Preparative isolation and purification of four flavonoids from the petals of Nelumbo nucifera by high-speed counter-current chromatography.

PubMed

Xingfeng, Guo; Daijie, Wang; Wenjuan, Duan; Jinhua, Du; Xiao, Wang

2010-01-01

Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high-speed counter-current chromatography (HSCCC). Following an initial clean-up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC-PAD, ESI-MS, (1)H-NMR and (13)C-NMR. The separation was performed using a two-phase solvent system composed of ethyl acetate-methanol-water-acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow-rate of 1.0 mL/min in the head-to-tail elution mode. Ultimately, 5.0 mg syringetin-3-O-beta-d-glucoside, 6.5 mg quercetin-3-O-beta-d-glucoside, 12.8 mg isorhamnetin-3-O-beta-d-glucoside and 32.5 mg kaempferol-3-O-beta-d-glucoside were obtained from 125 mg crude sample. The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. (c) 2009 John Wiley & Sons, Ltd.

New trends and affinity tag designs for recombinant protein purification.

PubMed

Wood, David W

2014-06-01

Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Copyright © 2014 Elsevier Ltd. All rights reserved.

A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells

PubMed Central

Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang

2017-01-01

Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P < 0.05). There was no significant difference in islet viability among the four groups. The islet purity, function, yield, and cost of our method were superior to those of the Ficoll-400 and 1077 methods, but inferior to the handpicking method. However, the handpicking method may cause wrist injury and visual impairment in researchers during large-scale islet isolation (>1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized. PMID:28207765

A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

PubMed

Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li

2017-01-01

Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P < 0.05). There was no significant difference in islet viability among the four groups. The islet purity, function, yield, and cost of our method were superior to those of the Ficoll-400 and 1077 methods, but inferior to the handpicking method. However, the handpicking method may cause wrist injury and visual impairment in researchers during large-scale islet isolation (>1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

A novel method for purification of the endogenously expressed fission yeast Set2 complex.

PubMed

Suzuki, Shota; Nagao, Koji; Obuse, Chikashi; Murakami, Yota; Takahata, Shinya

2014-05-01

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8μm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. Copyright © 2014 Elsevier Inc. All rights reserved.

Starvation is more efficient than the washing technique for purification of rat Sertoli cells.

PubMed

Ghasemzadeh-Hasankolaei, Mohammad; Eslaminejad, Mohamadreza Baghaban; Sedighi-Gilani, Mohammadali; Mokarizadeh, Aram

2014-09-01

Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p < 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species.

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

PubMed

Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

2011-05-01

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

An improved method for purification of recombinant truncated heme oxygenase-1 by expanded bed adsorption and gel filtration.

PubMed

Hu, Hong-Bo; Wang, Wei; Han, Ling; Zhou, Wen-Pu; Zhang, Xue-Hong

2007-03-01

Recombinant truncated human heme oxygenase-1 (hHO-1) expressed in Escherichia coli was efficiently separated and purified from feedstock by DEAE-ion exchange expanded bed adsorption. Protocol optimization of hHO-1 on DEAE adsorbent resulted in adsorption in 0 M NaCl and elution in 150 mM NaCl at a pH of 8.5. The active enzyme fractions separated from the expanded bed column were further purified by a Superdex 75 gel filtration step. The specific hHO-1 activity increased from 0.82 +/- 0.05 to 24.8 +/- 1.8 U/mg during the whole purification steps. The recovery and purification factor of truncated hHO-1 of the whole purification were 72.7 +/- 4.7 and 30.2 +/- 2.3%, respectively. This purification process can decrease the demand on the preparation of feedstock and simplify the purification process.

Purification of recombinant Aβ(1-42) and pGlu-Aβ(3-42) using preparative SDS-PAGE.

PubMed

Spahn, Claudia; Wermann, Michael; Eichentopf, Rico; Hause, Gerd; Schlenzig, Dagmar; Schilling, Stephan

2017-08-01

Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was  >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2001-02-01

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

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

PubMed Central

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

2001-01-01

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

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

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

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. 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. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

PubMed

Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

2008-11-01

A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

Combustion water purification techniques influence on OBT analysing using liquid scintillation counting method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varlam, C.; Vagner, I.; Faurescu, I.

In order to determine organically bound tritium (OBT) from environmental samples, these must be converted into water, measurable by liquid scintillation counting (LSC). For this purpose we conducted some experiments to determine OBT level of a grass sample collected from an uncontaminated area. The studied grass sample was combusted in a Parr bomb. However usual interfering phenomena were identified: color or chemical quench, chemiluminescence, overlap over tritium spectrum because of other radionuclides presence as impurities ({sup 14}C from organically compounds, {sup 36}Cl as chloride and free chlorine, {sup 40}K as potassium cations) and emulsion separation. So the purification of themore » combustion water before scintillation counting appeared to be essential. 5 purification methods were tested: distillation with chemical treatment (Na{sub 2}O{sub 2} and KMnO{sub 4}), lyophilization, chemical treatment (Na{sub 2}O{sub 2} and KMnO{sub 4}) followed by lyophilization, azeotropic distillation with toluene and treatment with a volcanic tuff followed by lyophilization. After the purification step each sample was measured and the OBT measured concentration, together with physico-chemical analysis of the water analyzed, revealed that the most efficient method applied for purification of the combustion water was the method using chemical treatment followed by lyophilization.« less

A gas circulation and purification system for gas-cell-based low-energy RI-beam production.

