using E. coli. Surprisingly, streptavidin produced by S. lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling. We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance.

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Directory of Open Access Journals (Sweden)

P. Hauk

2011-04-01

Full Text Available Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272 was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272 per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272 produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira.

Science.gov (United States)

Hauk, P; Carvalho, E; Ho, P L

2011-04-01

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Purification and characterization of amine oxidase from soybean seedlings.

Science.gov (United States)

Vianello, F; Di Paolo, M L; Stevanato, R; Gasparini, R; Rigo, A

1993-11-15

A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.

MHC class II tetramers made from isolated recombinant α and β chains refolded with affinity-tagged peptides

DEFF Research Database (Denmark)

Braendstrup, Peter; Justesen, Sune Frederik Lamdahl; Osterbye, Thomas

2013-01-01

Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking...... effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding...... a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides...

The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

Energy Technology Data Exchange (ETDEWEB)

Briers, Yves [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Schmelcher, Mathias; Loessner, Martin J. [Institute of Food Science and Nutrition, ETH Zuerich, Schmelzbergstrasse 7, CH-8092 Zuerich (Switzerland); Hendrix, Jelle; Engelborghs, Yves [Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200G, B-3001 Leuven (Belgium); Volckaert, Guido [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Lavigne, Rob, E-mail: rob.lavigne@biw.kuleuven.be [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium)

2009-05-29

The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled receptors.

Science.gov (United States)

Whitehurst, Charles E; Annis, D Allen

2008-07-01

Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on mass spectrometry to identify compounds that preferentially bind to a protein target. Of the many affinity selection-mass spectrometry techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine high throughput screening platforms. Because affinity selection-mass spectrometry techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by employing mass spectrometry for ligand detection, these techniques enable high throughput screening of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein coupled receptors yield the highest percentage of therapeutically effective drugs. In this manuscript, we present the emerging application of affinity selection-mass spectrometry to the high throughput screening of G protein coupled receptors. We also review how affinity selection-mass spectrometry can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target-ligand binding interactions, enabling the classification of orthosteric and allosteric binders.

Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

Science.gov (United States)

Keefe, Andrew J.; Jiang, Shaoyi

2012-01-01

Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from Tetrahymena thermophila.

OpenAIRE

Lu, Q; Schierer, T; Kang, S G; Henderson, E

1998-01-01

G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most high...

Molecular electron affinities

International Nuclear Information System (INIS)

Fukuda, E.K.

1983-01-01

Molecular electron affinities have historically been difficult quantities to measure accurately. These difficulties arise from differences in structure between the ion and neutral as well as the existence of excited negative ion states. To circumvent these problems, relative electron affinities were determined in this dissertation by studying equilibrium electron transfer reactions using a pulsed ion cyclotron resonance (ICR) spectrometer. Direct measurement of ion and neutral concentrations for reactions of the general type, A - + B = B - + A, allow calculation of the equilibrium constant and, therefore, the free energy change. The free energy difference is related to the difference in electron affinities between A and B. A relative electron affinity scale covering a range of about 45 kcal/mol was constructed with various substituted p-benzoquinones, nitrobenzenes, anhydrides, and benzophenones. To assign absolute electron affinities, various species with accurately known electron affinities are tied to the scale via ion-cyclotron double resonance bracketing techniques. After the relative scale is anchored to these species with well-known electron affinities, the scale is then used as a check on other electron affinity values as well as generating new electron affinity values. Many discrepancies were found between the electron affinities measured using the ICR technique and previous literature determinations

Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

International Nuclear Information System (INIS)

Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

1987-01-01

The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

Science.gov (United States)

Plaga, W; Lottspeich, F; Oesterhelt, D

1992-04-01

An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production

Directory of Open Access Journals (Sweden)

Endo Yaeta

2008-03-01

Full Text Available Abstract Background The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. Results We designed four ligation independent cloning (LIC vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Conclusion Four newly

Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

Science.gov (United States)

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2016-02-01

Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression, purification, crystallization and preliminary X-ray diffraction studies of the human keratin 4-binding domain of serine-rich repeat protein 1 from Streptococcus agalactiae

International Nuclear Information System (INIS)

Sundaresan, Ramya; Samen, Ulrike; Ponnuraj, Karthe

2011-01-01

Expression, purification and crystallization of Srr-1-K4BD, a human keratin 4-binding domain of serine-rich repeat protein 1 from S. agalactiae, was carried out. Native crystals of Srr-1-K4BD diffracted to 3.8 Å resolution using synchrotron radiation. Serine-rich repeat protein 1 (Srr-1) is a surface protein from Streptococcus agalactiae. A 17 kDa region of this protein has been identified to bind to human keratin 4 (K4) and is termed the Srr-1 K4-binding domain (Srr-1-K4BD). Recombinant Srr-1-K4BD was overexpressed in Escherichia coli BL21 (DE3) cells. Native and selenomethionine-substituted proteins were prepared using Luria–Bertani (LB) and M9 minimal media, respectively. A two-step purification protocol was carried out to obtain a final homogenous sample of Srr-1-K4BD. Crystals of native Srr-1-K4BD were obtained using PEG 3350 as a precipitant. The crystals diffracted to 3.8 Å resolution using synchrotron radiation and belonged to space group P2 1 , with unit-cell parameters a = 47.56, b = 59.48, c = 94.71 Å, β = 93.95°

The utility of affine variables and affine coherent states

International Nuclear Information System (INIS)

Klauder, John R

2012-01-01

Affine coherent states are generated by affine kinematical variables much like canonical coherent states are generated by canonical kinematical variables. Although all classical and quantum formalisms normally entail canonical variables, it is shown that affine variables can serve equally well for many classical and quantum studies. This general purpose analysis provides tools to discuss two major applications: (1) the completely successful quantization of a nonrenormalizable scalar quantum field theory by affine techniques, in complete contrast to canonical techniques which only offer triviality; and (2) a formulation of the kinematical portion of quantum gravity that favors affine kinematical variables over canonical kinematical variables, and which generates a framework in which a favorable analysis of the constrained dynamical issues can take place. All this is possible because of the close connection between the affine and the canonical stories, while the few distinctions can be used to advantage when appropriate. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’. (review)

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

International Nuclear Information System (INIS)

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

1990-01-01

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 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- 125 I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein

Radioligand purification prior to routine receptor assays

International Nuclear Information System (INIS)

Le Goff, J.-M.; Berthois, Y.; Martin, P.-M.

1988-01-01

The need to repurify the commercially available radioligands [ 3 H]estradiol and [ 3 H]testosterone before use in routine assays was investigated. Storage of these products for 2 months after delivery led to appreciable degradation of [ 3 H]estradiol compared to [ 3 H]testosterone. Unexpectedly, TLC and even HPLC procedures were ineffective in completely restoring the purity of [ 3 H]-estradiol and the unremoved polar products induced important variations in our estrogen receptor assays. An increase in non-specific binding and a concomitant decrease in total binding were observed resulting in an underestimation of specific binding sites and of the affinity constant. In some cases Scatchard analysis was not possible. The authors therefore strongly recommend the repurification of low-stability radioligands and propose an economic time-saving procedure for the purification of [ 3 H]estradiol by solvent differential partition which requires no high-cost investment in apparatus. (author)

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

Science.gov (United States)

Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

2018-04-01

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

International Nuclear Information System (INIS)

Iourin, Oleg; Harlos, Karl; El Omari, Kamel; Lu, Weixian; Kadlec, Jan; Iqbal, Munir; Meier, Christoph; Palmer, Andrew; Jones, Ian; Thomas, Carole; Brownlie, Joe; Grimes, Jonathan M.; Stuart, David I.

2012-01-01

The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the ectodomain of BVDV E2 are described. Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress

Expression, purification and crystallization of a lyssavirus matrix (M) protein

Energy Technology Data Exchange (ETDEWEB)

Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

2008-04-01

The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

Expression, purification and crystallization of a lyssavirus matrix (M) protein

International Nuclear Information System (INIS)

Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

2008-01-01

The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

Report: Affinity Chromatography.

Science.gov (United States)

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)

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

Directory of Open Access Journals (Sweden)

Yuichi Ikeda

Full Text Available 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.

A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate

DEFF Research Database (Denmark)

Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose

2014-01-01

, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover...... was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity...... purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism....

Automated Purification and Suspension Array Detection of 16S rRNA from Soil and Sediment Extracts by Using Tunable Surface Microparticles

OpenAIRE

Chandler, Darrell P.; Jarrell, Ann E.

2004-01-01

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and...

Information encoded in non-native states drives substrate-chaperone pairing.

Science.gov (United States)

Mapa, Koyeli; Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik

2012-09-05

Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

International Nuclear Information System (INIS)

White, Tommi A.; Tanner, John J.

2005-01-01

Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2 1 2 1 2 1 , with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

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

Science.gov (United States)

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

2015-01-01

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

Purification of Anthocyanins with o-Dihydroxy Arrangement by Sorption in Cationic Resins Charged with Fe(III

Directory of Open Access Journals (Sweden)

Araceli Castañeda-Ovando

2014-01-01

Full Text Available In the present work, a new purification method of anthocyanins with o-dihydroxy arrangement is proposed. This method is based on a ligand-exchange mechanism, using a cationic exchange resin loaded with metallic ions in order to increase the affinity of the resin to the anthocyanin(s with o-dihydroxy arrangement. This method was used to purify the main anthocyanin (cyanidin-3-glucoside; Cy-3-glc from the anthocyanic methanolic extract of blue corn. The best sorption result was using Fe(III in its ion form. The purification procedure begins with the formation of a metal-anthocyanin complex (Cy-3-glc-Fe which was optimal at pH 5, followed by a NaOH 0.1 M elution process in order to eliminate anthocyanins without o-dihydroxy arrangement, sugars, and organic acids. Finally, the pure anthocyanin is obtained by adding HCl 0.1 M which breaks the metal-anthocyanin complex.

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

Science.gov (United States)

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

In vivo biotinylation of recombinant beta-glucosidase enables simultaneous purification and immobilization on streptavidin coated magnetic particles

DEFF Research Database (Denmark)

Alftrén, Johan; Ottow, Kim Ekelund; Hobley, Timothy John

2013-01-01

Beta-glucosidase from Bacillus licheniformis was in vivo biotinylated in Escherichia coli and subsequently immobilized directly from cell lysate on streptavidin coated magnetic particles. In vivo biotinylation was mediated by fusing the Biotin Acceptor Peptide to the C-terminal of beta......-glucosidase and co-expressing the BirA biotin ligase. The approach enabled simultaneous purification and immobilization of the enzyme from crude cell lysate on magnetic particles because of the high affinity and strong interaction between biotin and streptavidin. After immobilization of the biotinylated beta...

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

Science.gov (United States)

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

Directory of Open Access Journals (Sweden)

Zhiqiang Yan

Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

Science.gov (United States)

Rehan, Shahid; Jaakola, Veli-Pekka

2015-10-01

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

Science.gov (United States)

Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

2012-01-01

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. PMID:22520159

Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

Science.gov (United States)

Liau, Nicholas P D; Laktyushin, Artem; Babon, Jeffrey J

2017-01-01

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

Effect of charcoal on water purification

OpenAIRE

Suzuki, Hirotaka; Kawahigashi, Tatsuo

2014-01-01

[Abstract] A natural basin system purifies water through self-purification, but the water pollution load of a river might exceed its self-purification capacity. Charcoal, which is used for other uses aside from heating, such as air purification, was evaluated experimentally for water quality purification. The experiment described herein is based on simple water quality measurements. Some experimentally obtained results are discussed.

Hirota's solitons in the affine and the conformal affine Toda models

International Nuclear Information System (INIS)

Aratyn, H.; Constantinidis, C.P.; Ferreira, L.A.; Gomes, J.F.; Zimerman, A.H.

1993-01-01

We use Hirota's method formulated as a recursive scheme to construct a complete set of soliton solutions for the affine Toda field theory based on an arbitrary Lie algebra. Our solutions include a new class of solitons connected with two different types of degeneracies encountered in Hirota's perturbation approach. We also derive an universal mass formula for all Hirota's solutions to the affine Toda model valid for all underlying Lie groups. Embedding of the affine Toda model in the conformal affine Toda model plays a crucial role in this analysis. (orig.)

The bubble method of water purification

Science.gov (United States)

Smirnov, B. M.; Babaeva, N. Yu.; Naidis, G. V.; Panov, V. A.; Saveliev, A. S.; Son, E. E.; Tereshonok, D. V.

2018-02-01

The processes of water purification from admixture molecules are analyzed. The purification rate is limited due to a low diffusion coefficient of the admixture molecules in water. At non-small concentrations of the admixture molecules, the water purication can proceed through association of molecules in condensed nanoparticles which fall on the bottom of the water volume. The rate of association may be increased in an external electric field, but in reality this cannot change significantly the rate of the purification process. The bubble method of water purification is considered, where air bubbles formed at the bottom of the water volume, transfer admixture molecules to the interface. This method allows one to clean small water volumes fast. This mechanism of water purification is realized experimentally and exhibits the promises of the bubble purification method.

Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

Directory of Open Access Journals (Sweden)

Seetha Ram Kotra

2014-03-01

Full Text Available Objective:Synthetic cationic antimicrobial peptide (SC-AMP is an important and upcoming therapeutic molecule against onventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods:The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method. Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3 and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain - intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a oncentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC of the purified SC-AMP was studied against both Gram positive and Gram negative microorganisms. Conclusion: For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of enzymatic cleavage. J Microbiol Infect Dis 2014;4(1:13-19

The solutions of affine and conformal affine Toda field theory

International Nuclear Information System (INIS)

Papadopoulos, G.; Spence, B.

1994-02-01

We give new formulations of the solutions of the field equations of the affine Toda and conformal affine Toda theories on a cylinder and two-dimensional Minkowski space-time. These solutions are parameterised in terms of initial data and the resulting covariant phase spaces are diffeomorphic to the Hamiltonian ones. We derive the fundamental Poisson brackets of the parameters of the solutions and give the general static solutions for the affine theory. (authors). 10 refs

Statistical and Judgmental Criteria for Scale Purification

DEFF Research Database (Denmark)

Wieland, Andreas; Durach, Christian F.; Kembro, Joakim

2017-01-01

of scale purification, to critically analyze the current state of scale purification in supply chain management (SCM) research and to provide suggestions for advancing the scale-purification process. Design/methodology/approach A framework for making scale-purification decisions is developed and used...

Aqueous two-phase system purification for superoxide dismutase induced by menadione from Phanerochaete chrysosporium.

Science.gov (United States)

Kavakcıoğlu, Berna; Tongul, Burcu; Tarhan, Leman

2017-03-01

In the present work, the partitioning behavior of menadione-induced superoxide dismutase (SOD; EC 1.15.1.1), an antioxidant enzyme that has various applications in the medical and cosmetic industries, from the white rot fungus Phanerochaete chrysosporium has been characterized on different types of aqueous two-phase systems (ATPSs) (poly(ethylene glycol)/polypropylene glycol (PEG/PPG)-dextran, PEG-salt and PPG-salt). PEG-salt combinations were found most optimal systems for the purification of SOD. The best partition conditions were found using the PEG-3350 24% and K 2 HPO 4 5% (w/w) with pH 7.0 at 25 °C. The partition coefficient of total SOD activity and total protein concentration observed in this system were 0.17 and 6.65, respectively, with the recovery percentage as 78.90% in the bottom phase and 13.17% in the top phase. The highest purification fold for SOD from P. chrysosporium was found as 6.04 in the bottom phase of PEG 3350%24 - K 2 HPO 4 %5 (w/w) system with pH 7.0. SOD purified from P. chrysosporium was determined to be a homodimer in its native state with a molecular weight of 60  ± 4 kDa. Consequently, simple and only one step PEG-salt ATPS system was developed for SOD purification from P. chrysosporium.

Lectin affinity electrophoresis.

Science.gov (United States)

Kobayashi, Yuka

2014-01-01

An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

Purification and characterization of Escherichia coli MreB protein.

Science.gov (United States)

Nurse, Pearl; Marians, Kenneth J

2013-02-01

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μM.

Purification and Characterization of Escherichia coli MreB Protein*

Science.gov (United States)

Nurse, Pearl; Marians, Kenneth J.

2013-01-01

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm. PMID:23235161

DYNAMIC MODELLING AND ADVANCED PREDICTIVE CONTROL OF A CONTINUOUS PROCESS OF ENZYME PURIFICATION

Directory of Open Access Journals (Sweden)

Dechechi E.C.

1997-01-01

Full Text Available A dynamic mathematical model, simulation and computer control of a Continuous Affinity Recycle Extraction (CARE process, a protein purification technique based on protein adsorption on solid-phase adsorbents is described in this work. This process, consisting of three reactors, is a multivariable process with considerable time delay in the on-line analyses of the controlled variable. An advanced predictive control configuration, specifically the Dynamic Matrix Control (DMC, was applied. The DMC algorithm was applied in process schemes where the aim was to maintain constant the enzyme concentration in the outlet of the third reactor. The performance of the DMC controller was analyzed in the feed-flow disturbances and the results are presented.

Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

Energy Technology Data Exchange (ETDEWEB)

White, Tommi A.; Tanner, John J., E-mail: tannerjj@missouri.edu [Departments of Chemistry and Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States)

2005-08-01

Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

Studies on Brucella interferon: Chromatographic behaviour and purification

International Nuclear Information System (INIS)

Bousquet-Ucla, C.; Wietzerbin, J.; Falcoff, E.

1980-01-01

Interferon was induced by infecting mice with Brucella suis. Serum containing interferon activity was analyzed by chromatography on Concanavalin A-Sepharose and Phenyl-Sepharose CL-4B columns. Antiviral activity was completely retained by the lectin column indicating that all the interferon molecules are glycosylated. The chromatographic behaviour of Brucella interferon on Phenyl-Sepharose CL-4B show that, like other interferons, Brucella interferon displays hydrophobic properties. However, the hydrophobicity of the interferon molecule was masked in the crude preparation and was only detectable when purified Brucella interferon was used for chromatography. The antigenic properties of Brucella interferon provided the means for developing an affinity chromatographic method resulting in about 60.000 fold purification. As in the case of viral interferon, treatment of L cells with Brucella interferon induced specific enhanced in vitro phosphorylation of a 67.000 molecular weight protein after incubation of cell extracts with doublestranded RNA and [γ- 32 p]ATP. (auth.)

Enhanced binding by dextran-grafting to Protein A affinity chromatographic media.

Science.gov (United States)

Zhao, Lan; Zhu, Kai; Huang, Yongdong; Li, Qiang; Li, Xiunan; Zhang, Rongyue; Su, Zhiguo; Wang, Qibao; Ma, Guanghui

2017-04-01

Dextran-grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose-based matrix, followed by epoxy-activation and Protein A coupling site-directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran-grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction-dried gel, increased by 24% compared with that of the non-grafted medium. The binding capacity of dextran-grafted medium decreased about 7% after 40 cleaning-in-place cycles, much lower than that of the non-grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran-grafted medium faster than that of non-grafted one. Atomic force microscopy showed that this dextran-grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non-grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high-performance dextran-grafted Protein A affinity chromatographic medium has promising applications in large-scale antibody purification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dataset concerning GroEL chaperonin interaction with proteins

Directory of Open Access Journals (Sweden)

V.V. Marchenkov

2016-03-01

Full Text Available GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020 [1], and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin and denatured (lysozyme, α-lactalbumin and pepsin proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

Purification of Water by Aquatic Plants

OpenAIRE

Morimitsu, Katsuhito; Kawahigashi, Tatsuo

2013-01-01

[Abstract] Water quality purification of many water systems including those occurring in rivers depends to a great degree on water quality purification activities of aquatic plants and microbes. This paper presents a discussion of results, based on laboratory experiments, of purification by aquatic plants.

Adsorption of endotoxins on Ca2+ -iminodiacetic acid by metal ion affinity chromatography.

Science.gov (United States)

Lopes, André Moreni; Romeu, Jorge Sánchez; Meireles, Rolando Páez; Perera, Gabriel Marquez; Morales, Rolando Perdomo; Pessoa, Adalberto; Cárdenas, Lourdes Zumalacárregui

2012-11-01

Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

Lp-dual affine surface area

Science.gov (United States)

Wei, Wang; Binwu, He

2008-12-01

According to the notion of Lp-affine surface area by Lutwak, in this paper, we introduce the concept of Lp-dual affine surface area. Further, we establish the affine isoperimetric inequality and the Blaschke-Santaló inequality for Lp-dual affine surface area. Besides, the dual Brunn-Minkowski inequality for Lp-dual affine surface area is presented.