PubMed

Sonoda, T; Tsubota, T; Wada, M; Katayama, I; Kojima, T M; Reponen, M

2016-06-01

A gas circulation and purification system was developed at the RIKEN Radioactive Isotope Beam Factory that can be used for gas-cell-based low-energy RI-beam production. A high-flow-rate gas cell filled with one atmosphere of buffer gas (argon or helium) is used for the deceleration and thermalization of high-energy RI-beams. The exhausted buffer gas is efficiently collected using a compact dry pump and returned to the gas cell with a recovery efficiency of >97%. The buffer gas is efficiently purified using two gas purifiers as well as collision cleaning, which eliminates impurities in the gas. An impurity level of one part per billion is achieved with this method.

A gas circulation and purification system for gas-cell-based low-energy RI-beam production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sonoda, T.; Wada, M.; Katayama, I.

A gas circulation and purification system was developed at the RIKEN Radioactive Isotope Beam Factory that can be used for gas-cell-based low-energy RI-beam production. A high-flow-rate gas cell filled with one atmosphere of buffer gas (argon or helium) is used for the deceleration and thermalization of high-energy RI-beams. The exhausted buffer gas is efficiently collected using a compact dry pump and returned to the gas cell with a recovery efficiency of >97%. The buffer gas is efficiently purified using two gas purifiers as well as collision cleaning, which eliminates impurities in the gas. An impurity level of one part permore » billion is achieved with this method.« less

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya.

PubMed

Tsai, Chi-Chu; Shih, Huei-Chuan; Ko, Ya-Zhu; Wang, Ren-Huang; Li, Shu-Ju; Chiang, Yu-Chung

2016-09-24

Papaya (Carica papaya L.) is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique-based on DNA analysis-was developed for detecting male-hermaphrodite-specific markers to examine the papaya's sex type. This method is based on the loop-mediated isothermal amplification (LAMP) and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya's sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source.

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya

PubMed Central

Tsai, Chi-Chu; Shih, Huei-Chuan; Ko, Ya-Zhu; Wang, Ren-Huang; Li, Shu-Ju; Chiang, Yu-Chung

2016-01-01

Papaya (Carica papaya L.) is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique—based on DNA analysis—was developed for detecting male-hermaphrodite-specific markers to examine the papaya’s sex type. This method is based on the loop-mediated isothermal amplification (LAMP) and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya’s sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source. PMID:27669237

Preparative isolation, fast centrifugal partition chromatography purification and biological activity of cajaflavanone from Derris ferruginea stems.

PubMed

Morel, Sylvie; Landreau, Anne; Nguyen, Van Hung; Derbré, Séverine; Grellier, Philippe; Pape, Patrice Le; Pagniez, Fabrice; Litaudon, Marc; Richomme, Pascal

2012-01-01

The Derris genus is known to contain flavonoid derivatives, including prenylated flavanones and isoflavonoids such as rotenoids, which are generally associated with significant biological activity. To develop an efficient preparative isolation procedure for bioactive cajaflavanone. Fast centrifugal partition chromatography (FCPC) was optimised to purify cajaflavanone from Derris ferruginea stems in a single step as compared to fractionation from the cyclohexane extract by successive conventional solid-liquid chromatography procedures. The purification yield, purity, time and solvent consumption per procedure are described. The anti-fungal, anti-bacterial, anti-leishmanial, anti-plasmodial, anti-oxidant activities and the inhibition of advanced glycation end-products (AGEs) by cajaflavanone accumulation are described. FCPC enabled cajaflavanone purification in a single separation step, yielding sufficient quantities to perform in vitro biological screening. Interestingly, cajaflavanone had an inhibitory effect on the formation of AGEs, without displaying any in vitro anti-oxidant activity. A simple and efficient procedure, in comparison with other preparative methods, for bioactive cajaflavone purification has been developed using FCPC. Copyright © 2011 John Wiley & Sons, Ltd.

Improving the large scale purification of the HIV microbicide, griffithsin.

PubMed

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 griffithsin. The methodology can be readily scaled to the bench top or industry and process components can be used for purification of additional proteins based on biophysical characteristics.

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

PubMed

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.

Purification of Plant Receptor Kinases from Plant Plasma Membranes.

PubMed

Lee, Jin Suk

2017-01-01

Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein. Here we describe two different optimized protein purification protocols, batch and on-chip immunoprecipitation, which efficiently isolate plant membrane receptor kinases for functional analysis.

Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene.

PubMed

Hashimoto, Haruo; Mizushima, Tomoko; Chijiwa, Tsuyoshi; Nakamura, Masato; Suemizu, Hiroshi

2017-06-15

The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction. Copyright © 2017 Elsevier B.V. All rights reserved.