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

Science.gov (United States)

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

2013-11-01

Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHproduct variants using the combined pH and salt based elution gradient and removal of the host cell impurities in flow-through are the key novel features of the developed multimodal chromatographic purification step. Copyright © 2013 Elsevier B.V. All rights reserved.

In vivo phosphorylation of a peptide tag for protein purification.

Science.gov (United States)

Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

2016-05-01

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

Comparing Russian and Finnish standards of water purification

OpenAIRE

Maria, Pupkova

2012-01-01

The subject of this thesis is water purification. The first aim of this thesis is to consider different ways of water purification. The second aim is to compare Finnish and Russian standards of water purification. The third one is to show water purification methods on the pattern of Mikkeli water purification plan. Water purification methods of water intended for human consumption will be described.Combined tables will be done according to the quality requirement of drinking water of both,...

Native fruit traits may mediate dispersal competition between native and non-native plants

Directory of Open Access Journals (Sweden)

Clare Aslan

2012-02-01

Full Text Available Seed disperser preferences may mediate the impact of invasive, non-native plant species on their new ecological communities. Significant seed disperser preference for invasives over native species could facilitate the spread of the invasives while impeding native plant dispersal. Such competition for dispersers could negatively impact the fitness of some native plants. Here, we review published literature to identify circumstances under which preference for non-native fruits occurs. The importance of fruit attraction is underscored by several studies demonstrating that invasive, fleshy-fruited plant species are particularly attractive to regional frugivores. A small set of studies directly compare frugivore preference for native vs. invasive species, and we find that different designs and goals within such studies frequently yield contrasting results. When similar native and non-native plant species have been compared, frugivores have tended to show preference for the non-natives. This preference appears to stem from enhanced feeding efficiency or accessibility associated with the non-native fruits. On the other hand, studies examining preference within existing suites of co-occurring species, with no attempt to maximize fruit similarity, show mixed results, with frugivores in most cases acting opportunistically or preferring native species. A simple, exploratory meta-analysis finds significant preference for native species when these studies are examined as a group. We illustrate the contrasting findings typical of these two approaches with results from two small-scale aviary experiments we conducted to determine preference by frugivorous bird species in northern California. In these case studies, native birds preferred the native fruit species as long as it was dissimilar from non-native fruits, while non-native European starlings preferred non-native fruit. However, native birds showed slight, non-significant preference for non-native fruit

Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin.

Science.gov (United States)

Narahari, Akkaladevi; Swamy, Musti J

2010-04-21

The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% alpha-helix, 35.8% beta-sheet, 22.5% beta-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4-70 degrees C, but a sharp decrease was seen between 75 and 85 degrees C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 degrees C, indicating that it is a relatively stable protein.

Purification and Partial Characterization of Catalase from Chicken Erythrocytes and the Effect of Various Inhibitors on Enzyme Activity

OpenAIRE

AYDEMİR, Tülin; KURU, Kevser

2003-01-01

Catalase plays a major role in the protection of tissues from the toxic effects of H2O2 and partially reduced oxygen species. A nearly 136-fold enzyme purification was obtained from chicken erythrocyte by acetone precipitation, ethanol-chloroform treatment, CM-cellulose and Sephadex G-200 chromatography. The specific activity of purified enzyme was 42,556 U/mg. The molecular weight of the native chicken erythrocyte catalase was estimated at 240 kDa by gel filtration. SDS-gel electr...

Rapid purification of recombinant histones.

Science.gov (United States)

Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

2014-01-01

The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

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

Energy Technology Data Exchange (ETDEWEB)

Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Elkak, Assem [Laboratory of “Valorisation des Ressources Naturelles et Produits de Santé (VRNPS)”, Doctoral School of Sciences and Technology, Lebanese University, Rafic Hariri University Campus, Hadath (Lebanon); Denizli, Adil, E-mail: denizli@hacettepe.edu [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

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 pH 7.4. 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). - Highlights: • Cu(II)-ions were coupled to PHEMA using PEI as the chelating ligand. • Cu(II) chelated [PHEMA/PEI] cryogels for IgG separation were produced. • Maximum IgG adsorption capacity

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

International Nuclear Information System (INIS)

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

2016-01-01

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 pH 7.4. 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). - Highlights: • Cu(II)-ions were coupled to PHEMA using PEI as the chelating ligand. • Cu(II) chelated [PHEMA/PEI] cryogels for IgG separation were produced. • Maximum IgG adsorption capacity

Ligand binding to G protein-coupled receptors in tethered cell membranes

DEFF Research Database (Denmark)

Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

2003-01-01

for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

Affinity separation based on hydrogen bonding

NARCIS (Netherlands)

Gruijters, B.W.T.

2007-01-01

The purification - work up and separation from other compounds - of chemical reactions is a crucial step in the synthesis of organic molecules. Therefore, organic chemists have developed a variety of work up and purification techniques throughout the last centuries, and novel methods are being

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Thermotoga neapolitana β-glucosidase B

Energy Technology Data Exchange (ETDEWEB)

Turner, Pernilla [Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Pramhed, Anna [Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Kanders, Erik; Hedström, Martin; Karlsson, Eva Nordberg, E-mail: eva.nordberg-karlsson@biotek.lu.se [Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Logan, Derek T., E-mail: eva.nordberg-karlsson@biotek.lu.se [Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden)

2007-09-01

Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. β-Glucosidases belong to families 1, 3 and 9 of the glycoside hydrolases and act on cello-oligosaccharides. Family 1 and 3 enzymes are retaining and are reported to have transglycosylation activity, which can be used to produce oligosaccharides and glycoconjugates. Family 3 enzymes are less well characterized than their family 1 homologues and to date only two crystal structures have been solved. Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. Crystals of selenomethionine-substituted protein have also been grown. The crystals belong to space group C222{sub 1}, with unit-cell parameters a = 74.9, b = 127.0, c = 175.2 Å. Native data have been collected to 2.4 Å resolution and the structure has been solved to 2.7 Å using the selenomethionine MAD method. Model building and refinement of the structure are under way.

Petunia peroxidase a: isolation, purification and characteristics.

Science.gov (United States)

Hendriks, T; Wijsman, H J; van Loon, L C

1991-07-01

The fast-moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia (Petunia hybrida). Over 1300-fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio (A405 nm/A280 nm) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin-A--Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties. The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at -20 degrees C with H2O2. The addition of 1 mol H2O2/mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H2O2. The pH optimum of PRXa for the reaction with H2O2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin-like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross-linking of lignin in the plant cell wall.

Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum

DEFF Research Database (Denmark)

Christensen, Michelle Brønniche; Sørensen, Jens Christian; Jacobsen, Stine

2013-01-01

BACKGROUND: Serum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species......-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials...... for purification of equine SAA based on biochemical properties.Freeze dried equine acute phase serum was dissolved in 70% 2-propanol, 8 M urea, and milli-Q water, respectively. Supercritical fluid extraction (SFE), size-exclusive chromatography (FPLC-SEC), and preparative isoelectric focusing (IEF) were performed...

Non-Native & Native English Teachers

Directory of Open Access Journals (Sweden)

İrfan Tosuncuoglu

2017-12-01

Full Text Available In many countries the primary (mother tongue language is not English but there is a great demand for English language teachers all over the world. The demand in this field is try to be filled largely by non-native English speaking teachers who have learned English in the country or abroad, or from another non native English peaking teachers. In some countries, particularly those where English speaking is a a sign of status, the students prefer to learn English from a native English speaker. The perception is that a non-native English speaking teacher is a less authentic teacher than a native English speaker and their instruction is not satifactory in some ways. This paper will try to examine the literature to explore whether there is a difference in instructional effectiveness between NNESTs and native English teachers.

Continuous affine processes

DEFF Research Database (Denmark)

Buchardt, Kristian

2016-01-01

Affine processes possess the property that expectations of exponential affine transformations are given by a set of Riccati differential equations, which is the main feature of this popular class of processes. In this paper we generalise these results for expectations of more general transformati...

Lysine purification with cation exchange resin

International Nuclear Information System (INIS)

Khayati, GH.; Mottaghi Talab, M.; Hamooni Hagheeghat, M.; Fatemi, M.

2003-01-01

L-lysine is an essential amino acid for the growth most of animal species and the number one limiting amino acid for poultry. After production and biomass removal by filtration and centrifugation, the essential next step is the lysine purification and recovery. There are different methods for lysine purification. The ion exchange process is one of the most commonly used purification methods. Lysine recovery was done from broth by ion exchange resin in three different ways: repeated passing, resin soaking and the usual method. Impurities were isolated from the column by repeated wash with distilled water. Recovery and purification was done with NH 4 OH and different alcohol volumes respectively. The results showed that repeated passing is the best method for lysine absorption (maximum range 86.21 %). Washing with alkali solution revealed that most of lysine is obtained in the first step of washing. The highest degree of lysine purification was achieved with the use of 4 volumes of alcohol

Expression, purification, and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris.

Science.gov (United States)

Tang, Xiang-Shan; Tang, Zhi-Ru; Wang, Sheng-Ping; Feng, Ze-Meng; Zhou, Dong; Li, Tie-Jun; Yin, Yu-Long

2012-02-01

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N(1) domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA-LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA-LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA-LFA-LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA-LFC peptide was purified via two-step column chromatography and identified by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA-LFC. The fermentation supernate and the purified LFA-LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA-LFC in P. pastoris and may have potential practical applications in microbial peptide production.

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

Directory of Open Access Journals (Sweden)

Weonu Choe

2016-12-01

Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

The metric-affine gravitational theory as the gauge theory of the affine group

International Nuclear Information System (INIS)

Lord, E.A.

1978-01-01

The metric-affine gravitational theory is shown to be the gauge theory of the affine group, or equivalently, the gauge theory of the group GL(4,R) of tetrad deformations in a space-time with a locally Minkowskian metric. The identities of the metric-affine theory, and the relationship between them and those of general relativity and Sciama-Kibble theory, are derived. (Auth.)

Partial Purification and Characterization of Extracellular Protease ...

African Journals Online (AJOL)

Nigerian Journal of Basic and Applied Sciences ... Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium sulphate precipitation gave 2.26 fold increase in purification with specific activity of 46.13 units/mg protein while purification on Sephadex CM50 resulted in reduced ...

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

Science.gov (United States)

Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

2016-09-01

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of high-affinity calmodulin-binding proteins in rat liver

International Nuclear Information System (INIS)

Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

1987-01-01

The Ca 2+ -dependent binding of [ 125 I] calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca 2+ -dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca 2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver

Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Gimble, F S; Thorner, J

1993-10-15

The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

Directory of Open Access Journals (Sweden)

Gao-Feng Qiu

2009-01-01

Full Text Available Cathepsin C (CTSC is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen is localized exclusively in cortical rods (CRs of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37∘C for 40 hours under native conditions, the recombinant CTSC (rCTSC exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

A Generalized Affine Isoperimetric Inequality

OpenAIRE

Chen, Wenxiong; Howard, Ralph; Lutwak, Erwin; Yang, Deane; Zhang, Gaoyong

2004-01-01

A purely analytic proof is given for an inequality that has as a direct consequence the two most important affine isoperimetric inequalities of plane convex geometry: The Blaschke-Santalo inequality and the affine isoperimetric inequality of affine differential geometry.

Novel precursor-derived Si-C-N ceramic material for purification application.

Science.gov (United States)

Roh, Changhyun; Lee, Sea-Hoon; Francois, Villatte

2008-02-01

Metagenomic DNA (gDNA) is an important research topic because uncultivated microorganisms represent an interesting reservoir of genes with potential biotechnological applications. Here we describe a novel Si-C-N chromatography approach of gDNA extraction from environmental samples. Amorphous Si-C-N ceramic was obtained by the pyrolysis of a polymer precursor (polycarbosilazane) at 1350 degrees C in Ar. The purified gDNA fragments were at least 12 kilo base pairs in size and were sufficiently free of contaminants, thus were applicable to both restriction enzyme digestion using five different enzymes and polymerase chain reaction amplification for detecting phylogenetic groups of native microorganisms in environmental samples. This Si-C-N material produces pure gDNA that can be utilized in some of the most common molecular biological procedures applied in the purification step.

Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

DEFF Research Database (Denmark)

Vorum, H; Pedersen, A O; Honoré, B

1992-01-01

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture...... of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...... and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture...

Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment-Based Screening.

Science.gov (United States)

Göth, Melanie; Badock, Volker; Weiske, Jörg; Pagel, Kevin; Kuropka, Benno

2017-08-08

Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention in recent years. So far, only a few studies have discussed mass spectrometry as a screening technology for fragments. Herein, we report the application of native electrospray ionization mass spectrometry (MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screening conducted with a thermal shift assay (TSA) and represented different binding categories. Our data indicated that, beside specific complex formation, many fragments show extensive multiple binding and also charge-state shifts. Both of these factors complicate automated data analysis and decrease the attractiveness of native MS as a primary screening tool for fragments. A comparison of the hits identified by native MS and TSA showed good agreement for two of the proteins. Furthermore, we discuss general challenges, including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS to be a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

Science.gov (United States)

Winge, Stefan; Yderland, Louise; Kannicht, Christoph; Hermans, Pim; Adema, Simon; Schmidt, Torben; Gilljam, Gustav; Linhult, Martin; Tiemeyer, Maya; Belyanskaya, Larisa; Walter, Olaf

2015-11-01

Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Lp-mixed affine surface area

Science.gov (United States)

Wang, Weidong; Leng, Gangsong

2007-11-01

According to the three notions of mixed affine surface area, Lp-affine surface area and Lp-mixed affine surface area proposed by Lutwak, in this article, we give the concept of ith Lp-mixed affine surface area such that the first and second notions of Lutwak are its special cases. Further, some Lutwak's results are extended associated with this concept. Besides, applying this concept, we establish an inequality for the volumes and dual quermassintegrals of a class of star bodies.

In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

Directory of Open Access Journals (Sweden)

Aytak Novinrooz

Full Text Available E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC, and fatal hemolytic uremic syndrome (HUS and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA, and B subunit of E. coli heat labile enterotoxin (LTB which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+ expression vector and transferred to E. coli BL21(DE3 cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3 cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG. The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient

Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions

International Nuclear Information System (INIS)

Kiser, Philip D.; Lodowski, David T.; Palczewski, Krzysztof

2007-01-01

A 3.02 Å crystal structure of native GroEL from E. coli is presented. GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 Å crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code http://www.rcsb.org/pdb/explore.do?structureId), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution

Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

International Nuclear Information System (INIS)

Campos, Samuel K.; Barry, Michael A.

2006-01-01

The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon

Entanglement purification of multi-mode quantum states

International Nuclear Information System (INIS)

Clausen, J; Knoell, L; Welsch, D-G

2003-01-01

An iterative random procedure is considered allowing entanglement purification of a class of multi-mode quantum states. In certain cases, complete purification may be achieved using only a single signal state preparation. A physical implementation based on beam splitter arrays and non-linear elements is suggested. The influence of loss is analysed in the example of purification of entangled N-mode coherent states

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

Science.gov (United States)

Savardashtaki, Amir; Sarkari, Bahador; Arianfar, Farzane; Mostafavi-Pour, Zohreh

2017-06-01

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

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

Science.gov (United States)

Konami, Y; Yamamoto, K; Osawa, T

1991-02-01

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

Analysis of protein interactions at native chloroplast membranes by ellipsometry.

Directory of Open Access Journals (Sweden)

Verena Kriechbaumer

Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

The various sodium purification techniques

International Nuclear Information System (INIS)

Courouau, J.L.; Masse, F.; Rodriguez, G.; Latge, C.; Redon, B.

1997-01-01

In the framework of sodium waste treatment, the sodium purification phase plays an essential role in the chain of operations leading to the transformation of the active sodium, considered as waste, into a stable sodium salt. The objectives of the purification operations are: To keep a low impurity level, particularly a low concentration in oxygen and hydrogen, in order to allow its transfer to a processing plant, and in order to avoid risks of plugging and/or corrosion in sodium facilities; To reduce the sodium activity in order to limit the dose rate close to the facilities, and in order to reduce the activity of the liquid and gaseous effluents. After a recall of the different kind of impurities that can be present in sodium, and of the different purification methods that could be associated with, the following points are highlighted: (i) Oxygen and hydrogen purification needs, and presentation of some selection criteria for a purification unit adapted to a sodium processing plant, as well as 2 cold trap concepts that are in accordance with these criteria: PSICHOS and PIRAMIDE. (ii) Tritium reduction in a bulk of liquid sodium by swamping, isotopic exchange, or permeation throughout a membrane. (iii) Caesium trapping on carbonaceous matrix. The main matrices used at present are R.V.C. (Reticulated Vitreous Carbon) and Actitex/Pica products. Tests in the laboratory and on an experimental device have demonstrated the performances of these materials, which are able to reduce sodium activity in Cs 134 and Cs 137 to very low values. The sodium purification processes as regards to the hydrogen, oxygen and caesium, that are aimed at facilitating the subsequent treatment of sodium, are therefore mastered operations. Regarding the operations associated with the reduction of the tritium activity, the methods are in the process of being qualified, or to be qualified. (author)

Entanglement of purification: from spin chains to holography

Science.gov (United States)

Nguyen, Phuc; Devakul, Trithep; Halbasch, Matthew G.; Zaletel, Michael P.; Swingle, Brian

2018-01-01

Purification is a powerful technique in quantum physics whereby a mixed quantum state is extended to a pure state on a larger system. This process is not unique, and in systems composed of many degrees of freedom, one natural purification is the one with minimal entanglement. Here we study the entropy of the minimally entangled purification, called the entanglement of purification, in three model systems: an Ising spin chain, conformal field theories holographically dual to Einstein gravity, and random stabilizer tensor networks. We conjecture values for the entanglement of purification in all these models, and we support our conjectures with a variety of numerical and analytical results. We find that such minimally entangled purifications have a number of applications, from enhancing entanglement-based tensor network methods for describing mixed states to elucidating novel aspects of the emergence of geometry from entanglement in the AdS/CFT correspondence.

Bioinspired Materials for Water Purification

Directory of Open Access Journals (Sweden)

Alfredo Gonzalez-Perez

2016-06-01

Full Text Available Water scarcity issues associated with inadequate access to clean water and sanitation is a ubiquitous problem occurring globally. Addressing future challenges will require a combination of new technological development in water purification and environmental remediation technology with suitable conservation policies. In this scenario, new bioinspired materials will play a pivotal role in the development of more efficient and environmentally friendly solutions. The role of amphiphilic self-assembly on the fabrication of new biomimetic membranes for membrane separation like reverse osmosis is emphasized. Mesoporous support materials for semiconductor growth in the photocatalytic degradation of pollutants and new carriers for immobilization of bacteria in bioreactors are used in the removal and processing of different kind of water pollutants like heavy metals. Obstacles to improve and optimize the fabrication as well as a better understanding of their performance in small-scale and pilot purification systems need to be addressed. However, it is expected that these new biomimetic materials will find their way into the current water purification technologies to improve their purification/removal performance in a cost-effective and environmentally friendly way.

Mapping Affinities in Academic Organizations

Directory of Open Access Journals (Sweden)

Dario Rodighiero

2018-02-01

Full Text Available Scholarly affinities are one of the most fundamental hidden dynamics that drive scientific development. Some affinities are actual, and consequently can be measured through classical academic metrics such as co-authoring. Other affinities are potential, and therefore do not leave visible traces in information systems; for instance, some peers may share interests without actually knowing it. This article illustrates the development of a map of affinities for academic collectives, designed to be relevant to three audiences: the management, the scholars themselves, and the external public. Our case study involves the School of Architecture, Civil and Environmental Engineering of EPFL, hereinafter ENAC. The school consists of around 1,000 scholars, 70 laboratories, and 3 institutes. The actual affinities are modeled using the data available from the information systems reporting publications, teaching, and advising scholars, whereas the potential affinities are addressed through text mining of the publications. The major challenge for designing such a map is to represent the multi-dimensionality and multi-scale nature of the information. The affinities are not limited to the computation of heterogeneous sources of information; they also apply at different scales. The map, thus, shows local affinities inside a given laboratory, as well as global affinities among laboratories. This article presents a graphical grammar to represent affinities. Its effectiveness is illustrated by two actualizations of the design proposal: an interactive online system in which the map can be parameterized, and a large-scale carpet of 250 square meters. In both cases, we discuss how the materiality influences the representation of data, in particular the way key questions could be appropriately addressed considering the three target audiences: the insights gained by the management and their consequences in terms of governance, the understanding of the scholars’ own

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Directory of Open Access Journals (Sweden)

Yingying Cai

Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Science.gov (United States)

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Venturi purification device and its application in purification of gaseous waste of nuclear facilities

International Nuclear Information System (INIS)

Kong Jinsong; Yu Ren; Yang Huanlei

2013-01-01

The working principle of Venturi purification device and its purification of aerosol have been described. Then, taking the gaseous iodine as an example, the absorption process of insoluble gas pollutants is discussed, the calculation methods of the gas-liquid contact area, mass transfer rate and efficiency of mass transfer are educed, and the factors that affect the efficiency of mass transfer are analyzed. (authors)

Fractionation and purification of DNA methylases of Shigella sonnei

International Nuclear Information System (INIS)

Suchkov, S.V.; Nikol'skaya, I.I.; Debov, S.S.