Peg Precipitation Coupled with Chromatography is a New and Sufficient Method for the Purification of Botulinum Neurotoxin Type B

PubMed Central

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

2012-01-01

Clostridium botulinum neurotoxins are used to treat a variety of neuro-muscular disorders, as well as in cosmetology. The increased demand requires efficient methods for the production and purification of these toxins. In this study, a new purification process was developed for purifying type B neurotoxin. The kinetics of C.botulinum strain growth and neurotoxin production were determined for maximum yield of toxin. The neurotoxin was purified by polyethylene glycol (PEG) precipitation and chromatography. Based on design of full factorial experiment, 20% (w/v) PEG-6000, 4°C, pH 5.0 and 0.3 M NaCl were optimal conditions to obtain a high recovery rate of 87% for the type B neurotoxin complex, as indicated by a purification factor of 61.5 fold. Furthermore, residual bacterial cells, impurity proteins and some nucleic acids were removed by PEG precipitation. The following purification of neurotoxin was accomplished by two chromatography techniques using Sephacryl™ S-100 and phenyl HP columns. The neurotoxin was recovered with an overall yield of 21.5% and the purification factor increased to 216.7 fold. In addition, a mouse bioassay determined the purified neurotoxin complex possessed a specific toxicity (LD50) of 4.095 ng/kg. PMID:22761863

Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes

PubMed Central

Watson, Dionysios C.; Yung, Bryant C.; Bergamaschi, Cristina; Chowdhury, Bhabadeb; Bear, Jenifer; Stellas, Dimitris; Morales-Kastresana, Aizea; Jones, Jennifer C.; Felber, Barbara K.; Chen, Xiaoyuan; Pavlakis, George N.

2018-01-01

ABSTRACT The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. PMID:29535850

Efficient purification and concentration of viruses from a large body of high turbidity seawater.

PubMed

Sun, Guowei; Xiao, Jinzhou; Wang, Hongming; Gong, Chaowen; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

2014-01-01

Marine viruses are the most abundant entities in the ocean and play crucial roles in the marine ecological system. However, understanding of viral diversity on large scale depends on efficient and reliable viral purification and concentration techniques. Here, we report on developing an efficient method to purify and concentrate viruses from large body of high turbidity seawater. The developed method characterizes with high viral recovery efficiency, high concentration factor, high viral particle densities and high-throughput, and is reliable for viral concentration from high turbidity seawater. Recovered viral particles were used directly for subsequent analysis by epifluorescence microscopy, transmission electron microscopy and metagenomic sequencing. Three points are essential for this method:•The sampled seawater (>150 L) was initially divided into two parts, water fraction and settled matter fraction, after natural sedimentation.•Both viruses in the water fraction concentrated by tangential flow filtration (TFF) and viruses isolated from the settled matter fraction were considered as the whole viral community in high turbidity seawater.•The viral concentrates were re-concentrated by using centrifugal filter device in order to obtain high density of viral particles.

Ion-exchange chromatography purification of extracellular vesicles.

PubMed

Kosanović, Maja; Milutinović, Bojana; Goč, Sanja; Mitić, Ninoslav; Janković, Miroslava

2017-08-01

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

Methods of cell purification: a critical juncture for laboratory research and translational science.

PubMed

Amos, Peter J; Cagavi Bozkulak, Esra; Qyang, Yibing

2012-01-01

Research in cell biology and the development of translational technologies are driven by competition, public expectations, and regulatory oversight, putting these fields at a critical juncture. Success in these fields is quickly becoming dependent on the ability of researchers to identify and isolate specific cell populations from heterogeneous mixtures accurately and efficiently. Many methods for cell purification have been developed, and each has advantages and disadvantages that must be considered in light of the intended application. Current cell separation strategies make use of surface proteins, genetic expression, and physics to isolate specific cells by phenotypic traits. Cell purification is also dependent on the cellular reagents available for use and the intended application, as these factors may preclude certain mechanisms used in the processes of labeling and sorting cells. Copyright © 2011 S. Karger AG, Basel.

Advanced purification of petroleum refinery wastewater by catalytic vacuum distillation.

PubMed

Yan, Long; Ma, Hongzhu; Wang, Bo; Mao, Wei; Chen, Yashao

2010-06-15

In our work, a new process, catalytic vacuum distillation (CVD) was utilized for purification of petroleum refinery wastewater that was characteristic of high chemical oxygen demand (COD) and salinity. Moreover, various common promoters, like FeCl(3), kaolin, H(2)SO(4) and NaOH were investigated to improve the purification efficiency of CVD. Here, the purification efficiency was estimated by COD testing, electrolytic conductivity, UV-vis spectrum, gas chromatography-mass spectrometry (GC-MS) and pH value. The results showed that NaOH promoted CVD displayed higher efficiency in purification of refinery wastewater than other systems, where the pellucid effluents with low salinity and high COD removal efficiency (99%) were obtained after treatment, and the corresponding pH values of effluents varied from 7 to 9. Furthermore, environment estimation was also tested and the results showed that the effluent had no influence on plant growth. Thus, based on satisfied removal efficiency of COD and salinity achieved simultaneously, NaOH promoted CVD process is an effective approach to purify petroleum refinery wastewater. Copyright 2010 Elsevier B.V. All rights reserved.

Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).

PubMed

Guan, Dongli; Chen, Zhilei

2017-01-01

Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn 2+ ) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.

Continuous Purification of Colloidal Quantum Dots in Large-Scale Using Porous Electrodes in Flow Channel.