1986-01-01

A comparative study was made of the possibilities of various types of column chromatography and isoelectrofocusing for the analysis of the set of DNA methylases of the strains Shigella sonnei 47. On the basic of cation-exchange chromatography, a simple, rapid, and convenient method has been developed for the production of a highly active summary preparations of DNA methylases. It has been shown that affinity chromatography on heparin-Sepharose provides for the separation of methylases according to the characteristic of specificity for the nitrogen base. The presence of six individuals enzymes of DNA methylation, differing in value of the isoelectric point, was demonstrated in Shigella sonnei 47 cells by the IEF* method. Increased ability of the DNA methylases of Shigella sonnei 47 for nonspecific aggregation during the process of fractionation was demonstrated, which makes the use of column chromatography unsuitable for the isolation and purification of individual methylating enzymes of the investigated strain. The advantages of the IEF method, as well as its potentialities from the standpoint of studying various molecular forms of site-specific enzymes, are discussed

Nonequivalence of alpha-bungarotoxin binding sites in the native nicotinic receptor molecule

International Nuclear Information System (INIS)

Conti-Tronconi, B.M.; Tang, F.; Walgrave, S.; Gallagher, W.

1990-01-01

In the native, membrane-bound form of the nicotinic acetylcholine receptor (M-AcChR) the two sites for the cholinergic antagonist alpha-bungarotoxin (alpha-BGT) have different binding properties. One site has high affinity, and the M-AcChR/alpha-BGT complexes thus formed dissociate very slowly, similar to the complexes formed with detergent-solubilized AcChR (S-AcChR). The second site has much lower affinity (KD approximately 59 +/- 35 nM) and forms quickly reversible complexes. The nondenaturing detergent Triton X-100 is known to solubilize the AcChR in a form unable, upon binding of cholinergic ligands, to open the ion channel and to become desensitized. Solubilization of the AcChR in Triton X-100 affects the binding properties of this second site and converts it to a high-affinity, slowly reversible site. Prolonged incubation of M-AcChR at 4 degrees C converts the low-affinity site to a high-affinity site similar to those observed in the presence of Triton X-100. Although the two sites have similar properties when the AcChR is solubilized in Triton X-100, their nonequivalence can be demonstrated by the effect on alpha-BGT binding of concanavalin A, which strongly reduces the association rate of one site only. The Bmax of alpha-BGT to either Triton-solubilized AcChR or M-AcChR is not affected by the presence of concanavalin A. Occupancy of the high-affinity, slowly reversible site in M-AcChR inhibits the Triton X-100 induced conversion to irreversibility of the second site. At difference with alpha-BGT, the long alpha-neurotoxin from Naja naja siamensis venom (alpha-NTX) binds with high affinity and in a very slowly reversible fashion to two sites in the M-AcChR. We confirm here that Triton-solubilized AcChR or M-AcChR binds in a very slowly reversible fashion the same amount of alpha-NTX

Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

Directory of Open Access Journals (Sweden)

Sujatha P Koduvayur

Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

Photocatalytic materials and technologies for air purification.

Science.gov (United States)

Ren, Hangjuan; Koshy, Pramod; Chen, Wen-Fan; Qi, Shaohua; Sorrell, Charles Christopher

2017-03-05

Since there is increasing concern for the impact of air quality on human health, the present work surveys the materials and technologies for air purification using photocatalytic materials. The coverage includes (1) current photocatalytic materials for the decomposition of chemical contaminants and disinfection of pathogens present in air and (2) photocatalytic air purification systems that are used currently and under development. The present work focuses on five main themes. First, the mechanisms of photodegradation and photodisinfection are explained. Second, system designs for photocatalytic air purification are surveyed. Third, the photocatalytic materials used for air purification and their characteristics are considered, including both conventional and more recently developed photocatalysts. Fourth, the methods used to fabricate these materials are discussed. Fifth, the most significant coverage is devoted to materials design strategies aimed at improving the performance of photocatalysts for air purification. The review concludes with a brief consideration of promising future directions for materials research in photocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification.

Science.gov (United States)

Liu, Hui F; McCooey, Beth; Duarte, Tiago; Myers, Deanna E; Hudson, Terry; Amanullah, Ashraf; van Reis, Robert; Kelley, Brian D

2011-09-28

purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes. Copyright © 2011 Elsevier B.V. All rights reserved.

Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

DEFF Research Database (Denmark)

Kristensen, Janni; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

2005-01-01

We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23...... therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only...... proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome....

Fundamentals of affinity cell separations.

Science.gov (United States)

Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

2018-03-01

Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface processes during purification of InP quantum dots

Directory of Open Access Journals (Sweden)

Natalia Mordvinova

2014-08-01

Full Text Available Recently, a new simple and fast method for the synthesis of InP quantum dots by using phosphine as phosphorous precursor and myristic acid as surface stabilizer was reported. Purification after synthesis is necessary to obtain samples with good optical properties. Two methods of purification were compared and the surface processes which occur during purification were studied. Traditional precipitation with acetone is accompanied by a small increase in photoluminescence. It occurs that during the purification the hydrolysis of the indium precursor takes place, which leads to a better surface passivation. The electrophoretic purification technique does not increase luminescence efficiency but yields very pure quantum dots in only a few minutes. Additionally, the formation of In(OH3 during the low temperature synthesis was explained. Purification of quantum dots is a very significant part of postsynthetical treatment that determines the properties of the material. But this subject is not sufficiently discussed in the literature. The paper is devoted to the processes that occur at the surface of quantum dots during purification. A new method of purification, electrophoresis, is investigated and described in particular.

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Science.gov (United States)

Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

2013-01-01

Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

Science.gov (United States)

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

Purification of immunoreactive radiolabeled moniclonal antibodies with anti-iodiotypic moniclonal antibodies

International Nuclear Information System (INIS)

Temponi, M.; Pupa, S.; Ferrone, S.

1990-01-01

A method is described to purify immunoreactive moniclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified an elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yeald was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies. (author). 12 refs.; 4 figs.; 1 tab

Purification of neuraminidase from Influenza virus subtype H5N1

Directory of Open Access Journals (Sweden)

Simson Tariga

2009-03-01

Full Text Available Influenza-virus neuraminidase plays vital role in the survival of the organisms. Vaccination of animals with this glycoprotein confers immune responses so that enable it to protect the animals from incoming infection. Supplementation of conventional vaccines with this glycoprotein increases the protection and longevity of the vaccine. Purified neuraminidase can also be used to develop serological tests for differentiation of serologically positive animals due to infection or to vaccination. In this study purification of neuraminidase from influenza virus subtype H5N1 was described. Triton x-100 and Octyl β-D-glucopyranoside were used to extract and diluted the glycoprotein membrane. The enzymatic activity of the neuraminidase was assayed using a fluorochrome substrate, 4-methylumbelliferyl-a-D-N-acetyl neuraminic acid, which was found to be simple, sensitive and suitable for the purification purpose. The neuraminidase was absorbed selectively on an oxamic-acid agarose column. The purity of neuraminidase eluted from this affinity column was high. A higher purity of the neuraminidase was obtained by further separation with gel filtration on Superdex-200. The purified neuraminidase was enzymatically active and did not contain any detectable haemagglutinin, either by haemagglutination assay or by monospecific antibodies raised against H5N1 hemagglutinin. The purified neuraminidase was recognized strongly by antibodies raised against an internal but only weakly by that against C-terminal regions of the neuraminidase protein of H5N1-influenza virus. The purified neuraminidase was in tetrameric forms but dissociated into monomeric form on reducing condition, or mostly dimeric form on non-reducing SDS-PAGE.

Affine field theories

International Nuclear Information System (INIS)

Cadavid, A.C.

1989-01-01

The author constructs a non-Abelian field theory by gauging a Kac-Moody algebra, obtaining an infinite tower of interacting vector fields and associated ghosts, that obey slightly modified Feynman rules. She discusses the spontaneous symmetry breaking of such theory via the Higgs mechanism. If the Higgs particle lies in the Cartan subalgebra of the Kac-Moody algebra, the previously massless vectors acquire a mass spectrum that is linear in the Kac-Moody index and has additional fine structure depending on the associated Lie algebra. She proceeds to show that there is no obstacle in implementing the affine extension of supersymmetric Yang-Mills theories. The result is valid in four, six and ten space-time dimensions. Then the affine extension of supergravity is investigated. She discusses only the loop algebra since the affine extension of the super-Poincare algebra appears inconsistent. The construction of the affine supergravity theory is carried out by the group manifold method and leads to an action describing infinite towers of spin 2 and spin 3/2 fields that interact subject to the symmetries of the loop algebra. The equations of motion satisfy the usual consistency check. Finally, she postulates a theory in which both the vector and scalar fields lie in the loop algebra of SO(3). This theory has an expanded soliton sector, and corresponding to the original 't Hooft-Polyakov solitonic solutions she now finds an infinite family of exact, special solutions of the new equations. She also proposes a perturbation method for obtaining an arbitrary solution of those equations for each level of the affine index

Water purification in Borexino

Energy Technology Data Exchange (ETDEWEB)

Giammarchi, M. [Infn Milano (Italy); Balata, M.; Ioannucci, L.; Nisi, S. [Laboratori Nazionali del Gran Sasso (Italy); Goretti, A.; Ianni, A. [Princeton University (United States); Miramonti, L. [Dip. di Fisica dell' Università di Milano e Infn (Italy)

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.

Radiation-adsorption purification of effluents containing pesticides

International Nuclear Information System (INIS)

Brusentseva, S.A.; Shubin, V.N.; Nikonorova, G.K.; Zorin, D.M.; Sosnovskaya, A.A.; Petryaev, E.P.; Vlasova, V.I.; Edimicheva, I.P.; Subbotina, N.N.; Belorusskij Gosudarstvennyj Univ., Minsk)

1986-01-01

The radiation-adsorption purification is one of the new direction in the radiation purification of natural wastes and effluents containing pesticides. This method combines the conventional adsorption purification with radiation treatment of the sorbent, and the result the protection time of the sorbent increases due to the radiation regeneration of carbon. In present work the method was used for purification of effluents from pesticides, such as 4,4'Dichlorodiphenyltrichloroethane /DDT/, 1,2,3,4,5,6-hexachlorocyclohexane /HCCH/, dimethyl 2,2-dichlorovinylphosphate /DDVF/ and petroleum products (a mixture of kerosene and xylene in ratio 7:1). Such effluents are formed at factories producing an insecticide aerosol 'Prime-71'. Three investigations were carried out on model with a solution similar composition to industrial effluents. (author)

Electron affinities: theoretical

International Nuclear Information System (INIS)

Kaufman, J.J.

1976-01-01

A brief description is given of the conceptual background and formalism of the various ab-initio and semi-ab-initio quantum computational techniques for calculating atomic and molecular electron affinities: Hartree--Fock--Roothaan SCF, configuration interaction (CI), multiconfiguration SCF (MC-SCF), Bethe--Goldstone, superposition of configurations (SOC), ab-initio effective core model potentials, Xα-MS, plus other less common methods. Illustrative and comparative examples of electron affinities calculated by these various methods are presented

Hemoglobin affinity in Andean rodents

Directory of Open Access Journals (Sweden)

HRVOJ OSTOJIC

2002-01-01

Full Text Available Blood hemoglobin oxygen affinity (P50 was measured in three Andean species and in the laboratory rat (control, all raised near sea level. Chinchilla lanigera (Molina, 1792 has an altitudinal habitat range from low Andean slopes up to 3000 m., while Chinchilla brevicaudata (Waterhouse, 1848 has an altitudinal range from 3000 to 5000 m. The laboratory type guinea pig, wild type guinea pig (Cavia porcellus, (Waterhouse, 1748, and laboratory rat (Rattus norvegicus were also raised at sea level. The Andean species had high hemoglobin oxygen affinities (low P50 compared with the rat. Chinchilla brevicaudata had a higher affinity than Chinchilla lanigera. The wild type guinea pig had a higher affinity than the laboratory type. As has been shown in other species, this is another example of an inverse correlation between the altitude level and the P50 values. This is the first hemoglobin oxygen affinity study in Chinchilla brevicaudata.

Interactions between Cellulolytic Enzymes with Native, Autohydrolysis, and Technical Lignins and the Effect of a Polysorbate Amphiphile in Reducing Nonproductive Binding.

Science.gov (United States)

Fritz, Consuelo; Ferrer, Ana; Salas, Carlos; Jameel, Hasan; Rojas, Orlando J

2015-12-14

Understanding enzyme-substrate interactions is critical in designing strategies for bioconversion of lignocellulosic biomass. In this study we monitored molecular events, in situ and in real time, including the adsorption and desorption of cellulolytic enzymes on lignins and cellulose, by using quartz crystal microgravimetry and surface plasmon resonance. The effect of a nonionic surface active molecule was also elucidated. Three lignin substrates relevant to the sugar platform in biorefinery efforts were considered, namely, hardwood autohydrolysis cellulolytic (HWAH), hardwood native cellulolytic (MPCEL), and nonwood native cellulolytic (WSCEL) lignin. In addition, Kraft lignins derived from softwoods (SWK) and hardwoods (HWK) were used as references. The results indicated a high affinity between the lignins with both, monocomponent and multicomponent enzymes. More importantly, the addition of nonionic surfactants at concentrations above their critical micelle concentration reduced remarkably (by over 90%) the nonproductive interactions between the cellulolytic enzymes and the lignins. This effect was hypothesized to be a consequence of the balance of hydrophobic and hydrogen bonding interactions. Moreover, the reduction of surface roughness and increased wettability of lignin surfaces upon surfactant treatment contributed to a lower affinity with the enzymes. Conformational changes of cellulases were observed upon their adsorption on lignin carrying preadsorbed surfactant. Weak electrostatic interactions were determined in aqueous media at pH between 4.8 and 5.5 for the native cellulolytic lignins (MPCEL and WSCEL), whereby a ∼20% reduction in the enzyme affinity was observed. This was mainly explained by electrostatic interactions (osmotic pressure effects) between charged lignins and cellulases. Noteworthy, adsorption of nonionic surfactants onto cellulose, in the form cellulose nanofibrils, did not affect its hydrolytic conversion. Overall, our results

Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

Science.gov (United States)

Wade, James H; Jones, Joshua D; Lenov, Ivan L; Riordan, Colleen M; Sligar, Stephen G; Bailey, Ryan C

2017-08-22

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

21 CFR 876.5665 - Water purification system for hemodialysis.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Water purification system for hemodialysis. 876... SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is a...

A simple photolytic reactor employing Ag-doped ZnO nanowires for water purification

Energy Technology Data Exchange (ETDEWEB)

Udom, Innocent; Zhang, Yangyang [Clean Energy Research Center, College of Engineering, University of South Florida, Tampa, FL 33620 (United States); Ram, Manoj K., E-mail: mkram@usf.edu [Clean Energy Research Center, College of Engineering, University of South Florida, Tampa, FL 33620 (United States); Stefanakos, Elias K. [Clean Energy Research Center, College of Engineering, University of South Florida, Tampa, FL 33620 (United States); Hepp, Aloysius F. [Department of Chemical and Biomedical Engineering, University of South Florida, Tampa, Fl 33620 (United States); Elzein, Radwan; Schlaf, Rudy [Department of Electrical Engineering, University of South Florida, Tampa, Fl 33620 (United States); Goswami, D. Yogi [NASA Glenn Research Center, Research and Technology Directorate, MS 302-1, 21000 Brookpark Road, Cleveland, OH 44135 (United States)

2014-08-01

Well-aligned native zinc oxide (ZnO) and silver-doped ZnO (Ag-ZnO) films were deposited on borosilicate glass via a simple, low-cost, low-temperature, scalable hydrothermal process. The as-synthesized ZnO and Ag-ZnO films were characterized by X-ray diffraction; scanning electron microscopy, UV–visible spectroscopy, and Fourier transform infrared spectroscopy. A simple photolytic reactor was fabricated and later used to find the optimum experimental conditions for photocatalytic performance. The photodegradation of methyl orange in water was investigated using as-prepared ZnO and Ag-ZnO nanowires, and was compared to P25 (a commercial photocatalyst) in both visible and UV radiations. The P25 and Ag-ZnO showed a similar photodegradation performance under UV light, but Ag-ZnO demonstrated superior photocatalytic activity under visible irradiation. The optimized doping of Ag in Ag-ZnO enhanced photocatalytic activity in a simple reactor design and indicated potential applicability of Ag-ZnO for large-scale purification of water under solar irradiation. - Highlights: • Well-aligned zinc oxide (ZnO) and silver-doped ZnO (Ag-ZnO) nanowires were developed. • Simple and effective photolytic reactor was fabricated for water purification. • Ag-ZnO demonstrated superior photocatalytic activity under visible irradiation. • Amount of Ag atoms in Ag-ZnO nanowires is a key to increase photocatalytic activity.

A simple photolytic reactor employing Ag-doped ZnO nanowires for water purification

International Nuclear Information System (INIS)

Udom, Innocent; Zhang, Yangyang; Ram, Manoj K.; Stefanakos, Elias K.; Hepp, Aloysius F.; Elzein, Radwan; Schlaf, Rudy; Goswami, D. Yogi

2014-01-01

Well-aligned native zinc oxide (ZnO) and silver-doped ZnO (Ag-ZnO) films were deposited on borosilicate glass via a simple, low-cost, low-temperature, scalable hydrothermal process. The as-synthesized ZnO and Ag-ZnO films were characterized by X-ray diffraction; scanning electron microscopy, UV–visible spectroscopy, and Fourier transform infrared spectroscopy. A simple photolytic reactor was fabricated and later used to find the optimum experimental conditions for photocatalytic performance. The photodegradation of methyl orange in water was investigated using as-prepared ZnO and Ag-ZnO nanowires, and was compared to P25 (a commercial photocatalyst) in both visible and UV radiations. The P25 and Ag-ZnO showed a similar photodegradation performance under UV light, but Ag-ZnO demonstrated superior photocatalytic activity under visible irradiation. The optimized doping of Ag in Ag-ZnO enhanced photocatalytic activity in a simple reactor design and indicated potential applicability of Ag-ZnO for large-scale purification of water under solar irradiation. - Highlights: • Well-aligned zinc oxide (ZnO) and silver-doped ZnO (Ag-ZnO) nanowires were developed. • Simple and effective photolytic reactor was fabricated for water purification. • Ag-ZnO demonstrated superior photocatalytic activity under visible irradiation. • Amount of Ag atoms in Ag-ZnO nanowires is a key to increase photocatalytic activity

Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Aryal, Uma K.; Lin, Chiann Tso; Kim, Jong Seo; Heibeck, Tyler H.; Wang, Jun; Qian, Weijun; Lin, Yuehe

2012-04-20

Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The Inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we performed immunoaffinity purification and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. The exact phosphorylation site of BChE was confirmed on Serine 198 by MS/MS with a 108 Da modification mass and accurately measured parent ion masses. The phosphorylated BChE peptide was also successfully detected in the immunoaffinity purified sample from paraoxon treated human plasma. Thus, immunoaffinity purification combined with mass spectrometry represents a viable approach for the detection of paraoxon-modified BChE and other forms of modified BChE as exposure biomarkers of organophosphates and nerve agents.

Sodium purification in Rapsodie; La purification du sodium a Rapsodie

Energy Technology Data Exchange (ETDEWEB)

Giraud, B [Commissariat a l' Energie Atomique, Dir. des Piles Atomiques, Cadarache (France). Centre d' Etudes Nucleaires

1968-07-01

This report is one of a series of publications presenting the main results of tests carried out during the start-up of the first french fast neutron reactor: Rapsodie. The article presents the sodium purification techniques used in the reactor cooling circuits both from the constructional point of view and with respect to results obtained during the first years working. (author) [French] Ce rapport fait partie d'une serie de publications presentant l'essentiel des resultats des essais effectues a l'occasion du demarrage du premier reacteur francais a neutrons rapides: RAPSODIE. Cet article expose les techniques de la purification du sodium utilise dans les circuits de refroidissement du reacteur tant au point de vue de leur realisation technologique, que des resultats obtenus pendant la premiere annee de fonctionnement. (auteur)

Uranium hexafluoride purification

International Nuclear Information System (INIS)

Araujo, Eneas F. de

1986-01-01

Uranium hexafluoride might contain a large amount of impurities after manufacturing or handling. Three usual methods of purification of uranium hexafluoride were presented: selective sorption, sublimation, and distillation. Since uranium hexafluoride usually is contaminated with hydrogen fluoride, a theoretical study of the phase equilibrium properties was performed for the binary system UF 6 -HF. A large deviation from the ideal solution behaviour was observed. A purification unity based on a constant reflux batch distillation process was developed. A procedure was established in order to design the re boiler, condenser and packed columns for the UF 6 -HF mixture separation. A bench scale facility for fractional distillation of uranium hexafluoride was described. Basic operations for that facility and results extracted from several batches were discussed. (author)

Purification of functionalized DNA origami nanostructures.