PubMed

Lim, Hosub; Woo, Ju Young; Lee, Doh C; Lee, Jinkee; Jeong, Sohee; Kim, Duckjong

2017-02-27

Colloidal quantum dots (QDs) afford huge potential in numerous applications owing to their excellent optical and electronic properties. After the synthesis of QDs, separating QDs from unreacted impurities in large scale is one of the biggest issues to achieve scalable and high performance optoelectronic applications. Thus far, however, continuous purification method, which is essential for mass production, has rarely been reported. In this study, we developed a new continuous purification process that is suitable to the mass production of high-quality QDs. As-synthesized QDs are driven by electrophoresis in a flow channel and captured by porous electrodes and finally separated from the unreacted impurities. Nuclear magnetic resonance and ultraviolet/visible/near-infrared absorption spectroscopic data clearly showed that the impurities were efficiently removed from QDs with the purification yield, defined as the ratio of the mass of purified QDs to that of QDs in the crude solution, up to 87%. Also, we could successfully predict the purification yield depending on purification conditions with a simple theoretical model. The proposed large-scale purification process could be an important cornerstone for the mass production and industrial use of high-quality QDs.

Continuous Purification of Colloidal Quantum Dots in Large-Scale Using Porous Electrodes in Flow Channel

NASA Astrophysics Data System (ADS)

Lim, Hosub; Woo, Ju Young; Lee, Doh Chang; Lee, Jinkee; Jeong, Sohee; Kim, Duckjong

2017-11-01

Colloidal Quantum dots (QDs) afford huge potential in numerous applications owing to their excellent optical and electronic properties. After the synthesis of QDs, separating QDs from unreacted impurities in large scale is one of the biggest issues to achieve scalable and high performance optoelectronic applications. Thus far, however, continuous purification method, which is essential for mass production, has rarely been reported. In this study, we developed a new continuous purification process that is suitable to the mass production of high-quality QDs. As-synthesized QDs are driven by electrophoresis in a flow channel and captured by porous electrodes and finally separated from the unreacted impurities. Nuclear magnetic resonance and ultraviolet/visible/near-infrared absorption spectroscopic data clearly showed that the impurities were efficiently removed from QDs with the purification yield, defined as the ratio of the mass of purified QDs to that of QDs in the crude solution, up to 87%. Also, we could successfully predict the purification yield depending on purification conditions with a simple theoretical model. The proposed large-scale purification process could be an important cornerstone for the mass production and industrial use of high-quality QDs.

Efficient purification of Apolipoprotein A1 (ApoA1) from plasma by HEA HyperCel™: An alternative approach.

PubMed

G, Arun Govind; Kamalanathan, Agamudi Shivasankaran; Vijayalakshmi, Mookambeswaran Arunachalam; Venkataraman, Krishnan

2018-01-15

HDL-ApoA1 plays a pivotal role in the prevention of atherosclerosis and cardiovascular diseases. ApoA1 purification from blood plasma has always remained tedious, involving multiple steps, large volumes of plasma and substantial loss in the final yield of pure ApoA1. In this study, a two-step method has been developed and optimized for the purification of ApoA1 from plasma. Plasma was first subjected to 60% ammonium sulphate (NH 4 ) 2 SO 4 precipitation and subsequently, ApoA1 was recovered using mixed mode chromatographic sorbent, HEA HyperCel™. ApoA1 was found to be enriched in 60% (NH 4 ) 2 SO 4 supernatant that was dialyzed and injected onto HEA sorbent with 50 mM phosphate buffer pH 7.4. The bound proteins were eluted by decreasing the pH in step-gradient from pH 7.4 to pH 4.0 and subsequently to pH 3.5 using 50 mM sodium acetate buffer. Gel electrophoresis showed elution of homogeneous apoA1 at pH 3.5, with purity and yield of 63%. An interesting feature of this approach is that the purified ApoA1 was monomeric with a mass of 28,079.30 Da as confirmed by MS analysis. This simple and efficient method of purification of apoA1 serves as an alternative method which can be combined with traditional approaches and has a great potential for biochemical and clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Production of recombinant protein G through high-density fermentation of engineered bacteria as well as purification.

PubMed

Zhang, Hu-Cheng; Yang, Jun; Yang, Guo-Wei; Wang, Xiao-Jie; Fan, Hai-Tao

2015-08-01

Recombinant Streptococcus Protein G (PG) is a cell wall protein, which, when combined with mammal immunoglobulin, is used in separating antibody technology. High-density fermentation technologies using an engineered recombinant PG-producing bacteria as well as PG separation and purification technologies have a direct impact on the availability and application of PG. Through primary and secondary seed cultivation, a recombinant E. coli strain was subjected to high-density fermentation with controlled feed supplement concentration under stimulation with isopropyl β-D-1-thiogalactopyranoside. The present study investigated the effect of factors including inoculum size, oxygen levels, pH and the cultivating method on the fermentation process, as well as the effect of the separation and purification technologies, including ultrasonication, nickel column affinity chromatography, Sephadex G-25 gel filtration chromatography and diethylaminoethanol-sepharose fast flow ion exchange chromatography on the yield and purity of PG. The efficiency of extraction was detected using SDS-PAGE. High-density fermentation yielded 80-150 g/l of bacteria and 1 g PG was obtained from one liter broth. The present study delivered a highly efficient novel method via which PG can be obtained at a high concentration and a purity >95%.

Dynamic pH junction high-speed counter-current chromatography coupled with microwave-assisted extraction for online separation and purification of alkaloids from Stephania cepharantha.