Science.gov (United States)

Shaw, Alan; Benson, Erik; Högberg, Björn

2015-05-26

The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

Biodiesel separation and purification: A review

International Nuclear Information System (INIS)

Atadashi, I.M.; Aroua, M.K.; Aziz, A. Abdul

2011-01-01

Biodiesel as a biodegradable, sustainable and clean energy has worldwide attracted renewed and growing interest in topical years, chiefly due to development in biodiesel fuel and ecological pressures which include climatic changes. In the production of biodiesel from biomass, separation and purification of biodiesel is a critical technology. Conventional technologies used for biodiesel separation such as gravitational settling, decantation, filtration and biodiesel purification such as water washing, acid washing, and washing with ether and absorbents have proven to be inefficient, time and energy consumptive, and less cost effective. The involvement of membrane reactor and separative membrane shows great promise for the separation and purification of biodiesel. Membrane technology needs to be explored and exploited to overcome the difficulties usually encountered in the separation and purification of biodiesel. In this paper both conventional and most recent membrane technologies used in refining biodiesel have been critically reviewed. The effects of catalysts, free fatty acids, water content and oil to methanol ratios on the purity and quality of biodiesel are also examined. (author)

Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330

International Nuclear Information System (INIS)

Aggarwal, Nidhi; Mandal, P. K.; Gautham, Namasivayam; Chadha, Anju

2013-01-01

The expression, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies on C. parapsilosis carbonyl reductase are reported. The NAD(P)H-dependent carbonyl reductase from Candida parapsilosis ATCC 7330 catalyses the asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate to ethyl (R)-4-phenyl-2-hydroxybutanoate, a precursor of angiotensin-converting enzyme inhibitors such as Cilazapril and Benazepril. The carbonyl reductase was expressed in Escherichia coli and purified by GST-affinity and size-exclusion chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.86 Å resolution. The asymmetric unit contained two molecules of carbonyl reductase, with a solvent content of 48%. The structure was solved by molecular replacement using cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae as a search model

Purification, crystallization and preliminary X-ray analysis of haemoglobin from ostrich (Struthio camelus)

International Nuclear Information System (INIS)

Sundaresan, S. S.; Ramesh, P.; Sivakumar, K.; Ponnuswamy, M. N.

2009-01-01

Purification, crystallization and preliminary X-ray analysis of haemoglobin from ostrich (Struthio camelus) has been carried out under 293 K temperature conditions. The ostrich is a large flightless bird which contains inositol tetrakisphosphate in erythrocytes and its whole blood oxygen affinity is higher. Efforts have been made to explore the structure–function relationship of ostrich heamoglobin. Haemoglobin is a tetrameric protein that carries oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs. The oxygen-binding properties of haemoglobin are regulated through the binding of allosteric effectors. The respiratory system of avian species is unique and complex in nature when compared with that of mammals. In avian species, inositol pentaphosphate (inositol-P 5 ) is present in the erythrocytes of the adult and is thought to be the major factor responsible for the relatively high oxygen affinity of the whole blood. The ostrich (Struthio camelus) is a large flightless bird which contains inositol tetrakisphosphate (inositol-P 4 ) in its erythrocytes and its whole blood oxygen affinity is higher. Efforts have been made to explore the structure–function relationship of ostrich haemoglobin. Ostrich haemoglobin was purified using ion-exchange chromatography. Haemoglobin crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant in 50 mM phosphate buffer pH 7.2. Data were collected using a MAR345 image-plate detector system. The crystals of ostrich haemoglobin diffracted to 2.2 Å resolution. They belonged to the orthorhombic space group P2 1 2 1 2 1 with one whole biological molecule in the asymmetric unit; the unit-cell parameters were a = 80.93, b = 81.68, c = 102.05 Å

High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

Directory of Open Access Journals (Sweden)

Stefan Ryser

Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

2017 Guralp Affinity Digitizer Evaluation.

Energy Technology Data Exchange (ETDEWEB)

Merchant, Bion J.

2018-03-01

Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

Science.gov (United States)

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

2013-01-01

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

Non-Native Plant Invasion along Elevation and Canopy Closure Gradients in a Middle Rocky Mountain Ecosystem.

Directory of Open Access Journals (Sweden)

Joshua P Averett

Full Text Available Mountain environments are currently among the ecosystems least invaded by non-native species; however, mountains are increasingly under threat of non-native plant invasion. The slow pace of exotic plant invasions in mountain ecosystems is likely due to a combination of low anthropogenic disturbances, low propagule supply, and extreme/steep environmental gradients. The importance of any one of these factors is debated and likely ecosystem dependent. We evaluated the importance of various correlates of plant invasions in the Wallowa Mountain Range of northeastern Oregon and explored whether non-native species distributions differed from native species along an elevation gradient. Vascular plant communities were sampled in summer 2012 along three mountain roads. Transects (n = 20 were evenly stratified by elevation (~70 m intervals along each road. Vascular plant species abundances and environmental parameters were measured. We used indicator species analysis to identify habitat affinities for non-native species. Plots were ordinated in species space, joint plots and non-parametric multiplicative regression were used to relate species and community variation to environmental variables. Non-native species richness decreased continuously with increasing elevation. In contrast, native species richness displayed a unimodal distribution with maximum richness occurring at mid-elevations. Species composition was strongly related to elevation and canopy openness. Overlays of trait and environmental factors onto non-metric multidimensional ordinations identified the montane-subalpine community transition and over-story canopy closure exceeding 60% as potential barriers to non-native species establishment. Unlike native species, non-native species showed little evidence for high-elevation or closed-canopy specialization. These data suggest that non-native plants currently found in the Wallowa Mountains are dependent on open canopies and disturbance for

Non-Native Plant Invasion along Elevation and Canopy Closure Gradients in a Middle Rocky Mountain Ecosystem.

Science.gov (United States)

Averett, Joshua P; McCune, Bruce; Parks, Catherine G; Naylor, Bridgett J; DelCurto, Tim; Mata-González, Ricardo

2016-01-01

Mountain environments are currently among the ecosystems least invaded by non-native species; however, mountains are increasingly under threat of non-native plant invasion. The slow pace of exotic plant invasions in mountain ecosystems is likely due to a combination of low anthropogenic disturbances, low propagule supply, and extreme/steep environmental gradients. The importance of any one of these factors is debated and likely ecosystem dependent. We evaluated the importance of various correlates of plant invasions in the Wallowa Mountain Range of northeastern Oregon and explored whether non-native species distributions differed from native species along an elevation gradient. Vascular plant communities were sampled in summer 2012 along three mountain roads. Transects (n = 20) were evenly stratified by elevation (~70 m intervals) along each road. Vascular plant species abundances and environmental parameters were measured. We used indicator species analysis to identify habitat affinities for non-native species. Plots were ordinated in species space, joint plots and non-parametric multiplicative regression were used to relate species and community variation to environmental variables. Non-native species richness decreased continuously with increasing elevation. In contrast, native species richness displayed a unimodal distribution with maximum richness occurring at mid-elevations. Species composition was strongly related to elevation and canopy openness. Overlays of trait and environmental factors onto non-metric multidimensional ordinations identified the montane-subalpine community transition and over-story canopy closure exceeding 60% as potential barriers to non-native species establishment. Unlike native species, non-native species showed little evidence for high-elevation or closed-canopy specialization. These data suggest that non-native plants currently found in the Wallowa Mountains are dependent on open canopies and disturbance for establishment in low

Purification of crude biodiesel using dry washing and membrane technologies

OpenAIRE

Atadashi, I.M.

2015-01-01

Purification of crude biodiesel is mandatory for the fuel to meet the strict international standard specifications for biodiesel. Therefore, this paper carefully analyzed recently published literatures which deal with the purification of biodiesel. As such, dry washing technologies and the most recent membrane biodiesel purification process have been thoroughly examined. Although purification of biodiesel using dry washing process involving magnesol and ion exchange resins provides high-quali...

Two-parameter quantum affine algebra Ur,s(sln-circumflex), Drinfeld realization and quantum affine Lyndon basis

International Nuclear Information System (INIS)

Hu Naihong; Rosso, M.; Zhang Honglian

2006-12-01

We further find the defining structure of a two-parameter quantum affine algebra U r,s (sl n -circumflex) (n > 2) in the sense of Benkart-Witherspoon [BW1] after the work of [BGH1], [HS] and [BH], which turns out to be a Drinfeld double. Of more importance for the 'affine' cases is that we work out the compatible two-parameter version of the Drinfeld realization as a quantum affinization of U r,s (sl n ) and establish the Drinfeld isomorphism Theorem in the two-parameter setting via developing a new remarkable combinatorial approach - quantum 'affine' Lyndon basis with an explicit valid algorithm, based on the Drinfeld realization. (author)

Antimicrobial Peptide Production and Purification.

Science.gov (United States)

Suda, Srinivas; Field, Des; Barron, Niall

2017-01-01

Antimicrobial peptides (AMPs) are natural defense compounds which are synthesized as ribosomal gene-encoded pre-peptides and produced by all living organisms. AMPs are small peptides, usually cationic and typically have hydrophobic residues which interact with cell membranes and have either a narrow or broad spectrum of biological activity. AMPs are isolated from the natural host or heterologously expressed in other hosts such as Escherichia coli. The proto-typical lantibiotic Nisin is a widely used AMP that is produced by the food-grade organism Lactococcus lactis. Although AMP production and purification procedures require optimization for individual AMPs, the Nisin production and purification protocol outlined in this chapter can be easily applied with minor modifications for the production and purification of other lantibiotics or AMPs. While Nisin is produced and secreted into the supernatant, steps to recover Nisin from both cell-free supernatant and cell pellet are outlined in detail.

Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

Directory of Open Access Journals (Sweden)

Haddad Muhammad

2016-01-01

Full Text Available Camelid’ s heavy-chain antibody (HCAb consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody® was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG. Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.

Representations of affine Hecke algebras

CERN Document Server

Xi, Nanhua

1994-01-01

Kazhdan and Lusztig classified the simple modules of an affine Hecke algebra Hq (q E C*) provided that q is not a root of 1 (Invent. Math. 1987). Ginzburg had some very interesting work on affine Hecke algebras. Combining these results simple Hq-modules can be classified provided that the order of q is not too small. These Lecture Notes of N. Xi show that the classification of simple Hq-modules is essentially different from general cases when q is a root of 1 of certain orders. In addition the based rings of affine Weyl groups are shown to be of interest in understanding irreducible representations of affine Hecke algebras. Basic knowledge of abstract algebra is enough to read one third of the book. Some knowledge of K-theory, algebraic group, and Kazhdan-Lusztig cell of Cexeter group is useful for the rest

Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

Science.gov (United States)

Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

2013-06-01

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

Aquatic macroinvertebrate responses to native and non-native predators

Directory of Open Access Journals (Sweden)

Haddaway N. R.

2014-01-01

Full Text Available Non-native species can profoundly affect native ecosystems through trophic interactions with native species. Native prey may respond differently to non-native versus native predators since they lack prior experience. Here we investigate antipredator responses of two common freshwater macroinvertebrates, Gammarus pulex and Potamopyrgus jenkinsi, to olfactory cues from three predators; sympatric native fish (Gasterosteus aculeatus, sympatric native crayfish (Austropotamobius pallipes, and novel invasive crayfish (Pacifastacus leniusculus. G. pulex responded differently to fish and crayfish; showing enhanced locomotion in response to fish, but a preference for the dark over the light in response to the crayfish. P.jenkinsi showed increased vertical migration in response to all three predator cues relative to controls. These different responses to fish and crayfish are hypothesised to reflect the predators’ differing predation types; benthic for crayfish and pelagic for fish. However, we found no difference in response to native versus invasive crayfish, indicating that prey naiveté is unlikely to drive the impacts of invasive crayfish. The Predator Recognition Continuum Hypothesis proposes that benefits of generalisable predator recognition outweigh costs when predators are diverse. Generalised responses of prey as observed here will be adaptive in the presence of an invader, and may reduce novel predators’ potential impacts.

Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin

International Nuclear Information System (INIS)

Hillier, Brian J.; Sundaresan, Vidyasankar; Stout, C. David; Vacquier, Victor D.

2005-01-01

The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain

Antisymmetric tensor generalizations of affine vector fields.

Science.gov (United States)

Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

2016-02-01

Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

Manifolds with integrable affine shape operator

Directory of Open Access Journals (Sweden)

Daniel A. Joaquín

2005-05-01

Full Text Available This work establishes the conditions for the existence of vector fields with the property that theirs covariant derivative, with respect to the affine normal connection, be the affine shape operatorS in hypersurfaces. Some results are obtained from this property and, in particular, for some kind of affine decomposable hypersurfaces we explicitely get the actual vector fields.

Cytotoxic Effects of Native and Recombinant Frutalin, a Plant Galactose-Binding Lectin, on HeLa Cervical Cancer Cells

Directory of Open Access Journals (Sweden)

Carla Oliveira

2011-01-01

Full Text Available Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC50~100 μg/mL and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity.

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

Directory of Open Access Journals (Sweden)

Bing Xu

Full Text Available G protein-coupled receptors (GPCRs exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(--TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

RELIGION AND PURIFICATION OF SOUL

Directory of Open Access Journals (Sweden)

Azam Khodashenas Pelko

2010-11-01

Full Text Available The Jainism emphasizes three major teachings about the purification of the soul (jiva, Ahimsa, Aparigrapha and anekantwad. Jainism, The focus of this religion has been purification of the soul by means of right conduct, right faith and right knowledge. The ultimate goal of Hinduism is Moksha or liberation (total freedom. In Hinduism, purification of the soul is a goal that one must work to attain. The Buddhism is the science of pursuing the aim of making the human mind perfect, and of purifying the human soul. The knowledge of purifying of the soul and softening of the hearts is as essential for human. They having the correct motivations means purifying our souls from hypocrisy, caprice, and heedlessness. The primary goal of Taoism may be described as the mystical intuition of the Tao, which is the way, the undivided unity, and the ultimate Reality. According to the Christianity access to truth cannot be conceived without purity of the soul

Solvent-extraction purification of neptunium

International Nuclear Information System (INIS)

Kyser, E.A.; Hudlow, S.L.

2008-01-01

The Savannah River Site (SRS) has recovered 237 Np from reactor fuel that is currently being processed into NpO 2 for future production of 238 Pu. Several purification flowsheets have been utilized. An oxidizing solvent-extraction (SX) flowsheet was used to remove Fe, sulfate ion, and Th while simultaneously 237 Np, 238 Pu, u, and nonradioactive Ce(IV) was extracted into the tributyl phosphate (TBP) based organic solvent. A reducing SX flowsheet (second pass) removed the Ce and Pu and recovered both Np and U. The oxidizing flowsheet was necessary for solutions that contained excessive amounts of sulfate ion. Anion exchange was used to perform final purification of Np from Pu, U, and various non-actinide impurities. The Np(IV) in the purified solution was then oxalate-precipitated and calcined to an oxide for shipment to other facilities for storage and future target fabrication. Performance details of the SX purification and process difficulties are discussed. (authors)

Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.

Science.gov (United States)

Jha, Shweta; Agarwal, Saurabh; Sanyal, Indraneel; Amla, D V

2016-05-01

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

Application of enhanced electronegative multimodal chromatography as the primary capture step for immunoglobulin G purification.

Science.gov (United States)

Wang, Yanli; Chen, Quan; Xian, Mo; Nian, Rui; Xu, Fei

2018-06-01

In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.

Fluorescence spectroscopic investigation of the interaction of citrinin with native and chemically modified cyclodextrins

Energy Technology Data Exchange (ETDEWEB)

Poór, Miklós, E-mail: poor.miklos@pte.hu [Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Szigeti út 12, Pécs H-7624 (Hungary); Matisz, Gergely; Kunsági-Máté, Sándor [Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6, Pécs H-7624 (Hungary); János Szentágothai Research Center, Ifjúság útja 20, Pécs H-7624 (Hungary); Derdák, Diána [Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6, Pécs H-7624 (Hungary); Szente, Lajos [CycloLab Cyclodextrin Research & Development Laboratory Ltd., Illatos út 7, Budapest H-1097 (Hungary); and others

2016-04-15

Citrinin (CIT) is a nephrotoxic mycotoxin produced by several Aspergillus, Penicillium and Monascus species. CIT is unavoidable contaminant of different foods and drinks due to its wide occurrence and high thermal stability. For this reason, development of new, more sensitive analytical methods and decontamination strategies has high importance. In our study, the complex formation of CIT with native and chemically modified cyclodextrins was investigated using fluorescence spectroscopy. Furthermore, thermodynamic and molecular modeling studies were also performed for the deeper understanding of these host-guest interactions. Our results show that among the tested compounds methylated β-cyclodextrins form the most stable complexes with CIT and these derivatives cause the highest fluorescence enhancement of CIT as well. These observations recommend that some of the chemically modified derivatives show more favourable properties than the native cyclodextrin, and suggesting more promising analytical applicability and higher affinity as potential toxin binders.

Affine LIBOR Models with Multiple Curves

DEFF Research Database (Denmark)

Grbac, Zorana; Papapantoleon, Antonis; Schoenmakers, John

2015-01-01

are specified following the methodology of the affine LIBOR models and are driven by the wide and flexible class of affine processes. The affine property is preserved under forward measures, which allows us to derive Fourier pricing formulas for caps, swaptions, and basis swaptions. A model specification...... with dependent LIBOR rates is developed that allows for an efficient and accurate calibration to a system of caplet prices....

Lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS): a novel method to analyze protein-lipid interaction.

Science.gov (United States)

Bieberich, Erhard

2011-04-26

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane

Native excellence

International Nuclear Information System (INIS)

Bower, T.

1992-01-01

Syncrude Canada Ltd., operator of the oil sands mine and processing plant near Fort McMurray, Alberta, produces 11% of Canada's crude oil and is the country's largest private-sector employer of native Canadians. Syncrude has the goal of employing about 10% native Canadians, which is about the percentage of natives in the regional population. Examples are presented of successful native employment and entrepreneurship at Syncrude. Doreen Janvier, once employed at Syncrude's mine wash bays, was challenged to form her own company to contract out labor services. Her company, DJM Enterprises, now has a 2-year contract to operate three highly sophisticated wash bays used to clean mining equipment, and is looking to bid on other labor contracts. Mabel Laviolette serves as liaison between the oil containment and recovery team, who recover oil skimmed off Syncrude's tailings basin, and the area manager. The team approach and the seasonal nature of the employment fit in well with native cultural patterns. The excellence of native teamwork is also illustrated in the mine rescue team, one unit of which is entirely native Canadian. Part of Syncrude's aboriginal policy is to encourage development of aboriginal enterprises, such as native-owned Clearwater Welding and Fabricating Ltd., which has held welding and fabricating contracts with most major companies in the region and is a major supplier of skilled tradesmen to Syncrude. Syncrude also provides employment and training, encourages natives to continue their education, and promotes local community development. 4 figs

Purification of recombinant aprotinin produced in transgenic corn seed: separation from CTI utilizing ion-exchange chromatography

Directory of Open Access Journals (Sweden)

A. R. Azzoni

2005-09-01

Full Text Available Protein expression in transgenic plants is considered one of the most promising approaches for producing pharmaceutical proteins. As has happened with other recombinant protein production schemes, the downstream processing (dsp of these proteins produced in plants is key to the technical and economic success of large-scale applications. Since dsp of proteins produced transgenically in plants has not been extensively studied, it is necessary to broaden the investigation in this field in order to more precisely evaluate the commercial feasibility of this route of expression. In this work, we studied the substitution of an IMAC chromatographic step, described in previous work (Azzoni et al., 2002, with ion-exchange chromatography on SP Sepharose Fast Flow resin as the second step in the purification of recombinant aprotinin from transgenic maize seed. The main goal of this second purification step is to separate the recombinant aprotinin from the native corn trypsin inhibitor. Analysis of the adsorption isotherms determined at 25°C under different conditions allowed selection of 0.020 M Tris pH 8.5 as the adsorption buffer. The cation-exchange chromatographic process produced a high-purity aprotinin that was more than ten times more concentrated than that generated using an IMAC step.

Purification of rhamnolipid using colloidal magnetic nanoparticles ...

African Journals Online (AJOL)

Phospholipid-coated colloidal magnetic nanoparticles with mean magnetite core size of 9 nm are shown to be effective ion exchange media for the recovery and purification of Rhaminolipid from culture mixtures. These particles have high adsorption capacity for purification (an order of magnitude larger than the best ...

Effect of water purification process in radioactive content: analysis on small scale purification plants

International Nuclear Information System (INIS)

Lopez del Rio, H.; Quiroga S, J. C.; Davila R, J. I.; Mireles G, F.

2009-10-01

Water from small scale purification plants is a low cost alternative for consumers in comparison to the bottled commercial presentations. Because of its low cost per liter, the consumption of this product has increased in recent years, stimulating in turn the installation of purification systems for these small businesses. The purpose of this study was to estimate the efficiency of small scale purification systems located in the cities of Zacatecas and Guadalupe, Zacatecas, to reduce the radioactive content of water. It was measured the total alpha and beta activity in water samples of entry and exit to process, through the liquid scintillation technique. In general it was observed that the process is more efficient in removing alpha that beta activity. The fraction of total alpha activity removed varied between 27 and 100%, while between 0 and 77% of the total beta activity was removed by the analyzed plants. In all cases, the total radioactivity level was lower than the maximum permissible value settled by the official mexican standard for drinking water. (Author)

Affinity Programs and the Real Estate Brokerage Industry

OpenAIRE

G Stacy Sirmans; David A. Macpherson

2001-01-01

This study surveys active real estate brokers obtaining information on involvement in affinity programs and referral/relocation networks. Some results regarding affinity involvement are: (a) 13% of respondents reported affinity affilliations, 75% reported no affiliations, and 12% indicated plans to become involved within the next year; (b) about half having affinity affiliations were involved with 2-4 groups; (c) affinity relationships were most often with membership organizations, corporatio...

Vulnerability of freshwater native biodiversity to non-native ...

Science.gov (United States)

Background/Question/Methods Non-native species pose one of the greatest threats to native biodiversity. The literature provides plentiful empirical and anecdotal evidence of this phenomenon; however, such evidence is limited to local or regional scales. Employing geospatial analyses, we investigate the potential threat of non-native species to threatened and endangered aquatic animal taxa inhabiting unprotected areas across the continental US. We compiled distribution information from existing publicly available databases at the watershed scale (12-digit hydrologic unit code). We mapped non-native aquatic plant and animal species richness, and an index of cumulative invasion pressure, which weights non-native richness by the time since invasion of each species. These distributions were compared to the distributions of native aquatic taxa (fish, amphibians, mollusks, and decapods) from the International Union for the Conservation of Nature (IUCN) database. We mapped the proportion of species listed by IUCN as threatened and endangered, and a species rarity index per watershed. An overlay analysis identified watersheds experiencing high pressure from non-native species and also containing high proportions of threatened and endangered species or exhibiting high species rarity. Conservation priorities were identified by generating priority indices from these overlays and mapping them relative to the distribution of protected areas across the US. Results/Conclusion

Invasive non-native species' provision of refugia for endangered native species.

Science.gov (United States)

Chiba, Satoshi

2010-08-01

The influence of non-native species on native ecosystems is not predicted easily when interspecific interactions are complex. Species removal can result in unexpected and undesired changes to other ecosystem components. I examined whether invasive non-native species may both harm and provide refugia for endangered native species. The invasive non-native plant Casuarina stricta has damaged the native flora and caused decline of the snail fauna on the Ogasawara Islands, Japan. On Anijima in 2006 and 2009, I examined endemic land snails in the genus Ogasawarana. I compared the density of live specimens and frequency of predation scars (from black rats [Rattus rattus]) on empty shells in native vegetation and Casuarina forests. The density of land snails was greater in native vegetation than in Casuarina forests in 2006. Nevertheless, radical declines in the density of land snails occurred in native vegetation since 2006 in association with increasing predation by black rats. In contrast, abundance of Ogasawarana did not decline in the Casuarina forest, where shells with predation scars from rats were rare. As a result, the density of snails was greater in the Casuarina forest than in native vegetation. Removal of Casuarina was associated with an increased proportion of shells with predation scars from rats and a decrease in the density of Ogasawarana. The thick and dense litter of Casuarina appears to provide refugia for native land snails by protecting them from predation by rats; thus, eradication of rats should precede eradication of Casuarina. Adaptive strategies, particularly those that consider the removal order of non-native species, are crucial to minimizing the unintended effects of eradication on native species. In addition, my results suggested that in some cases a given non-native species can be used to mitigate the impacts of other non-native species on native species.

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

Science.gov (United States)

Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

2006-01-01

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

Credibility of native and non-native speakers of English revisited: Do non-native listeners feel the same?

OpenAIRE

Hanzlíková, Dagmar; Skarnitzl, Radek

2017-01-01

This study reports on research stimulated by Lev-Ari and Keysar (2010) who showed that native listeners find statements delivered by foreign-accented speakers to be less true than those read by native speakers. Our objective was to replicate the study with non-native listeners to see whether this effect is also relevant in international communication contexts. The same set of statements from the original study was recorded by 6 native and 6 nonnative speakers of English. 121 non-native listen...

A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate

DEFF Research Database (Denmark)

Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose

2014-01-01

P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic...... leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase......, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover...

Purification of crude biodiesel using dry washing and membrane technologies

Directory of Open Access Journals (Sweden)

I.M. Atadashi

2015-12-01

Full Text Available Purification of crude biodiesel is mandatory for the fuel to meet the strict international standard specifications for biodiesel. Therefore, this paper carefully analyzed recently published literatures which deal with the purification of biodiesel. As such, dry washing technologies and the most recent membrane biodiesel purification process have been thoroughly examined. Although purification of biodiesel using dry washing process involving magnesol and ion exchange resins provides high-quality biodiesel fuel, considerable amount of spent absorbents is recorded, besides the skeletal knowledge on its operating process. Further, recent findings have shown that biodiesel purification using membrane technique could offer high-quality biodiesel fuel with less wastewater discharges. Thus, both researchers and industries are expected to benefit from the development of membrane technique in purifying crude biodiesel. As well biodiesel purification via membranes has been shown to be environmentally friendly. For these reasons, it is important to explore and exploit membrane technology to purify crude biodiesel.

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

Directory of Open Access Journals (Sweden)

Zarei Saeed

2011-08-01

Full Text Available Abstract Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also

Using Affinity Diagrams to Evaluate Interactive Prototypes

DEFF Research Database (Denmark)

Lucero, Andrés

2015-01-01

our particular use of affinity diagramming in prototype evaluations. We reflect on a decade’s experience using affinity diagramming across a number of projects, both in industry and academia. Our affinity diagramming process in interaction design has been tailored and consists of four stages: creating...

Materials for Molybdenum 99 purification

International Nuclear Information System (INIS)

Wilkinson, M. Victoria; Mondino, Angel V.; Manzini, Alberto C.

2003-01-01

The National Atomic Energy Commission (CNEA) produces fission Mo 99, an isotope of wide use in nuclear medicine. In order to simplify the current Mo 99 production process, to shorten its duration and reduce impurities in the final product, alternative methods for purification steps were looked for. In this work a variety of new materials for the purification columns were designed, all of them with carbon. These materials were studied and a material which contribute with the best results for molybdenum retention, was selected. The preparation procedure and the working conditions were determined. (author)

Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry

Science.gov (United States)

Zhang, Jiang; Loo, Rachel R. Ogorzalek; Loo, Joseph A.

2017-09-01

Native mass spectrometry (MS) with electrospray ionization (ESI) has evolved as an invaluable tool for the characterization of intact native proteins and non-covalently bound protein complexes. Here we report the structural characterization by high resolution native top-down MS of human thrombin and its complex with the Bock thrombin binding aptamer (TBA), a 15-nucleotide DNA with high specificity and affinity for thrombin. Accurate mass measurements revealed that the predominant form of native human α-thrombin contains a glycosylation mass of 2205 Da, corresponding to a sialylated symmetric biantennary oligosaccharide structure without fucosylation. Native MS showed that thrombin and TBA predominantly form a 1:1 complex under near physiological conditions (pH 6.8, 200 mM NH4OAc), but the binding stoichiometry is influenced by the solution ionic strength. In 20 mM ammonium acetate solution, up to two TBAs were bound to thrombin, whereas increasing the solution ionic strength destabilized the thrombin-TBA complex and 1 M NH4OAc nearly completely dissociated the complex. This observation is consistent with the mediation of thrombin-aptamer binding through electrostatic interactions and it is further consistent with the human thrombin structure that contains two anion binding sites on the surface. Electron capture dissociation (ECD) top-down MS of the thrombin-TBA complex performed with a high resolution 15 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer showed the primary binding site to be at exosite I located near the N-terminal sequence of the heavy chain, consistent with crystallographic data. High resolution native top-down MS is complementary to traditional structural biology methods for structurally characterizing native proteins and protein-DNA complexes. [Figure not available: see fulltext.

Gel-Based Purification and Biochemical Study of Laccase Isozymes from Ganoderma sp. and Its Role in Enhanced Cotton Callogenesis

Directory of Open Access Journals (Sweden)

Krishna K. Sharma

2017-04-01

Full Text Available Basidiomycetous fungi, Ganoderma lucidum MDU-7 and Ganoderma sp. kk-02 secreted multiple laccase isozymes under diverse growth condition. Aromatic compounds and metal salts were also found to regulate the differential expression of laccase isozymes from both the Ganoderma sp. Laccase isozymes induced in the presence of copper from G. lucidum MDU-7 were purified by gel-based (native-PAGE purification method. The purity of laccase isozymes was checked by zymogram and SDS-PAGE. The SDS-PAGE of purified proteins confirmed the multimeric nature of laccase isozymes. The molecular mass of isozymes was found to be in the range of 40–66 kDa. Further, the purified laccase isozymes and their peptides were confirmed with the help of MALDI-TOF peptide fingerprinting. The biochemical characterization of laccase isozymes viz. Glac L2, Glac L3, Glac L4, and Glac L5 have shown the optimum temperature in the range of 30°–45°C and pH 3.0. The Km values of all the laccase isozymes determined for guaiacol were (96–281 μM, ABTS (15–83 μM and O-tolidine (78–724 μM. Further, laccase isozymes from G. lucidum whole genome were studied using bioinformatics tools. The molecular modeling and docking of laccase isozymes with different substrates showed a significant binding affinity, which further validates our experimental results. Interestingly, copper induced laccase of 40 U/ml in culture medium was found to significantly induce cotton callogenesis. Interestingly, all the laccase isozymes were found to have an antioxidative role and therefore capable in free radicals scavenging during callogenesis. This is the first detailed study on the biochemical characterization of all the laccase isozymes purified by a gel-based novel method.

Lapa vermelha IV Hominid 1: morphological affinities of the earliest known American

Directory of Open Access Journals (Sweden)

Neves Walter A.

1999-01-01

Full Text Available Several studies concerning the extra-continental morphological affinities of Paleo-Indian skeletons, carried out independently in South and North America, have indicated that the Americas were first occupied by non-Mongoloids that made their way to the New World through the Bering Strait in ancient times. The first South Americans show a clear resemblance to modern South Pacific and African populations, while the first North Americans seem to be at an unresolved morphological position between modern South Pacific and Europeans. In none of these analyses the first Americans show any resemblance to either northeast Asians or modern native Americans. So far, these studies have included affirmed and putative early skeletons thought to date between 8,000 and 10,000 years B.P. In this work the extra-continental morphological affinities of a Paleo-Indian skeleton well dated between 11,000 and 11,500 years B.P. (Lapa Vermelha IV Hominid 1, or "Luzia" is investigated, using as comparative samples Howells' (1989 world-wide modern series and Habgood's (1985 Old World Late Pleistocene fossil hominids. The comparison between Lapa Vermelha IV Hominid 1 and Howells' series was based on canonical variate analysis, including 45 size-corrected craniometric variables, while the comparison with fossil hominids was based on principal component analysis, including 16 size-corrected variables. In the first case, Lapa Vermelha IV Hominid 1 exhibited an undisputed morphological affinity firstly with Africans and secondly with South Pacific populations. In the second comparison, the earliest known American skeleton had its closest similarities with early Australians, Zhoukoudian Upper Cave 103, and Taforalt 18. The results obtained clearly confirm the idea that the Americas were first colonized by a generalized Homo sapiens population which inhabited East Asia in the Late Pleistocene, before the definition of the classic Mongoloid morphology.

The Cutting Edge of Affinity Electrophoresis Technology.

Science.gov (United States)

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

2015-03-18

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

Electron beam silicon purification

Energy Technology Data Exchange (ETDEWEB)

Kravtsov, Anatoly [SIA ' ' KEPP EU' ' , Riga (Latvia); Kravtsov, Alexey [' ' KEPP-service' ' Ltd., Moscow (Russian Federation)

2014-11-15

Purification of heavily doped electronic grade silicon by evaporation of N-type impurities with electron beam heating was investigated in process with a batch weight up to 50 kilos. Effective temperature of the melt, an indicative parameter suitable for purification process characterization was calculated and appeared to be stable for different load weight processes. Purified material was successfully approbated in standard CZ processes of three different companies. Each company used its standard process and obtained CZ monocrystals applicable for photovoltaic application. These facts enable process to be successfully scaled up to commercial volumes (150-300 kg) and yield solar grade silicon. (copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

NATIVE VS NON-NATIVE ENGLISH TEACHERS

Directory of Open Access Journals (Sweden)

Masrizal Masrizal

2013-02-01

Full Text Available Although the majority of English language teachers worldwide are non-native English speakers (NNS, no research was conducted on these teachers until recently. A pioneer research by Peter Medgyes in 1994 took quite a long time until the other researchers found their interests in this issue. There is a widespread stereotype that a native speaker (NS is by nature the best person to teach his/her foreign language. In regard to this assumption, we then see a very limited room and opportunities for a non native teacher to teach language that is not his/hers. The aim of this article is to analyze the differences among these teachers in order to prove that non-native teachers have equal advantages that should be taken into account. The writer expects that the result of this short article could be a valuable input to the area of teaching English as a foreign language in Indonesia.

Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis.

Science.gov (United States)

Skottrup, Peter Durand; Leonard, Paul; Kaczmarek, Jakub Zbigniew; Veillard, Florian; Enghild, Jan Johannes; O'Kennedy, Richard; Sroka, Aneta; Clausen, Rasmus Prætorius; Potempa, Jan; Riise, Erik

2011-08-15

Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection. Copyright © 2011 Elsevier Inc. All rights reserved.

Purification of a-galactosidase from seeds of Sesbania marginata

Directory of Open Access Journals (Sweden)

Falco A.L.P.

2000-01-01

Full Text Available Alpha-galactosidase taken from a raw extract of Sesbania marginata legume seeds was purified by partitioning in aqueous two-phase systems (ATPS. Initially, galactomannan/dextran 2,000,000 systems were used for the purification, and the partition coefficients of alpha -galactosidase varied from 1.5 to 4.0. However, mass transport in these systems was poor due to the high viscosity of the employed polymers. Therefore, partitioning in polyethyleneglycol (PEG/ sodium phosphate systems and the effect of sodium chloride upon the enzyme purification and the yield of alpha -galactosidase were also investigated. The purification achieved in a single-step was 5.7 with a recovery of 144% of alpha -galactosidase, possibly due to the removal of materials which inhibited alpha -galactosidase activity before the purification. The removal of the main protein contaminants and the highest yields were achieved in PEG 4,000/ sodium phosphate + 6% NaCl system at pH 5.0. Further purification by preparative on-exchange chromatography was also developed.

Necessity of purification during bacterial DNA extraction with environmental soils

Directory of Open Access Journals (Sweden)

Hyun Jeong Lim

2017-08-01

Full Text Available Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification. The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg] showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.

Connections between quantized affine algebras and superalgebras

International Nuclear Information System (INIS)

Zhang, R.B.

1992-08-01

Every affine superalgebra with a symmetrizable Cartan matrix is closely related to an ordinary affine algebra with the same Cartan matrix. It is shown that the quantum supergroup associated with the former is essentially isomorphic to the quantum group associated with the latter in an appropriate class of representations. At the classical level, each integrable irreducible highest weight representation of the affine superalgebra has a corresponding irreducible representation of the affine algebra, which has the same weight space decomposition. (author). 5 refs, 3 tabs

Mobile Technology Affinity in Renal Transplant Recipients.

Science.gov (United States)

Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

Technological assumptions for biogas purification.

Science.gov (United States)

Makareviciene, Violeta; Sendzikiene, Egle

2015-01-01

Biogas can be used in the engines of transport vehicles and blended into natural gas networks, but it also requires the removal of carbon dioxide, hydrogen sulphide, and moisture. Biogas purification process flow diagrams have been developed for a process enabling the use of a dolomite suspension, as well as for solutions obtained by the filtration of the suspension, to obtain biogas free of hydrogen sulphide and with a carbon dioxide content that does not exceed 2%. The cost of biogas purification was evaluated on the basis of data on biogas production capacity and biogas production cost obtained from local water treatment facilities. It has been found that, with the use of dolomite suspension, the cost of biogas purification is approximately six times lower than that in the case of using a chemical sorbent such as monoethanolamine. The results showed travelling costs using biogas purified by dolomite suspension are nearly 1.5 time lower than travelling costs using gasoline and slightly lower than travelling costs using mineral diesel fuel.

Introduced brown trout alter native acanthocephalan infections in native fish.

Science.gov (United States)

Paterson, Rachel A; Townsend, Colin R; Poulin, Robert; Tompkins, Daniel M

2011-09-01

1. Native parasite acquisition provides introduced species with the potential to modify native host-parasite dynamics by acting as parasite reservoirs (with the 'spillback' of infection increasing the parasite burdens of native hosts) or sinks (with the 'dilution' of infection decreasing the parasite burdens of native hosts) of infection. 2. In New Zealand, negative correlations between the presence of introduced brown trout (Salmo trutta) and native parasite burdens of the native roundhead galaxias (Galaxias anomalus) have been observed, suggesting that parasite dilution is occurring. 3. We used a multiple-scale approach combining field observations, experimental infections and dynamic population modelling to investigate whether native Acanthocephalus galaxii acquisition by brown trout alters host-parasite dynamics in native roundhead galaxias. 4. Field observations demonstrated higher infection intensity in introduced trout than in native galaxias, but only small, immature A. galaxii were present in trout. Experimental infections also demonstrated that A. galaxii does not mature in trout, although parasite establishment and initial growth were similar in the two hosts. Taken together, these results support the hypothesis that trout may serve as an infection sink for the native parasite. 5. However, dynamic population modelling predicts that A. galaxii infections in native galaxias should at most only be slightly reduced by dilution in the presence of trout. Rather, model exploration indicates parasite densities in galaxias are highly sensitive to galaxias predation on infected amphipods, and to relative abundances of galaxias and trout. Hence, trout presence may instead reduce parasite burdens in galaxias by either reducing galaxias density or by altering galaxias foraging behaviour. © 2011 The Authors. Journal of Animal Ecology © 2011 British Ecological Society.

Water Purification Characteristic of the Actual Constructed Wetland with Carex dispalata in a Cold Area

Science.gov (United States)

Tsuji, Morio; Yamada, Kazuhiro; Hiratsuka, Akira; Tsukada, Hiroko

Carex dispalata, a native plant species applied in cold districts for water purification in constructed wetlands, has useful characteristics for landscape creation and maintenance. In this study, seasonal differences in purification ability were verified, along with comparison of frozen and non-frozen periods' performance. A wetland area was constructed using a “hydroponics method” and a “coir fiber based method”. Results show that the removal rates of BOD, SS, and Chl-a were high. On this constructed wetland reduces organic pollution, mainly phytoplankton, but the removal of nitrogen and phosphorus was insufficient. The respective mean values of influent and treated water during three years were 26.6 mg/L and 12.2 mg/L for BOD, and 27.9 mg/L and 7.5 mg/L for SS. The mean value of the BOD removal rate for the non-frozen period was 2.99 g/m2/d that for the frozen period was 1.86 g/m2/d. The removal rate followed the rise of the BOD load rate. The removal rate limits were about 4 g/m2/d during the frozen period and 15 g/m2/d during the non-frozen period. For operations, energy was unnecessary. The required working hours were about 20 h annually for all maintenance and management during operations.