PubMed

Yuan, Zhiquan; Xiao, Xiaohua; Li, Gongke

2013-11-22

A simple and efficient dynamic pH junction high-speed counter-current chromatography method was developed and further applied to the online extraction, separation and purification of alkaloids from Stephania cepharantha by coupling with microwave-assisted extraction. Mineral acid and organic base were added into the mobile phase and the sample solution, respectively, leading to the formation of a dynamic pH junction in the column and causing focus of alkaloids. Selective focus of analytes can be achieved on the basis of velocity changes of the pH junction through appropriate selection of solvent systems and optimization of additive concentrations. The extract can be directly introduced into the HSCCC for the online extraction, separation and purification of alkaloids from S. cepharantha. Continuous separation can be easily achieved with the same solvent system. Under the optimum conditions, 6.0 g original sample was extracted with 60 mL of the upper phase of hexane-ethyl acetate-methanol-water (1:1:1:1, v/v/v/v) containing 10% triethylamine under 50 °C and 400 W irradiation power for 10 min, the extracts were directly separated and purified by high-speed counter-current chromatography. A total of 5.7 mg sinomenine, 8.3mg 6,7-di-O-acetylsinococuline, 17.9 mg berbamine, 12.7 mg isotetrandrine and 14.6 mg cepharanthine were obtained with purities of 96.7%, 93.7%, 98.7%, 97.3% and 99.3%, respectively. The online method provides good selectivity to ionizable compounds and improves the separation and purification efficiency of the high-speed counter-current chromatography technique. It has good potential for separation and purification of effective compounds from natural products. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of 2 Purification Methods for Isolation of Human Adipose-Derived Stem Cells Based on Red Blood Cell Lysis With Ammonium Chloride and Hypotonic Sodium Chloride Solution.

PubMed

Li, Sheng-Hong; Liao, Xuan; Zhou, Tian-En; Xiao, Li-Ling; Chen, Yuan-Wen; Wu, Fan; Wang, Jing-Ru; Cheng, Biao; Song, Jian-Xing; Liu, Hong-Wei

2017-01-01

The present study was conducted to compare 2 purification methods for isolation of human adipose-derived stromal vascular fraction or stem cells (ADSCs) based on red blood cell (RBC) lysis with 155 mM ammonium chloride (NH4Cl) and hypotonic sodium chloride (NaCl) solution, and try to develop a safe, convenient, and cost-effective purification method for clinical applications. Adipose-derived stem cells and RBC were harvested from the fatty and fluid portions of liposuction aspirates, respectively. The suitable concentration of hypotonic NaCl solution on RBC lysis for purification of ADSCs was developed by RBC osmotic fragility test and flow cytometry analysis. The effects of 155 mM NH4Cl or 0.3% NaCl solution on ADSCs proliferation and RBC lysis efficiency were examined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and lysis efficiency test, respectively. In addition, the adipogenic and osteogenic capabilities, phenotype and genetic stability of ADSCs were evaluated by oil red staining, alkaline phosphatase activity measurement, flow cytometry, and karyotype analysis, respectively. Sodium chloride solution in 0.3% concentration effectively removed RBCs and did not influence the survival of ADSCs in the 10-minute incubation time. The lysis efficiency did not differ significantly between 0.3% NaCl and 155 mM NH4Cl. Moreover, the adipogenic and osteogenic capabilities, surface marker expression and karyotype of the ADSCs were not affected by lysis solutions or by lysis per se. However, the proliferation capacity in the 0.3% NaCl group was superior to that in 155 mM NH4Cl group. Our data suggest that 0.3% NaCl solution is useful for isolating ADSCs from liposuction aspirate for clinical applications with safety, convenience, and cost-effect.

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

Gram-scale purification of aconitine and identification of lappaconitine in Aconitum karacolicum.

PubMed

Tarbe, M; de Pomyers, H; Mugnier, L; Bertin, D; Ibragimov, T; Gigmes, D; Mabrouk, K

2017-07-01

Aconitum karacolicum from northern Kyrgyzstan (Alatau area) contains about 0.8-1% aconitine as well as other aconite derivatives that have already been identified. In this paper, we compare several methods for the further purification of an Aconitum karacolicum extract initially containing 80% of aconitine. Reverse-phase flash chromatography, reverse-phase semi-preparative HPLC, centrifugal partition chromatography (CPC) and recrystallization techniques were evaluated regarding first their efficiency to get the highest purity of aconitine (over 96%) and secondly their applicability in a semi-industrial scale purification process (in our case, 150g of plant extract). Even if the CPC technique shows the highest purification yield (63%), the recrystallization remains the method of choice to purify a large amount of aconitine as i) it can be easily carried out in safe conditions; ii) an aprotic solvent is used, avoiding aconitine degradation. Moreover, this study led us to the identification of lappaconitine in Aconitum karacolicum, a well-known alkaloid never found in this Aconitum species. Copyright © 2017 Elsevier B.V. All rights reserved.

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum.