Expression, purification and crystallization of a lyssavirus matrix (M) protein

Science.gov (United States)

Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

2008-01-01

The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

Extraction and purification of plutonium by a tertiary amine; Extraction et purification du plutonium par une amine tertiaire

Energy Technology Data Exchange (ETDEWEB)

Trentinian, M de; Chesne, A [Commissariat a l' Energie Atomique, Fontenay aux Roses, Section de Chimie des Actimides (France).Centre d' Etudes Nucleaires; Commissariat a l' Energie Atomique, Saclay (France).Centre d' Etudes Nucleaires

1960-07-01

Trilaurylamine diluted with a paraffinic solvent (dodecane) was studied as part of the research dealing with the separation and purification of plutonium. The physical properties (solubility of nitrates in the amine as a function of temperature) and the resistance to radiations of this substance were examined. The extraction characteristics of nitric solutions of plutonium, uranium and certain fission products are given as a function of the following factors: concentration of the various ions in solution, valency states. A method of plutonium purification based on these results is presented. (author) [French] La trilaurylamine diluee par un solvant paraffinique (dodecane) a ete etudiee dans le cadre des recherches concernant la separation et la purification du plutonium. Une etude des caracteres physiques (solubilite des nitrates dans l'amine en fonction de la temperature) s'ajoute a celle de la tenue aux radiations de ce corps. Les caracteristiques d'extraction de solutions nitriques de plutonium, uranium, et certains produits de fission, sont donnes en fonction des facteurs suivants: concentration des differents ions en solution, etats de valence. On presente une methode de purification du plutonium basee sur ces resultats. (auteur)

The Cutting Edge of Affinity Electrophoresis Technology

Science.gov (United States)

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

2015-01-01

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

Affine Fullerene C60 in a GS-Quasigroup

Directory of Open Access Journals (Sweden)

Vladimir Volenec

2014-01-01

Full Text Available It will be shown that the affine fullerene C60, which is defined as an affine image of buckminsterfullerene C60, can be obtained only by means of the golden section. The concept of the affine fullerene C60 will be constructed in a general GS-quasigroup using the statements about the relationships between affine regular pentagons and affine regular hexagons. The geometrical interpretation of all discovered relations in a general GS-quasigroup will be given in the GS-quasigroup C(1/2(1+5.

Optimization of laboratory scale production and purification of ...

African Journals Online (AJOL)

Microcystin content is however highly variable and optimised culture conditions are essential to produce viable yields of microcystin for purification. We describe the optimization of culture conditions and evaluation of various purification methods to enhance the yield of microcystin from laboratory scale culture.

Affinity Spaces and 21st Century Learning

Science.gov (United States)

Gee, James Paul

2017-01-01

This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

Hydrogen purification by periodic adsorption

Energy Technology Data Exchange (ETDEWEB)

Barg, Christian; Secchi, Argimiro R.; Trierweiler, Jorge O. [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Dept. de Engenharia Quimica]. E-mail: cbarg@enq.ufrgs.br; arge@enq.ufrgs.br; jorge@enq.ufrgs.br

2000-07-01

The periodic adsorption processes have been widely used for industrial applications, mainly because it spends less energy than the usual gas separation processes, like the cryogenic distillation. The largest commercial application of periodic adsorption processes is the pressure swing adsorption (PSA) applied to hydrogen purification. Although its wide use in the chemical and petrochemical industry, there are no reports in the open literature about complete modeling studies of a complex commercial unit, with multiple adsorbents and multiple beds and several feed components. This study has as objective the modeling, optimization and dynamical analysis of an industrial PSA unit for hydrogen purification. (author)

Extraction and purification of yellow cake

International Nuclear Information System (INIS)

Yousif, E.H.

2006-01-01

This dissertation has reviewed current studies on production and purification of yellow cake from uranium ores by both acid and alkaline leaching processes. It comprises three chapters, the first one deal with uranium minerals, uranium deposits, geology of uranium and uranium isotopes. The second chapter covers mining and milling methods, uranium leaching chemistry, precipitation, and purification of uranium concentrate by solvent extraction and possible impurities that commonly interfered with yellow cake. The last chapter presented ongoing literature review.(Author)

Rapid purification of radioiodinated peptides with Sep-Pak reversed phase cartridges and HPLC

International Nuclear Information System (INIS)

Miller, J.J.; Schultz, G.S.; Levy, R.S.

1984-01-01

A simple, rapid method is described for the purification of radioiodinated peptides for use in radioimmuno- and in radioreceptor assays. Iodinated reaction mixtures are applied directly onto Sep-Pak disposable, reversed phase cartridges equilibrated with phosphate buffer. Unreacted 125-iodide and other non-peptide reaction components are eluted with buffer. The peptide fraction is then eluted with 70% buffer:30% acetonitrile. The peptide fraction is further purified by reversed phase high pressure liquid chromatography to separate the native peptide and the mono- and diiodo-derivatives. In this study the method is used to prepare 125-iodide-labeled monoiodo-leucine enkephalin and monoiodo-angiotensin II, which are free of the parent peptides and diiodo-derivatives and are of maximum obtainable specific radioactivity. The usefulness of these labeled peptides in radioimmuno- and radioreceptor assays is demonstrated by their binding to specific antibodies and receptors, respectively. (author)

Microbiological and technical aspects of anaerobic waste water purification

International Nuclear Information System (INIS)

Aivasidis, A.

1994-01-01

Anaerobic waste water purification is likely to be another example of how innovations can result from the joint use of biological and technical concepts. No matter how far the optimization of oxygen input with aerobic waste water purification advances it will still be the less a real competitor for anaerobic techniques the more polluted the waste water is. The principle of carrier fixation to avoid their washing out, too, has often been observed in nature with sessile microorganisms. With highly polluted water, anaerobic purification does not only work at no expenditure of energy but it can also make excess energy available for use in other processes. Another important argument for anaerobic methods of waste water purification is probably the clearly reduced production of excess sludge. (orig.) [de

Multipartite electronic entanglement purification with charge detection

Energy Technology Data Exchange (ETDEWEB)

Sheng Yubo [Department of Physics, Tsinghua University, Beijing 100084 (China); Deng, Fu-Guo [Department of Physics, Beijing Normal University, Beijing 100875 (China); Long Guilu, E-mail: gllong@tsinghua.edu.c [Department of Physics, Tsinghua University, Beijing 100084 (China); Key Laboratory for Atomic and Molecular NanoSciences, Tsinghua University, Beijing 100084 (China); Tsinghua National Laboratory for Information Science and Technology, Beijing 100084 (China)

2011-01-17

We present a multipartite entanglement purification scheme in a Greenberger-Horne-Zeilinger state for electrons based on their spins and their charges. This scheme works for purification with two steps, i.e., bit-flip error correction and phase-flip error correction. By repeating these two steps, the parties in quantum communication can get some high-fidelity multipartite entangled electronic systems.

Purification of bentonitics of the city Cubati-PB to obtaining organoclays for use in oil-based drilling fluids

International Nuclear Information System (INIS)

Costa, J.M.R.; Vitorino, I.J.F.; Silva, I.A.; Neves, G.A.; Ferreira, H.C.

2011-01-01

In Boa Vista, PB, are located deposits of bentonite clays commonly used in the preparation of drilling fluids. The disorderly exploitation of the deposits of Boa Vista is causing the depletion of these clays, which will cause a very serious problem for the national oil industry. This work aims to characterize new deposits of bentonite clays Cubati, PB, for the development of organoclay from its refining using a hydrocyclone for use in oil based drilling fluids. The characterization of samples of the clays was performed through the techniques: AG, XRD, EDX, TGA and DTA. The characterization is typical of bentonite for the purification process was determined the best configuration of the hydrocyclone, and the environment organic diesel fuel, the best affinity was obtained with clays organophilizated with surfactant Praepagen WB. (author)

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

International Nuclear Information System (INIS)

Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

1988-01-01

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe -1 , Val 1 , Asn 2 , Gln 3 , His 4 , Ser 8 , His 9 , Glu 12 , Tyr 15 , Leu 16 ]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln 3 , Ala 4 ] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr 15 , Leu 16 ] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln 3 , Ala 4 , Tyr 15 ,Leu 16 ]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

On the structure of self-affine convex bodies

Energy Technology Data Exchange (ETDEWEB)

Voynov, A S [M. V. Lomonosov Moscow State University, Faculty of Mechanics and Mathematics, Moscow (Russian Federation)

2013-08-31

We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

Science.gov (United States)

Park, S H; Strobel, G A

1994-01-05

Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

21 CFR 884.6170 - Assisted reproduction water and water purification systems.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high quality...

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco1

Science.gov (United States)

Fujita, Satoshi; Uchimura, Seiichi; Noguchi, Masahiro; Demura, Taku

2016-01-01

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures. PMID:26747285

New Combined Electron-Beam Methods of Wastewater Purification

International Nuclear Information System (INIS)

Pikaev, A.K.; Makarov, I.E.; Ponomarev, A.V.; Kartasheva, L.I.; Podzorova, E.A.; Chulkov, V.N.; Han, B.; Kim, D.K.

1999-01-01

The paper is a brief review of the results obtained with the participation of the authors from the study on combined electron-beam methods for purification of some wastewaters. The data on purification of wastewaters containing dyes or hydrogen peroxide and municipal wastewater in the aerosol flow are considered

Polynomials associated with equilibria of affine Toda-Sutherland systems

International Nuclear Information System (INIS)

Odake, S; Sasaki, R

2004-01-01

An affine Toda-Sutherland system is a quasi-exactly solvable multi-particle dynamics based on an affine simple root system. It is a 'cross' between two well-known integrable multi-particle dynamics, an affine Toda molecule (exponential potential, periodic nearest-neighbour interaction) and a Sutherland system (inverse sine-square interaction). Polynomials describing the equilibrium positions of affine Toda-Sutherland systems are determined for all affine simple root systems

Alteration of the α1β2/α2β1 subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice

Energy Technology Data Exchange (ETDEWEB)

Inoguchi, Noriko; Mizuno, Nobuhiro; Baba, Seiki; Kumasaka, Takashi; Natarajan, Chandrasekhar; Storz, Jay F.; Moriyama, Hideaki; Permyakov, Eugene A.

2017-03-31

Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules. The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.

Different endothelin receptor affinities in dog tissues

International Nuclear Information System (INIS)

Loeffler, B.M.L.; Loehrer, W.

1991-01-01

Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here the authors report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl2, 1 mM EDTA, pH 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 micrograms of protein) were incubated at 25 degrees C for 180 min in the presence of 20 pM 125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 - 80 pM, Bmax 60 - 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 pM, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 pM, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 - 6 nM

Duals of Affine Grassmann Codes and Their Relatives

DEFF Research Database (Denmark)

Beelen, P.; Ghorpade, S. R.; Hoholdt, T.

2012-01-01

Affine Grassmann codes are a variant of generalized Reed-Muller codes and are closely related to Grassmann codes. These codes were introduced in a recent work by Beelen Here, we consider, more generally, affine Grassmann codes of a given level. We explicitly determine the dual of an affine...... Grassmann code of any level and compute its minimum distance. Further, we ameliorate the results by Beelen concerning the automorphism group of affine Grassmann codes. Finally, we prove that affine Grassmann codes and their duals have the property that they are linear codes generated by their minimum......-weight codewords. This provides a clean analogue of a corresponding result for generalized Reed-Muller codes....

A Novel Vertex Affinity for Community Detection

Energy Technology Data Exchange (ETDEWEB)

Yoo, Andy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sanders, Geoffrey [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Henson, Van [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

2015-10-05

We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases.

Science.gov (United States)

Vermeiren, Céline; Motte, Philippe; Viot, Delphine; Mairet-Coello, Georges; Courade, Jean-Philippe; Citron, Martin; Mercier, Joël; Hannestad, Jonas; Gillard, Michel

2018-02-01

Lilly/Avid's AV-1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimer's disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [ 18 F]AV-1451 uptake in Alzheimer's disease may not be possible. The aim of the present study was to characterize the in vitro binding of AV-1451 to understand and identify potential off-target binding that could explain the poor discrimination observed in PSP patients. [ 3 H]AV-1451 and AV-1451 were characterized in in vitro binding assays using recombinant and native proteins/tissues from postmortem samples of controls and Alzheimer's disease and PSP patients. [ 3 H]AV-1451 binds to multiple sites with nanomolar affinities in brain homogenates and to tau fibrils isolated from Alzheimer's disease or PSP patients. [ 3 H]AV-1451 also binds with similarly high affinities in brain homogenates devoid of tau pathology. This unexpected binding was demonstrated to be because of nanomolar affinities of [ 3 H]AV-1451 for monoamine oxidase A and B enzymes. High affinity of AV-1451 for monoamine oxidase proteins may limit its utility as a tau PET tracer in PSP and Alzheimer's disease because of high levels of monoamine oxidase expression in brain regions also affected by tau deposition, especially if monoamine oxidase levels change over time or with a treatment intervention. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

Automated multi-dimensional purification of tagged proteins.

Science.gov (United States)

Sigrell, Jill A; Eklund, Pär; Galin, Markus; Hedkvist, Lotta; Liljedahl, Pia; Johansson, Christine Markeland; Pless, Thomas; Torstenson, Karin

2003-01-01

The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. AKTA 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His6- or GST-tagged proteins per day and can produce 1-50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.

On affine non-negative matrix factorization

DEFF Research Database (Denmark)

Laurberg, Hans; Hansen, Lars Kai

2007-01-01

We generalize the non-negative matrix factorization (NMF) generative model to incorporate an explicit offset. Multiplicative estimation algorithms are provided for the resulting sparse affine NMF model. We show that the affine model has improved uniqueness properties and leads to more accurate id...

Automated high-throughput protein purification using an ÄKTApurifier and a CETAC autosampler.

Science.gov (United States)

Yoo, Daniel; Provchy, Justin; Park, Cynthia; Schulz, Craig; Walker, Kenneth

2014-05-30

As the pace of drug discovery accelerates there is an increased focus on screening larger numbers of protein therapeutic candidates to identify those that are functionally superior and to assess manufacturability earlier in the process. Although there have been advances toward high throughput (HT) cloning and expression, protein purification is still an area where improvements can be made to conventional techniques. Current methodologies for purification often involve a tradeoff between HT automation or capacity and quality. We present an ÄKTA combined with an autosampler, the ÄKTA-AS, which has the capability of purifying up to 240 samples in two chromatographic dimensions without the need for user intervention. The ÄKTA-AS has been shown to be reliable with sample volumes between 0.5 mL and 100 mL, and the innovative use of a uniquely configured loading valve ensures reliability by efficiently removing air from the system as well as preventing sample cross contamination. Incorporation of a sample pump flush minimizes sample loss and enables recoveries ranging from the low tens of micrograms to milligram quantities of protein. In addition, when used in an affinity capture-buffer exchange format the final samples are formulated in a buffer compatible with most assays without requirement of additional downstream processing. The system is designed to capture samples in 96-well microplate format allowing for seamless integration of downstream HT analytic processes such as microfluidic or HPLC analysis. Most notably, there is minimal operator intervention to operate this system, thereby increasing efficiency, sample consistency and reducing the risk of human error. Copyright © 2014 Elsevier B.V. All rights reserved.

Do native brown trout and non-native brook trout interact reproductively?

Science.gov (United States)

Cucherousset, J.; Aymes, J. C.; Poulet, N.; Santoul, F.; Céréghino, R.

2008-07-01

Reproductive interactions between native and non-native species of fish have received little attention compared to other types of interactions such as predation or competition for food and habitat. We studied the reproductive interactions between non-native brook trout ( Salvelinus fontinalis) and native brown trout ( Salmo trutta) in a Pyrenees Mountain stream (SW France). We found evidence of significant interspecific interactions owing to consistent spatial and temporal overlap in redd localizations and spawning periods. We observed mixed spawning groups composed of the two species, interspecific subordinate males, and presence of natural hybrids (tiger trout). These reproductive interactions could be detrimental to the reproduction success of both species. Our study shows that non-native species might have detrimental effects on native species via subtle hybridization behavior.

Improving image segmentation by learning region affinities

Energy Technology Data Exchange (ETDEWEB)

Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.

2010-11-03

We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

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

Science.gov (United States)

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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Scintillator Purification System for the Borexino Solar Neutrino Detector

OpenAIRE

Benziger, J.; Cadonati, L.; Calaprice, F.; Chen, M.; Corsi, A.; Dalnoki-Veress, F.; Fernholz, R.; Ford, R.; Galbiati, C.; Goretti, A.; Harding, E.; Ianni, Aldo; Ianni, Andrea; Kidner, S.; Leung, M.

2007-01-01

Purification of the 278 tons of liquid scintillator and 889 tons of buffer shielding for the Borexino solar neutrino detector was performed with a system that combined distillation, water extraction, gas stripping and filtration. The purification of the scintillator achieved unprecedented low backgrounds for the large scale liquid scintillation detector. This paper describes the principles of operation, design, construction and commissioning of the purification system, and reviews the require...

Improving the large scale purification of the HIV microbicide, griffithsin.

Science.gov (United States)

Fuqua, Joshua L; Wanga, Valentine; Palmer, Kenneth E

2015-02-22

Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery. We achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2 mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained. The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of

Two-step purification and partial characterization of an extra cellular α-amylase from Bacillus licheniformis

Directory of Open Access Journals (Sweden)

Zare Mirakabadi, A.

2012-11-01

Full Text Available The aim of this study was production and partial purification of α-amylase enzyme by Bacillus licheniformis. B. Licheniformis was allowed to grow in broth culture for purpose of inducing α-amylase enzyme. Optimal conditions for amylase production by B. Licheniformis are incubation period of 120 h, temperature of 37 °C and pH 7.0. The α-amylase enzyme was purified by ion exchange chromatography on DEAE-sepharose CL-6B and sephadex G-100 gel filtration with a 19.1-fold increase in specific activity as compared to the concentrated supernatant and with a specific activity of 926.47 U/mg. The α-amylase had the highest activity at pH 7.0 and 65 °C. According to the data on native polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was 72 kDa.

The Protein Maker: an automated system for high-throughput parallel purification

International Nuclear Information System (INIS)

Smith, Eric R.; Begley, Darren W.; Anderson, Vanessa; Raymond, Amy C.; Haffner, Taryn E.; Robinson, John I.; Edwards, Thomas E.; Duncan, Natalie; Gerdts, Cory J.; Mixon, Mark B.; Nollert, Peter; Staker, Bart L.; Stewart, Lance J.

2011-01-01

The Protein Maker instrument addresses a critical bottleneck in structural genomics by allowing automated purification and buffer testing of multiple protein targets in parallel with a single instrument. Here, the use of this instrument to (i) purify multiple influenza-virus proteins in parallel for crystallization trials and (ii) identify optimal lysis-buffer conditions prior to large-scale protein purification is described. The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications

Selection of imprinted nanoparticles by affinity chromatography.

Science.gov (United States)

Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

2009-04-15

Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

The ultrastructural morphology of native hepatitis B virus.

Science.gov (United States)

Kaito, Masahiko; Ohba, Hiroyoshi; Chiba, Joe; Kohara, Michinori; Tanaka, Hideaki; Fujita, Naoki; Gabazza, Esteban Cesar; Watanabe, Shozo; Konishi, Masayoshi; Adachi, Yukihiko

2006-09-01

Cell lines (2.2.15 cells) capable of supporting the replication of hepatitis B virus (HBV) DNA and intact viral particles have been established by HBV DNA transfection into HepG2 cells. The purpose of this study was to determine the ultrastructural morphology of native HBV particles without purification in the culture supernatants and in sera from patients. Electron microscopy (EM) and immunogold EM of the samples were carried out using polyclonal and monoclonal anti-hepatitis B surface antigen antibodies. HBV particles in the purified samples from the culture supernatants by density-gradient centrifugation were examined to compare the morphology with that of unpurified samples. EM and immunogold EM studies demonstrated the presence of Dane particles (41.8 nm in diameter), cobra-shaped (head diameter, 42.4 nm), and horn-shaped (head diameter, 43.5 nm) particles in the culture supernatants and in the sera from two patients. The tail of the cobra-like particles had a diameter of 21.0 nm and a length of 214 nm. The hornlike particles had a long branch 20.1 nm in diameter with a length of 189 nm, and a short branch 21.4 nm in diameter with a length of 112 nm. The ratio of Dane particles and cobra- and horn-shaped particles in the supernatants was 5 : 4 : 1. After ultracentrifugation, the cobra- and horn-shaped particles completely disappeared; there were only Dane particles together with spheres of 22 nm and filaments. In conclusion, this study showed for the first time that the native replicative form of HBV is cobra- and horn-shaped.

Contractions of affine spherical varieties

International Nuclear Information System (INIS)

Arzhantsev, I V

1999-01-01

The language of filtrations and contractions is used to describe the class of G-varieties obtainable as the total spaces of the construction of contraction applied to affine spherical varieties, which is well-known in invariant theory. These varieties are local models for arbitrary affine G-varieties of complexity 1 with a one-dimensional categorical quotient. As examples, reductive algebraic semigroups and three-dimensional SL 2 -varieties are considered

Simplified riboprobe purification using translucent straws as gel tubes.

Science.gov (United States)

Kol, S; Ben-Shlomo, I; Adashi, E Y; Rohan, R M

1996-01-01

Gel purification of radioactive riboprobes enhances the quality of the ribonuclease protection assay. A simple and effective method for riboprobe purification is described. The method uses acrylamide gels in plastic tubes to achieve electrophoretic separation of the RNA polymerase products.

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

Directory of Open Access Journals (Sweden)

Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Identification of Novel Surface-Exposed Proteins of Rickettsia rickettsii by Affinity Purification and Proteomics

Science.gov (United States)

Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

2014-01-01

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens. PMID:24950252

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

Directory of Open Access Journals (Sweden)

Wenping Gong

Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - Gi complex

International Nuclear Information System (INIS)

Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S.

1991-01-01

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K d of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the α and β subunits of G i , respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects

Purification and Characterization of Lipase from Aspergillus flavus ...

African Journals Online (AJOL)

USER

Abstract. Lipase from Aspergillus flavus was purified in a single step purification using MnFeO4 magnetic nano particles to achieve a 20.53- fold purification with specific activity of. 11.29 U/mg and a 59% recovery yield. SDS-PAGE of lipase showed a single pure band with corresponding molecular weight of 35 kDa.

Purification and Characterization of Lipase from Aspergillus flavus ...

African Journals Online (AJOL)

Lipase from Aspergillus flavus was purified in a single step purification using MnFeO4 magnetic nano particles to achieve a 20.53- fold purification with specific activity of 11.29 U/mg and a 59% recovery yield. SDS-PAGE of lipase showed a single pure band with corresponding molecular weight of 35 kDa. The optimal ...

Virus purification by CsCl density gradient using general centrifugation.

Science.gov (United States)

Nasukawa, Tadahiro; Uchiyama, Jumpei; Taharaguchi, Satoshi; Ota, Sumire; Ujihara, Takako; Matsuzaki, Shigenobu; Murakami, Hironobu; Mizukami, Keijirou; Sakaguchi, Masahiro

2017-11-01

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Laboratory of minerals purification

International Nuclear Information System (INIS)

2002-01-01

The laboratory of minerals purification was organized in 1962 where with application of modern physical and chemical methods were investigated the mechanism of flotation reagents interaction with minerals' surface, was elaborated technologies on rising complexity of using of republic's minerals

Waste water biological purification plants of dairy products industry and energy management

Science.gov (United States)

Stepanov, Sergey; Solkina, Olga; Stepanov, Alexander; Zhukova, Maria

2017-10-01

The paper presents results of engineering and economical comparison of waste water biological purification plants of dairy products industry. Three methods of purification are compared: traditional biological purification with the use of secondary clarifiers and afterpurification through granular-bed filters, biomembrane technology and physical-and-chemical treatment together with biomembrane technology for new construction conditions. The improvement of the biological purification technology using nitro-denitrification and membrane un-mixing of sludge mixture is a promising trend in this area. In these calculations, an energy management which is widely applied abroad was used. The descriptions of the three methods are illustrated with structural schemes. Costs of equipment and production areas are taken from manufacturers’ data. The research is aimed at an engineering and economical comparison of new constructions of waste water purification of dairy products industry. The experiment demonstrates advantages of biomembrane technology in waste water purification. This technology offers prospects of 122 million rubles cost saving during 25 years of operation when compared with of the technology of preparatory reagent flotation and of 13.7 million rubles cost saving compared to the option of traditional biological purification.

Periphyton density is similar on native and non-native plant species

NARCIS (Netherlands)

Grutters, B.M.C.; Gross, Elisabeth M.; van Donk, E.; Bakker, E.S.

2017-01-01

Non-native plants increasingly dominate the vegetation in aquatic ecosystems and thrive in eutrophic conditions. In eutrophic conditions, submerged plants risk being overgrown by epiphytic algae; however, if non-native plants are less susceptible to periphyton than natives, this would contribute to

Purification of human platelet-derived growth factor

International Nuclear Information System (INIS)

Raines, E.W.; Ross, R.

1985-01-01

The paper describes a method for purification of human platelet-derived growth factor (PDGF) from outdated platelet-rich plasma (PRP) using commonly available laboratory reagents and yielding a mitogen purified 800,000-fold over the starting material. [ 3 H]thymidine incorporation into DNA of cultured cells responsive to PDGF represents the most readily available method to follow its purification and define the biological activity of a purified preparation. Other assays to quantitate PDGF include radioreceptor assay and radioimmunoassay

Native and Non-native English Teachers' Perceptions of their Professional Identity: Convergent or Divergent?

Directory of Open Access Journals (Sweden)

Zia Tajeddin

2016-10-01

Full Text Available There is still a preference for native speaker teachers in the language teaching profession, which is supposed to influence the self-perceptions of native and nonnative teachers. However, the status of English as a globalized language is changing the legitimacy of native/nonnative teacher dichotomy. This study sought to investigate native and nonnative English-speaking teachers’ perceptions about native and nonnative teachers’ status and the advantages and disadvantages of being a native or nonnative teacher. Data were collected by means of a questionnaire and a semi-structured interview. A total of 200 native and nonnative teachers of English from the UK and the US, i.e. the inner circle, and Turkey and Iran, the expanding circle, participated in this study. A significant majority of nonnative teachers believed that native speaker teachers have better speaking proficiency, better pronunciation, and greater self-confidence. The findings also showed nonnative teachers’ lack of self-confidence and awareness of their role and status compared with native-speaker teachers, which could be the result of existing inequities between native and nonnative English-speaking teachers in ELT. The findings also revealed that native teachers disagreed more strongly with the concept of native teachers’ superiority over nonnative teachers. Native teachers argued that nonnative teachers have a good understanding of teaching methodology whereas native teachers are more competent in correct language. It can be concluded that teacher education programs in the expanding-circle countries should include materials for teachers to raise their awareness of their own professional status and role and to remove their misconception about native speaker fallacy.

Intensification of oily waste waters purification by means of liquid atomization

Science.gov (United States)

Eskin, A. A.; Tkach, N. S.; Kim, M. I.; Zakharov, G. A.

2017-10-01

In this research, a possibility of using liquid atomization for improving the efficiency of purification of wastewater by different methods has been studied. By the introduced method and an experimental setup for wastewater purification, saturation rate increases with its purification by means of dissolved air flotation. Liquid atomization under excess pressure allows to gain a large interfacial area between the saturated liquid and air, which may increase the rate of purified liquid saturation almost twice, compared to the existing methods of saturation. Current disadvantages of liquid atomization used for intensification of wastewater purification include high energy cost and secondary emulsion of polluting agents. It is also known that by means of liquid atomization a process of ozonizing can be intensified. Large contact surface between the purified liquid and ozone-air mixture increases the oxidizing efficiency, which allows to diminish ozone discharge. Liquid atomization may be used for purification of wastewaters by ultraviolet radiation. Small drops of liquid will be proportionally treated by ultraviolet, which makes it possible to do purification even of turbid wastewaters. High-speed liquid motion will prevent the pollution of quartz tubes of ultraviolet lamps.

Recent Advances in Nanoporous Membranes for Water Purification

Directory of Open Access Journals (Sweden)

Zhuqing Wang

2018-01-01

Full Text Available Nanoporous materials exhibit wide applications in the fields of electrocatalysis, nanodevice fabrication, energy, and environmental science, as well as analytical science. In this review, we present a summary of recent studies on nanoporous membranes for water purification application. The types and fabrication strategies of various nanoporous membranes are first introduced, and then the fabricated nanoporous membranes for removing various water pollutants, such as salt, metallic ions, anions, nanoparticles, organic chemicals, and biological substrates, are demonstrated and discussed. This work will be valuable for readers to understand the design and fabrication of various nanoporous membranes, and their potential purification mechanisms towards different water pollutants. In addition, it will be helpful for developing new nanoporous materials for quick, economic, and high-performance water purification.

Global affine differential geometry of hypersurfaces

CERN Document Server

Li, An-Min; Zhao, Guosong; Hu, Zejun

2015-01-01

This book draws a colorful and widespread picture of global affine hypersurface theory up to the most recent state. Moreover, the recent development revealed that affine differential geometry- as differential geometry in general- has an exciting intersection area with other fields of interest, like partial differential equations, global analysis, convex geometry and Riemann surfaces.

Purification of radiolabeled RNA using sephadex G-15 or G-50 chromatography

International Nuclear Information System (INIS)

Yoo, Beong Gyu; Lee, Jong Seok

1998-01-01

We attempted to purify radiolabeled RNA using Sephadex G-15 and G-50 chromatography instead of commercial RNA purification kit. In the Sephadex G-15 chromatography the major portion of RNA was eluted in the fractions ranging from 3rd to 5th whereas broad elution profile of RNA was obtained from the Sephadex G-50 chromatography. The elution profile and purity of RNA obtained from Sephadex G-15 chromatography was very similar to that by commercial RNA purification kit. Furthermore, operating time required for purification of RNA by Sephadex G-15 was rather smaller than that by commercial kit. Overall results suggest that the purification of radiolabeled RNA using Sephadex G-15 is more money and time saving than using commercial RNA purification kit

Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135.

Science.gov (United States)

De Luca, Maria Antonietta; Castelli, M Paola; Loi, Barbara; Porcu, Alessandra; Martorelli, Mariella; Miliano, Cristina; Kellett, Kathryn; Davidson, Colin; Stair, Jacqueline L; Schifano, Fabrizio; Di Chiara, Gaetano

2016-06-01

In order to investigate the in vivo dopamine (DA) stimulant properties of selected 3rd generation Spice/K2 cannabinoids, BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, their in vitro affinity and agonist potency at native rat and mice CB1 receptors was studied. The compounds bind with high affinity to CB1 receptors in rat cerebral cortex homogenates and stimulate CB1-induced [(35)S]GTPγS binding with high potency and efficacy. BB-22 and 5F-PB-22 showed the lowest Ki of binding to CB1 receptors (0.11 and 0.13 nM), i.e., 30 and 26 times lower respectively than that of JWH-018 (3.38 nM), and a potency (EC50, 2.9 and 3.7 nM, respectively) and efficacy (Emax, 217% and 203%, respectively) as CB1 agonists higher than JWH-018 (EC50, 20.2 nM; Emax, 163%). 5F-AKB-48 and STS-135 had higher Ki for CB1 binding, higher EC50 and lower Emax as CB1 agonists than BB-22 and 5F-PB-22 but still comparatively more favourable than JWH-018. The agonist properties of all the compounds were abolished or drastically reduced by the CB1 antagonist/inverse agonist AM251 (0.1 μM). No activation of G-protein was observed in CB1-KO mice. BB-22 (0.003-0.01 mg/kg i.v.) increased dialysate DA in the accumbens shell but not in the core or in the medial prefrontal cortex, with a bell shaped dose-response curve and an effect at 0.01 mg/kg and a biphasic time-course. Systemic AM251 (1.0 mg/kg i.p.) completely prevented the stimulant effect of BB-22 on dialysate DA in the NAc shell. All the other compounds increased dialysate DA in the NAc shell at doses consistent with their in vitro affinity for CB1 receptors (5F-PB-22, 0.01 mg/kg; 5F-AKB-48, 0.1 mg/kg; STS-135, 0.15 mg/kg i.v.). 3rd generation cannabinoids can be even more potent and super-high CB1 receptor agonists compared to JWH-018. Future research will try to establish if these properties can explain the high toxicity and lethality associated with these compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reanalysis and semantic persistence in native and non-native garden-path recovery.

Science.gov (United States)

Jacob, Gunnar; Felser, Claudia

2016-01-01

We report the results from an eye-movement monitoring study investigating how native and non-native speakers of English process temporarily ambiguous sentences such as While the gentleman was eating the burgers were still being reheated in the microwave, in which an initially plausible direct-object analysis is first ruled out by a syntactic disambiguation (were) and also later on by semantic information (being reheated). Both participant groups showed garden-path effects at the syntactic disambiguation, with native speakers showing significantly stronger effects of ambiguity than non-native speakers in later eye-movement measures but equally strong effects in first-pass reading times. Ambiguity effects at the semantic disambiguation and in participants' end-of-trial responses revealed that for both participant groups, the incorrect direct-object analysis was frequently maintained beyond the syntactic disambiguation. The non-native group showed weaker reanalysis effects at the syntactic disambiguation and was more likely to misinterpret the experimental sentences than the native group. Our results suggest that native language (L1) and non-native language (L2) parsing are similar with regard to sensitivity to syntactic and semantic error signals, but different with regard to processes of reanalysis.

Purification and characterization of three laccase isozymes from the ...

African Journals Online (AJOL)

2012-04-17

Apr 17, 2012 ... improve wine quality by removing fermentation inhibitors so as to increase yield of ethanol (Baldrian, 2006). They have also been used .... Summary of purification of laccase isozymes from Trametes sp. HS-03a. Purification .... and kinetics of a thermostable laccase from Pycnoporus sanguineus. (SCC 108).

Comparative study of peroxidase purification from apple and orange ...

African Journals Online (AJOL)

This paper reports the isolation and purification of peroxidase from low cost material; moreover, no significant work has been done on the isolation and purification of peroxidase from such cost effective sources (apple and orange seeds). Peroxidases had attracted considerable interest in recent years because of their ...

Development of Purification Protocol Specific for Bacteriocin 105B

Science.gov (United States)

2017-02-09

Bacillus anthracis. As the current application of broad-spectrum antimicrobials promotes the development of multi- drug resistant microorganisms...SPECTRUM TARGETED ANTIMICROBIALS ASSAYS PURIFICATION BACILLUS ANTHRACIS DRUG- RESISTANT MICROORGANISMS...through the purification procedure. The wide-spread use of broad-spectrum antimicrobial agents has led to the development of drug resistant

The elution of certain protein affinity tags with millimolar concentrations of diclofenac.

Science.gov (United States)

Baliova, Martina; Juhasova, Anna; Jursky, Frantisek

2015-12-01

Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents. Copyright © 2015 Elsevier B.V. All rights reserved.

Community-level plant-soil feedbacks explain landscape distribution of native and non-native plants.

Science.gov (United States)

Kulmatiski, Andrew

2018-02-01

Plant-soil feedbacks (PSFs) have gained attention for their potential role in explaining plant growth and invasion. While promising, most PSF research has measured plant monoculture growth on different soils in short-term, greenhouse experiments. Here, five soil types were conditioned by growing one native species, three non-native species, or a mixed plant community in different plots in a common-garden experiment. After 4 years, plants were removed and one native and one non-native plant community were planted into replicate plots of each soil type. After three additional years, the percentage cover of each of the three target species in each community was measured. These data were used to parameterize a plant community growth model. Model predictions were compared to native and non-native abundance on the landscape. Native community cover was lowest on soil conditioned by the dominant non-native, Centaurea diffusa , and non-native community cover was lowest on soil cultivated by the dominant native, Pseudoroegneria spicata . Consistent with plant growth on the landscape, the plant growth model predicted that the positive PSFs observed in the common-garden experiment would result in two distinct communities on the landscape: a native plant community on native soils and a non-native plant community on non-native soils. In contrast, when PSF effects were removed, the model predicted that non-native plants would dominate all soils, which was not consistent with plant growth on the landscape. Results provide an example where PSF effects were large enough to change the rank-order abundance of native and non-native plant communities and to explain plant distributions on the landscape. The positive PSFs that contributed to this effect reflected the ability of the two dominant plant species to suppress each other's growth. Results suggest that plant dominance, at least in this system, reflects the ability of a species to suppress the growth of dominant competitors

Ionic behavior of treated water at a water purification plant

OpenAIRE

Yanagida, Kazumi; Kawahigashi, Tatsuo

2012-01-01

[Abstract] Water at each processing stage in a water purification plant was extracted and analyzed to investigate changes of water quality. Investigations of water at each processing stage at the water purification plant are discussed herein.

New unitary affine-Virasoro constructions

International Nuclear Information System (INIS)

Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M.; Yamron, J.P.

1990-01-01

This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g

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

Science.gov (United States)

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

2012-01-01

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

Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

Energy Technology Data Exchange (ETDEWEB)

Sun, Junfen, E-mail: junfensun@dhu.edu.cn [State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, North People Road 2999, Shanghai 201620 (China); Wu, Lishun [Department of Chemistry and Chemical Engineering, Heze University, Daxue Road 2269, Heze, Shandong Province 274015 (China)

2014-07-01

This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

Native Speakers' Perception of Non-Native English Speech

Science.gov (United States)

Jaber, Maysa; Hussein, Riyad F.

2011-01-01

This study is aimed at investigating the rating and intelligibility of different non-native varieties of English, namely French English, Japanese English and Jordanian English by native English speakers and their attitudes towards these foreign accents. To achieve the goals of this study, the researchers used a web-based questionnaire which…

Response surface methodology optimization of partitioning of xylanase form Aspergillus Niger by metal affinity polymer-salt aqueous two-phase systems.

Science.gov (United States)

Fakhari, Mohamad Ali; Rahimpour, Farshad; Taran, Mojtaba

2017-09-15

Aqueous two phase affinity partitioning system using metal ligands was applied for partitioning and purification of xylanase produced by Aspergillus Niger. To minimization the number of experiments for the design parameters and develop predictive models for optimization of the purification process, response surface methodology (RSM) with a face-centered central composite design (CCF) has been used. Polyethylene glycol (PEG) 6000 was activated using epichlorohydrin, covalently linked to iminodiacetic acid (IDA), and the specific metal ligand Cu was attached to the polyethylene glycol-iminodiacetic acid (PEG-IDA). The influence of some experimental variables such as PEG (10-18%w/w), sodium sulfate (8-12%), PEG-IDA-Cu 2+ concentration (0-50% w/w of total PEG), pH of system (4-8) and crude enzyme loading (6-18%w/w) on xylanase and total protein partitioning coefficient, enzyme yield and enzyme specific activity were systematically evaluated. Two optimal point with high enzyme partitioning factor 10.97 and yield 79.95 (including 10% PEG, 12% Na 2 SO 4 , 50% ligand, pH 8 and 6% crude enzyme loading) and high specific activity in top phase 42.21 (including 14.73% PEG, 8.02% Na 2 SO 4 , 28.43% ligand, pH 7.7 and 6.08% crude enzyme loading) were attained. The adequacy of the RSM models was verified by a good agreement between experimental and predicted results. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Gupta, Vibha; Gupta, Rakesh K.; Khare, Garima; Salunke, Dinakar M.; Tyagi, Anil K.

2008-01-01

The cloning, purification and crystallization of a bacterioferritin from M. tuberculosis together with preliminary X-ray characterization of its crystals are reported. Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein product was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 Å resolution and belonged to space group P4 2 , with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 Å

Purification and fluorescent labeling of the human serotonin transporter

DEFF Research Database (Denmark)

Rasmussen, Søren G F; Gether, Ulrik

2005-01-01

To establish a purification procedure for the human serotonin transporter (hSERT) we expressed in Sf9 insect cells an epitope-tagged version of the transporter containing a FLAG epitope at the N-terminus and a polyhistidine tail at the C-terminus (FLAG-hSERT-12H). For purification, the transporter...

An intergeneric hybrid of a native minnow, the golden shiner, and an exotic minnow, the rudd

Science.gov (United States)

Burkhead, N.M.; Williams, J.D.

1991-01-01

The hybrid golden shiner Notemigonus crysoleucas × rudd Scardinius erythrophthalmus is the first known nonsalmonid, intergeneric hybrid of an exotic species and a North American native species. The cross is also the first valid record of a viable hybrid involving the native golden shiner. Meristic and mensural characters of 30 artificially produced hybrids of male golden shiners and female rudds were analyzed. Forty-seven percent of the meristic traits exhibited character states intermediate between those of parents. Twenty-seven percent of the meristic characters were supernumerary, suggesting developmental instability of the hybrid genome. Mensural hybrid characters were significantly skewed to the golden shiner phenotype. The skewed mensural inheritance and other skewed patterns of morphological inheritance also suggest problems in canalization of the hybrid phenome or atypical patterns of dominance. All hybrids were identifiable by intermediate squamation of the cultrate abdomen: the keel was mostly scaled but exhibited a small fleshy ridge posteriorly. This minnow hybrid allows general inferences to be made about the phylogenetic affinity of the golden shiner to other cultrate cyprinids of Eurasia. The hybrid cross has important management and conservation implications for fishes in North America. The hybrid is an example of how an exotic species may negatively affect a native species.