PubMed

Vázquez-Iglesias, Lorena; Estefanell-Ucha, Borja; Barcia-Castro, Leticia; Páez de la Cadena, María; Álvarez-Chaver, Paula; Ayude-Vázquez, Daniel; Rodríguez-Berrocal, Francisco Javier

2017-01-01

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum

PubMed Central

Estefanell-Ucha, Borja; Barcia-Castro, Leticia; Páez de la Cadena, María; Álvarez-Chaver, Paula; Ayude-Vázquez, Daniel; Rodríguez-Berrocal, Francisco Javier

2017-01-01

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot. PMID:28652930

Purification of crime scene DNA extracts using centrifugal filter devices

PubMed Central

2013-01-01

Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used. PMID:23618387

Air purification in industrial plants producing automotive rubber components in terms of energy efficiency

NASA Astrophysics Data System (ADS)

Grzebielec, Andrzej; Rusowicz, Artur; Szelągowski, Adam

2017-04-01

In automotive industry plants, which use injection molding machines for rubber processing, tar contaminates air to such an extent that air fails to enter standard heat recovery systems. Accumulated tar clogs ventilation heat recovery exchangers in just a few days. In the plant in which the research was conducted, tar contamination causes blockage of ventilation ducts. The effect of this phenomenon was that every half year channels had to be replaced with new ones, since the economic analysis has shown that cleaning them is not cost-efficient. Air temperature inside such plants is often, even in winter, higher than 30°C. The air, without any means of heat recovery, is discharged outside the buildings. The analyzed plant uses three types of media for production: hot water, cold water at 14°C (produced in a water chiller), and compressed air, generated in a unit with a rated power consumption of 180 kW. The aim of the study is to determine the energy efficiency improvement of this type of manufacturing plant. The main problem to solve is to provide an air purification process so that air can be used in heat recovery devices. The next problem to solve is to recover heat at such a temperature level that it would be possible to produce cold for technological purposes without air purification. Experimental studies have shown that air purification is feasible. By using one microjet head, a total of 75% of tar particles was removed from the air; by using 4 heads, a purification efficiency of 93% was obtained. This method of air purification causes air temperature to decrease from 35°C to 20°C, which significantly reduces the potential for heat recovery. The next step of the research was designing a cassette-plate heat exchanger to exchange heat without air purification. The economic analysis of such a solution revealed that replacing the heat exchanger with a new one even once a year was not cost-efficient. Another issue examined in the context of energy efficiency was the use of waste heat from the air compressor. Before any changes, the heat was picked up by a chilled water system. The idea was to use the heat for cold generation. Temperature of oil and air in the compressor exceeds 65°C, which makes it a perfect heat source for an adsorption refrigeration device. This solution reduced the cooling demand by 147 kW, thus reducing power consumption by 36.75 kW. This study shows that even in factories where air is heavily polluted with tar, there are huge potentials for energy recovery using existing technical solutions. It is important to note that problems of this kind should always be approached individually.

One-step purification of assembly-competent tubulin from diverse eukaryotic sources

PubMed Central

Widlund, Per O.; Podolski, Marija; Reber, Simone; Alper, Joshua; Storch, Marko; Hyman, Anthony A.; Howard, Jonathon; Drechsel, David N.

2012-01-01

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research. PMID:22993214

Chemical purification of lanthanides for low-background experiments

NASA Astrophysics Data System (ADS)

Boiko, R. S.

2017-10-01

There are many potentially active isotopes among the lanthanide elements which are possible to use for low-background experiments to search for double β decay, dark matter, to investigate rare α and β decays. These kind of experiments require very low level of radioactive contamination, but commercially available compounds of lanthanides are always contamined by uranium, thorium, radium, potassium, etc. A simple chemical method based on liquid-liquid extraction has been applied for the purification of CeO2, Nd2O3 and Gd˙2O˙3 from radioactive traces. Detailed schemes of purification procedure are described. Measurements by using HPGe spectrometry demonstrate high efficiency in K, Ra, Th, U contaminations reduction on at least one order of magnitude.

Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

PubMed Central

Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

2012-01-01

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

In situ synthesis of protein arrays.

PubMed

He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

2008-02-01

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

Analysis And Design Of A Water Purification System For The West African Area Of Operation

DTIC Science & Technology

2016-12-01

harmful metals and in disinfecting the water prior to human consumption . Research conducted proved that the BWS is more cost effective , efficient...and test a feasible and cost- effective prototype of a purification system to the BWS for improved capability. This study uses a design-based and...design. The prototype test results showed that the water purification system performed effectively and efficiently in accordance with the

Purification of pectinase from mango (Mangifera indica L. cv. Chokanan) waste using an aqueous organic phase system: a potential low cost source of the enzyme.

PubMed

Amid, Mehrnoush; Abdul Manap, Mohd Yazid; Mustafa, Shuhaimi

2013-07-15

As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%. Copyright © 2013 Elsevier B.V. All rights reserved.

Purification of Drug Loaded PLGA Nanoparticles Prepared by Emulsification Solvent Evaporation Using Stirred Cell Ultrafiltration Technique.

PubMed

Paswan, Suresh K; Saini, T R

2017-12-01

The emulsifiers in an exceedingly higher level are used in the preparation of drug loaded polymeric nanoparticles prepared by emulsification solvent evaporation method. This creates great problem to the formulator due to their serious toxicities when it is to be administered by parenteral route. The final product is therefore required to be freed from the used surfactants by the conventional purification techniques which is a cumbersome job. The solvent resistant stirred cell ultrafiltration unit (Millipore) was used in this study using polyethersulfone ultrafiltration membrane (Biomax®) having pore size of NMWL 300 KDa as the membrane filter. The purification efficiency of this technique was compared with the conventional centrifugation technique. The flow rate of ultrafiltration was optimized for removal of surfactant (polyvinyl alcohol) impurities to the acceptable levels in 1-3.5 h from the nanoparticle dispersion of tamoxifen prepared by emulsification solvent evaporation method. The present investigations demonstrate the application of solvent resistant stirred cell ultrafiltration technique for removal of toxic impurities of surfactant (PVA) from the polymeric drug nanoparticles (tamoxifen) prepared by emulsification solvent evaporation method. This technique offers added benefit of producing more concentrated nanoparticles dispersion without causing significant particle size growth which is observed in other purification techniques, e.g., centrifugation and ultracentrifugation.

A novel aqueous micellar two-phase system composed of surfactant and sorbitol for purification of pectinase enzyme from Psidium guajava and recycling phase components.

PubMed

Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

Photoswitchable method for the ordered attachment of proteins to surfaces

DOEpatents

Camarero, Julio A [Livermore, CA; DeYoreo, James J [Clayton, CA; Kwon, Youngeun [Livermore, CA

2011-07-05

Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

Photoswitchable method for the ordered attachment of proteins to surfaces

DOEpatents

Camarero, Julio A.; De Yoreo, James J.; Kwon, Youngeun

2010-04-20

Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

Isolation and purification of monosialotetrahexosylgangliosides from pig brain by extraction and liquid chromatography.

PubMed

Bian, Liujiao; Yang, Jianting; Sun, Yu

2015-10-01

Monosialotetrahexosylganglioside (GM1), one of glycosphingolipids containing sialic acid, plays particularly important role in fighting against paralysis, dementia and other diseases caused by brain and nerve damage. In this work, a simple and highly efficient method with high yield was developed for isolation and purification of GM1 from pig brain. The method consisted of an extraction by chloroform-methanol-water and a two-step chromatographic separation by DEAE-Sepharose Fast Flow anion-exchange medium and Sephacryl S-100 HR size-exclusion medium. The purified GM1 was proved to be homogeneous and had a purity of >98.0% by high-performance anion-exchange and size-exclusion chromatography. The molecular weight was 30.0 kDa by high-performance size-exclusion chromatography and 1546.9 Da by electrospray ionization mass spectrometry. The chromogenic reaction by resorcinol-hydrochloric acid solution indicated that the purified GM1 showed a specific chromogenic reaction of sialic acid. Through this isolation and purification program, ~1.0 mg of pure GM1 could be captured from 500 g wet pig brain tissue and the yield of GM1 was around 0.022%, which was higher than the yields by other methods. The method may provide an alternative for isolation and purification of GM1 in other biological tissues. Copyright © 2015 John Wiley & Sons, Ltd.

Compositions and Methods for Inhibiting Gene Expressions

NASA Technical Reports Server (NTRS)

Williams, Loren D. (Inventor); Hsiao, Chiaolong (Inventor); Fang, Po-Yu (Inventor); Williams, Justin (Inventor)

2018-01-01

A combined packing and assembly method that efficiently packs ribonucleic acid (RNA) into virus like particles (VLPs) has been developed. The VLPs can spontaneously assemble and load RNA in vivo, efficiently packaging specifically designed RNAs at high densities and with high purity. In some embodiments the RNA is capable of interference activity, or is a precursor of a RNA capable of causing interference activity. Compositions and methods for the efficient expression, production and purification of VLP-RNAs are provided. VLP-RNAs can be used for the storage of RNA for long periods, and provide the ability to deliver RNA in stable form that is readily taken up by cells.

Avian influenza virus RNA extraction

USDA-ARS?s Scientific Manuscript database

The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from expe...

Biocapture of CO2 by Different Microalgal-Based Technologies for Biogas Upgrading and Simultaneous Biogas Slurry Purification under Various Light Intensities and Photoperiods

PubMed Central

Guo, Pengfei; Zhang, Yuejin; Zhao, Yongjun

2018-01-01

Co-cultivation of microalgae and microbes for pollutant removal from sewage is considered as an effective wastewater treatment method. The aim of this study is to screen the optimal photoperiod, light intensity and microalgae co-cultivation method for simultaneously removing nutrients in biogas slurry and capturing CO2 in biogas. The microalgae–fungi pellets are deemed to be a viable option because of their high specific growth rate and nutrient and CO2 removal efficiency under the photoperiod of 14 h light:10 h dark. The order of both the biogas slurry purification and biogas upgrading is ranked the same, that is Chlorella vulgaris–Ganoderma lucidum > Chlorella vulgaris–activated sludge > Chlorella vulgaris under different light intensities. For all cultivation methods, the moderate light intensity of 450 μmol m−2 s−1 is regarded as the best choice. This research revealed that the control of photoperiod and light intensity can promote the biological treatment process of biogas slurry purification and biogas upgrading using microalgal-based technology. PMID:29543784

Biocapture of CO₂ by Different Microalgal-Based Technologies for Biogas Upgrading and Simultaneous Biogas Slurry Purification under Various Light Intensities and Photoperiods.

PubMed

Guo, Pengfei; Zhang, Yuejin; Zhao, Yongjun

2018-03-15

Abstract : Co-cultivation of microalgae and microbes for pollutant removal from sewage is considered as an effective wastewater treatment method. The aim of this study is to screen the optimal photoperiod, light intensity and microalgae co-cultivation method for simultaneously removing nutrients in biogas slurry and capturing CO₂ in biogas. The microalgae-fungi pellets are deemed to be a viable option because of their high specific growth rate and nutrient and CO 2 removal efficiency under the photoperiod of 14 h light:10 h dark. The order of both the biogas slurry purification and biogas upgrading is ranked the same, that is Chlorella vulga ris - Ganoderma lucidum > Chlorella vulga ris -activated sludge > Chlorella vulgaris under different light intensities. For all cultivation methods, the moderate light intensity of 450 μmol m -2 s -1 is regarded as the best choice. This research revealed that the control of photoperiod and light intensity can promote the biological treatment process of biogas slurry purification and biogas upgrading using microalgal-based technology.