DapE can function as an aspartyl peptidase in the presence of Mn2+.

Science.gov (United States)

Broder, Daniel H; Miller, Charles G

2003-08-01

Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn(2+). Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn(2+)-dependent peptidase is DapE (N-succinyl-L,L-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His(6)). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn(2+)-dependent aspartyl peptidase activity relative to the MPD dapE(+) strain. In addition, purified DapE-His(6) exhibited Mn(2+)-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn(2+) in the high-affinity site and Mn(2+) in the low-affinity site.

Dense Medium Plasma Water Purification Reactor (DMP WaPR), Phase I

Data.gov (United States)

National Aeronautics and Space Administration — The Dense Medium Plasma Water Purification Reactor offers significant improvements over existing water purification technologies used in Advanced Life Support...

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

Science.gov (United States)

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

State-of-the-art technocology in blood purification at present

Directory of Open Access Journals (Sweden)

Zhi-hong LIU

2011-02-01

Full Text Available Objective To review the recent advancement in clinical practices and studies on blood purification techniques,and to provide a guide for further studies on its application in military medicine.Methods Literature published in recent five years limited to blood purification field either in English or Chinese were retrieved by searching PubMed and CHKD.Analysis and summary were performed based on the literature.Results The advancements in blood purification in recent five years could be categorized into four fields,i.e.hemodialysis(HD,peritoneal dialysis(PD,continuous renal replacement therapy(CRRT,and adsorption therapy.The development in HD was aimed at promoting the ability of removal of toxic elements producing uremia and online monitor techniques,and PD was aimed at improvement of patients’ general condition and intervention to reduce the risk factors affecting long-term outcomes,and preparation of new PD solutions to improve the efficacy of PD.In regard to CRRT,the current progress had been focused on initiation time,dose and proposal of new hypothesis for high-volume hemofiltration(HVHF application.Adsorption therapy was another choice of blood purification.Domestic military medicine progress in blood purification in our armed forces was focused on techniques that could be used in treatment of casualties in war,including the basic and clinical study of extracorporeal circuit intervention(ECI for treatment of critically ill patients,problems arising from anticoagulation in ECI for patients with trauma,chemical agents poisoning,and adsorption technique.Conclusions Recently,the main advancement of blood purification technique is combined application of series techniques such as dialysis,hemofiltration,adsorption,and plasma exchange in treatment of critically ill patients.Studies on blood purification in domestic military medicine should be updated continuously to follow closely to the latest achievement in world,and translate these latest

Affine stochastic mortality

NARCIS (Netherlands)

Schrager, D.F.

2006-01-01

We propose a new model for stochastic mortality. The model is based on the literature on affine term structure models. It satisfies three important requirements for application in practice: analytical tractibility, clear interpretation of the factors and compatibility with financial option pricing

Small mammal use of native warm-season and non-native cool-season grass forage fields

Science.gov (United States)

Ryan L Klimstra,; Christopher E Moorman,; Converse, Sarah J.; Royle, J. Andrew; Craig A Harper,

2015-01-01

Recent emphasis has been put on establishing native warm-season grasses for forage production because it is thought native warm-season grasses provide higher quality wildlife habitat than do non-native cool-season grasses. However, it is not clear whether native warm-season grass fields provide better resources for small mammals than currently are available in non-native cool-season grass forage production fields. We developed a hierarchical spatially explicit capture-recapture model to compare abundance of hispid cotton rats (Sigmodon hispidus), white-footed mice (Peromyscus leucopus), and house mice (Mus musculus) among 4 hayed non-native cool-season grass fields, 4 hayed native warm-season grass fields, and 4 native warm-season grass-forb ("wildlife") fields managed for wildlife during 2 summer trapping periods in 2009 and 2010 of the western piedmont of North Carolina, USA. Cotton rat abundance estimates were greater in wildlife fields than in native warm-season grass and non-native cool-season grass fields and greater in native warm-season grass fields than in non-native cool-season grass fields. Abundances of white-footed mouse and house mouse populations were lower in wildlife fields than in native warm-season grass and non-native cool-season grass fields, but the abundances were not different between the native warm-season grass and non-native cool-season grass fields. Lack of cover following haying in non-native cool-season grass and native warm-season grass fields likely was the key factor limiting small mammal abundance, especially cotton rats, in forage fields. Retention of vegetation structure in managed forage production systems, either by alternately resting cool-season and warm-season grass forage fields or by leaving unharvested field borders, should provide refugia for small mammals during haying events.

Purification of cerium, neodymium and gadolinium for low background experiments

Directory of Open Access Journals (Sweden)

Boiko R.S.

2014-01-01

Full Text Available Cerium, neodymium and gadolinium contain double beta active isotopes. The most interesting are 150Nd and 160Gd (promising for 0ν2β search, 136Ce (2β+ candidate with one of the highest Q2β. The main problem of compounds containing lanthanide elements is their high radioactive contamination by uranium, radium, actinium and thorium. The new generation 2β experiments require development of methods for a deep purification of lanthanides from the radioactive elements. A combination of physical and chemical methods was applied to purify cerium, neodymium and gadolinium. Liquid-liquid extraction technique was used to remove traces of Th and U from neodymium, gadolinium and for purification of cerium from Th, U, Ra and K. Co-precipitation and recrystallization methods were utilized for further reduction of the impurities. The radioactive contamination of the samples before and after the purification was tested by using ultra-low-background HPGe gamma spectrometry. As a result of the purification procedure the radioactive contamination of gadolinium oxide (a similar purification efficiency was reached also with cerium and neodymium oxides was decreased from 0.12 Bq/kg to 0.007 Bq/kg in 228Th, from 0.04 Bq/kg to <0.006 Bq/kg in 226Ra, and from 0.9 Bq/kg to 0.04 Bq/kg in 40K. The purification methods are much less efficient for chemically very similar radioactive elements like actinium, lanthanum and lutetium.

Rank Two Affine Manifolds in Genus 3

OpenAIRE

Aulicino, David; Nguyen, Duc-Manh

2016-01-01

We complete the classification of rank two affine manifolds in the moduli space of translation surfaces in genus three. Combined with a recent result of Mirzakhani and Wright, this completes the classification of higher rank affine manifolds in genus three.

The Native Comic Book Project: native youth making comics and healthy decisions.

Science.gov (United States)

Montgomery, Michelle; Manuelito, Brenda; Nass, Carrie; Chock, Tami; Buchwald, Dedra

2012-04-01

American Indians and Alaska Natives have traditionally used stories and drawings to positively influence the well-being of their communities. The objective of this study was to describe the development of a curriculum that trains Native youth leaders to plan, write, and design original comic books to enhance healthy decision making. Project staff developed the Native Comic Book Project by adapting Dr. Michael Bitz's Comic Book Project to incorporate Native comic book art, Native storytelling, and decision-making skills. After conducting five train-the-trainer sessions for Native youth, staff were invited by youth participants to implement the full curriculum as a pilot test at one tribal community site in the Pacific Northwest. Implementation was accompanied by surveys and weekly participant observations and was followed by an interactive meeting to assess youth engagement, determine project acceptability, and solicit suggestions for curriculum changes. Six youths aged 12 to 15 (average age = 14) participated in the Native Comic Book Project. Youth participants stated that they liked the project and gained knowledge of the harmful effects of commercial tobacco use but wanted better integration of comic book creation, decision making, and Native storytelling themes. Previous health-related comic book projects did not recruit youth as active producers of content. This curriculum shows promise as a culturally appropriate intervention to help Native youth adopt healthy decision-making skills and healthy behaviors by creating their own comic books.

Native American nurse leadership.

Science.gov (United States)

Nichols, Lee A

2004-07-01

To identify which characteristics, wisdom, and skills are essential in becoming an effective Native American nurse leader. This will lead to the development of a curriculum suitable for Native American nurses. A qualitative, descriptive design was used for this study. Focus groups were conducted in Polson, Montana. A total of 67 Native and non-Native nurses participated. Sixty-seven percent of them were members of Indian tribes. Data were content analyzed using Spradley's ethnographic methodology. Three domains of analysis emerged: point of reference for the leader (individual, family, community), what a leader is (self-actualized, wise, experienced, political, bicultural, recognized, quiet presence, humble, spiritual, and visionary), and what a leader does (mentors, role models, communicates, listens, demonstrates values, mobilizes, and inspires). Native nurse leaders lead differently. Thus, a leadership curriculum suitable for Native nurses may lead to increased work productivity and therefore improved patient care for Native Americans.

Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

Science.gov (United States)

Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

2016-12-15

Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings. Copyright © 2016 Elsevier B.V. All rights reserved.

Cadmium purification with a vibrating reactor

International Nuclear Information System (INIS)

Torres, N.; Esna-Ashari, M.; Biallas, H.; Kangas, K.

1986-01-01

While electrolytically producing zinc from sulfide concentrates, purification is the most significant step. Impurities such as Co, Sn, Ge, Ni and Sb can cause extensive redissolution of the electrodeposited zinc, thus diminishing current efficiency. Other metals, particularly cadmium, lead and copper, can negatively affect zinc properties by deposition on the cathode. It is standard practice to use atomized zinc dust as a reducing agent in the purification process, either alone or combined with additives. In conventional operations, special facilities are necessary to produce zinc dust in an amount close to 8wt% of cathode production. This paper examines a technique which makes use of zinc granules instead of dust

Specialists' meeting on fast reactor cover gas purification

International Nuclear Information System (INIS)

1987-01-01

The tentative agenda was adopted by the participants without comment and was followed throughout the meeting. The following topics were discussed at the subsequent sessions of the meeting on 'Fast Reactor Cover Gas Purification': National Position Papers; Impurities: Sources and Measurement; Cover Gas Purification Techniques; Sodium Aerosol Trapping; Radiological Considerations. Based on the papers presented and the discussions following, session summaries and conclusions were prepared and are included in this report

Specialists' meeting on fast reactor cover gas purification

Energy Technology Data Exchange (ETDEWEB)

NONE

1987-07-01

The tentative agenda was adopted by the participants without comment and was followed throughout the meeting. The following topics were discussed at the subsequent sessions of the meeting on 'Fast Reactor Cover Gas Purification': National Position Papers; Impurities: Sources and Measurement; Cover Gas Purification Techniques; Sodium Aerosol Trapping; Radiological Considerations. Based on the papers presented and the discussions following, session summaries and conclusions were prepared and are included in this report.

A new helium gas recovery and purification system

International Nuclear Information System (INIS)

Yamamotot, T.; Suzuki, H.; Ishii, J.; Hamana, I.; Hayashi, S.; Mizutani, S.; Sanjo, S.

1974-01-01

A helium gas recovery and purification system, based on the principle of gas permeation through a membrane, is described. The system can be used for the purification of helium gas containing air as a contaminant. The apparatus, operating at ambient temperature does not need constant attention, the recovery ratio of helium gas is satisfactory and running costs are low. Gases other than helium can be processed with the apparatus. (U.K.)

Dynamics of Open Systems with Affine Maps

International Nuclear Information System (INIS)

Zhang Da-Jian; Liu Chong-Long; Tong Dian-Min

2015-01-01

Many quantum systems of interest are initially correlated with their environments and the reduced dynamics of open systems are an interesting while challenging topic. Affine maps, as an extension of completely positive maps, are a useful tool to describe the reduced dynamics of open systems with initial correlations. However, it is unclear what kind of initial state shares an affine map. In this study, we give a sufficient condition of initial states, in which the reduced dynamics can always be described by an affine map. Our result shows that if the initial states of the combined system constitute a convex set, and if the correspondence between the initial states of the open system and those of the combined system, defined by taking the partial trace, is a bijection, then the reduced dynamics of the open system can be described by an affine map. (paper)

Recovery and purification of ethylene

Science.gov (United States)

Reyneke, Rian [Katy, TX; Foral, Michael J [Aurora, IL; Lee, Guang-Chung [Houston, TX; Eng, Wayne W. Y. [League City, TX; Sinclair, Iain [Warrington, GB; Lodgson, Jeffery S [Naperville, IL

2008-10-21

A process for the recovery and purification of ethylene and optionally propylene from a stream containing lighter and heavier components that employs an ethylene distributor column and a partially thermally coupled distributed distillation system.

USING MICROSCALE THERMOPHORESIS TO EASILY MEASURE BINDING AFFINITY

Directory of Open Access Journals (Sweden)

Dennis Breitsprecher*

2018-03-01

Full Text Available While it’s very common for biologists and chemists to test whether or not two molecules interact with each other, it’s much more useful to gather information on the nature of that interaction. How strong is it? How long will it last? What does that mean for its biological function? One way to answer these questions is to study affinity. Binding affinity is defined as the strength of the binding interaction between a single biomolecule to its binding partner, or ligand, and it can be quantifiably measured, providing information on whether or not molecules are interacting, as well as assigning a value to the affinity. When measuring binding affinity, there are several parameters to look at, but the dissociation constant (Kd, which defines the likelihood that an interaction between two molecules will break, is a very common measurement. The smaller the dissociation constant, the more tightly bound the ligand is, and the higher the affinity is between the two molecules.

Purification and crystallization of a trimodular complex comprising the type II cohesin–dockerin interaction from the cellulosome of Clostridium thermocellum

International Nuclear Information System (INIS)

Adams, Jarrett J.; Pal, Gour; Yam, Katherine; Spencer, Holly L.; Jia, Zongchao; Smith, Steven P.

2004-01-01

A trimodular complex comprising the type II cohesin–dockerin interaction from the cellulosome of C. thermocellum has been purified and crystallized by the hanging-drop vapour-diffusion method. A native crystal and a selenomethionine derivative have been analyzed using X-ray diffraction. The high-affinity calcium-mediated type II cohesin–dockerin interaction is responsible for the attachment of the multi-enzyme cellulose-degrading complex, termed the cellulosome, to the cell surface of the thermophilic anaerobe Clostridium thermocellum. A trimodular 40 kDa complex comprising the SdbA type II cohesin and the the CipA type II dockerin–X module modular pair from the cellulosome of C. thermocellum has been crystallized. The crystals belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 45.21, b = 52.34, c = 154.69 Å. The asymmetric unit contains one molecule of the protein complex and native and selenomethionine-derivative crystals diffracted to 2.1 and 2.0 Å, respectively

Native listeners

NARCIS (Netherlands)

Cutler, A.

2002-01-01

Becoming a native listener is the necessary precursor to becoming a native speaker. Babies in the first year of life undertake a remarkable amount of work; by the time they begin to speak, they have perceptually mastered the phonological repertoire and phoneme co-occurrence probabilities of the

The Structure of Affine Buildings

CERN Document Server

Weiss, Richard M

2009-01-01

In The Structure of Affine Buildings, Richard Weiss gives a detailed presentation of the complete proof of the classification of Bruhat-Tits buildings first completed by Jacques Tits in 1986. The book includes numerous results about automorphisms, completions, and residues of these buildings. It also includes tables correlating the results in the locally finite case with the results of Tits's classification of absolutely simple algebraic groups defined over a local field. A companion to Weiss's The Structure of Spherical Buildings, The Structure of Affine Buildings is organized around the clas

The dynamics of metric-affine gravity

International Nuclear Information System (INIS)

Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

2011-01-01

Highlights: → The role and the dynamics of the connection in metric-affine theories is explored. → The most general second order action does not lead to a dynamical connection. → Including higher order invariants excites new degrees of freedom in the connection. → f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy

Biochemical and pharmacological studies of native and irradiated crotamine with gamma radiation of Co60

International Nuclear Information System (INIS)

Mitake, M.B.

2000-01-01

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide from South American rattlesnake venom, composed of 42 amino acid residues. It induces skeletal muscle spasms, leading to a spastic paralysis of hind limbs in mice. The objective was to carry out biochemical and pharmacological studies of native and irradiated crotamine with Co. Crotamine was purified from Crotalus durissus terrificus venom by Sephadex G-100 gel filtration followed by ion exchange chromatography, using a Fast performance Liquid Chromatography (FPLC) system. It was irradiated at 2 mg/ml in 0.15 m NaCl with 2.0 kGy gamma radiation emitted by a Co source. Native and irradiated crotamine were evaluated by biochemical characterization, toxic activity (LD50), and biodistribution. The native and irradiated crotamine were labeled with 29.6 MBq of I using chloramine T method and separated in a Sephadex G-50 column. Male Swiss mice (35 @ 5 g) were injected IP with 0.1 mL (2.4x10 cpm/mouse) of I native crotamine or with 0.4 mL (1.3 x 10 cpm/mouse) of I irradiated crotamine. The animals were sacrificed by ether inhalation at 0.08, 0.25, 0.5,1, 2, 3, 4, 8, 12, and 24 hours. Blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine radioactivity content. The results showed that gamma radiation did not change protein concentration, electrophoretic profile, or protein primary structure, although differences could be seen by spectroscopic techniques. Gamma radiation reduced crotamine toxicity, but did not eliminate bioactivity. Biodistribution studies showed that native and irradiated crotamine have hepatic metabolism and renal elimination. Native and irradiated crotamine have an affinity to skeletal muscle and did not cross the blood-brain barrier. (author)

Biochemical and pharmacological studies of native and irradiated crotamine with gamma radiation of 60Co

International Nuclear Information System (INIS)

Mitake, Malvina Boni

2000-01-01

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide from the South American rattlesnake venom, composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of this thesis was carry out biochemical and pharmacological studies of native and irradiated crotamine with 60 Co. Crotamine was purified from Crotalus durissus terrificus venom by Sephadex G-100 gel filtration followed by ion exchange chromatography, using a Fast Performance Liquid Chromatography (FPLC) system. It was irradiated at 2 mg/ml in 0.15 M Na Cl with 2.0 kGy gamma radiation emitted by a 60 Co source. The native and irradiated crotamine were evaluated by biochemical characterization, toxic activity (LD 50 and biodistribution. The native and irradiated crotamine were labelled with 29.6 MBq of 125 I using chloramine T method, and separated in a Sephadex G-50 column. Male Swiss mice (35± 5 g), were injected i.p. with o.1 mL (2.4 x 10 6 cpm/mouse) of 125 I native crotamine or with 0.4 mL (1.3 x 10 6 cpm/mouse) of 125 I irradiated crotamine. At 0.08; 0.25; 0.5; 1; 2; 4; 8; 12 and 24 hours the animal were killed by ether inhalation. Blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine radioactivity content. The results showed that gamma radiation did not change the protein concentration, the electroforetic profile or the primary structure of the protein, although differences were shown by spectroscopic techniques. The gamma radiation diminished the toxicity of crotamine, but it did not abolish bioactivity. Biodistribution studies showed that native and irradiated crotamine have hepatic metabolism and renal elimination. The native and irradiated crotamine have affinity by skeletal muscle and they did not pass the blood - brain

New concept of gas purification by electron attachment

International Nuclear Information System (INIS)

Tamon, Hajime; Mizota, Hirotoshi; Sano, Noriaki; Schulze, S.; Okazaki, Morio

1995-01-01

Recently, the public has become interested in the following types of gas purification: (1) removal of indoor air pollutants; (2) complete removal of dioxin from incineration plants; (3) complete removal of radioactive iodine compounds; (4) simultaneous removal of NOx and SOx in exhaust gases from cogeneration plants; (5) removal and decomposition of halocarbons; (6) ultrahigh purification of gas sued for semiconductor industries. A new concept of gas purification by electron attachment is proposed. Low-energy electrons generated in a corona-discharge reactor are captured by electronegative impurities, producing negative ions. The ions drift to the anode in the electric field and are removed at the anode of the reactor. Two types of reactors were used to remove the negative ions: a deposition-type reactor, which deposits negative ions at the anode surface; a sweep-out-type reactor, which sweeps out enriched electronegative impurities through the porous anode. Removals of dilute sulfur compounds, oxygen and iodine from nitrogen were conducted to verify the concept of gas purification. Simulation models were used to estimate removal efficiencies of these compounds, by taking into account electron attachment, and experimental constants of the models were determined. The removal efficiency correlated by the models agreed well with the experimental one

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes.

Science.gov (United States)

Merz, Michael; Eisele, Thomas; Berends, Pieter; Appel, Daniel; Rabe, Swen; Blank, Imre; Stressler, Timo; Fischer, Lutz

2015-06-17

Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the A. oryzae strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes.

Determinants of success in native and non-native listening comprehension: an individual differences approach

NARCIS (Netherlands)

Andringa, S.; Olsthoorn, N.; van Beuningen, C.; Schoonen, R.; Hulstijn, J.

2012-01-01

The goal of this study was to explain individual differences in both native and non-native listening comprehension; 121 native and 113 non-native speakers of Dutch were tested on various linguistic and nonlinguistic cognitive skills thought to underlie listening comprehension. Structural equation