Parallel scalability of Hartree-Fock calculations

NASA Astrophysics Data System (ADS)

Chow, Edmond; Liu, Xing; Smelyanskiy, Mikhail; Hammond, Jeff R.

2015-03-01

Quantum chemistry is increasingly performed using large cluster computers consisting of multiple interconnected nodes. For a fixed molecular problem, the efficiency of a calculation usually decreases as more nodes are used, due to the cost of communication between the nodes. This paper empirically investigates the parallel scalability of Hartree-Fock calculations. The construction of the Fock matrix and the density matrix calculation are analyzed separately. For the former, we use a parallelization of Fock matrix construction based on a static partitioning of work followed by a work stealing phase. For the latter, we use density matrix purification from the linear scaling methods literature, but without using sparsity. When using large numbers of nodes for moderately sized problems, density matrix computations are network-bandwidth bound, making purification methods potentially faster than eigendecomposition methods.

Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum.

PubMed

Robinson, Graham C; Vegunta, Yogesh; Gabus, Caroline; Gaubitz, Christl; Thore, Stéphane

2017-05-01

The Target of Rapamycin Complex is a central controller of cell growth and differentiation in eukaryotes. Its global architecture has been described by cryoelectron microscopy, and regions of its central TOR protein have been described by X-ray crystallography. However, the N-terminal region of this protein, which consists of a series of HEAT repeats, remains uncharacterised at high resolution, most likely due to the absence of a suitable purification procedure. Here, we present a robust method for the preparation of the HEAT-repeat domain, utilizing the thermophilic fungus Chaetomium thermophilum as a source organism. We describe construct design and stable expression in insect cells. An efficient two-step purification procedure is presented, and the purified product is characterised by SEC and MALDI-TOF MS. The methods described pave the way for a complete high-resolution characterisation of this elusive region of the TOR protein. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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.

Application of QUAL2K Model to Assess Ecological Purification Technology for a Polluted River

PubMed Central

Zhu, Wenting; Niu, Qian; Zhang, Ruibin; Ye, Rui; Qian, Xin; Qian, Yu

2015-01-01

Industrialization and urbanization have caused water pollution and ecosystem degradation, especially in urban canals and rivers in China; accordingly, effective water quality improvement programs are needed. In this study, the Tianlai River in Jiangsu, China was taken as a research site, and a combination of ecological purification technologies consisting of biological rope, phytoremediation, and activated carbon were applied in a laboratory-scale study to examine degradation coefficients under dynamic water conditions. Coefficients were then input into the QUAL2K model to simulate various hypothetical scenarios and determine the minimum density of ecological purification combination and hydraulic retention time (HRT) to meet Grade V or IV of the China standard for surface water. The minimum densities for Grade V and IV were 1.6 times and 2 times the experimental density, while the minimum HRTs for Grade V and IV were 2.4 day and 3 day. The results of this study should provide a practical and efficient design method for ecological purification programs. PMID:25689997

Preparative purification of the major anti-inflammatory triterpenoid esters from Marigold (Calendula officinalis).

PubMed

Hamburger, M; Adler, S; Baumann, D; Förg, A; Weinreich, B

2003-06-01

A method for the efficient preparative purification of faradiol 3-O-laurate, palmitate and myristate, the major anti-inflammatory triterpenoid esters in the flower heads of the medicinal plant Calendula officinalis has been developed. Gram quantities of the individual compounds were obtained with 96 to 98% purity by a combination of supercritical fluid extraction (SFE), normal-phase and reversed-phase column chromatography. During the work-up of the faradiol esters, accompanying minor compounds of the triterpene ester fraction were purified and identified by spectroscopic means as maniladiol 3-O-laurate and myristate.

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues.

PubMed

Meints, Lisa M; Poston, Anne W; Piligian, Brent F; Olson, Claire D; Badger, Katherine S; Woodruff, Peter J; Swarts, Benjamin M

2017-02-17

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a "green" biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18 F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Low Cost, Efficient Microcavity Plasma Ozone Generation for Water Remediation and Air Purification

DTIC Science & Technology

2012-06-01

Eliasson, and M. Hirth, “ Ozone Generation from Oxygen and Air: Discharge Physics and Reaction Mechanisms,” Ozone Sci. and Eng., vol. 10, pp. 367-378...Phase I Final Report: Low Cost, Efficient Microcavity Plasma Ozone Generation for Water Remediation and Air Purification...Contract Number: FA9550-11-C-0087 June 2012 Low Cost, Efficient Microcavity Plasma Ozone Generation for Water Remediation

Preparative isolation and purification of seven isoflavones from Belamcanda chinensis.

PubMed

Lee, Yeon Sil; Kim, Seon Ha; Kim, Jin Kyu; Lee, Sanghyun; Jung, Sang Hoon; Lim, Soon Sung

2011-01-01

Isoflavonoids from Belamcanda chinensis are known to have a number of physiological benefits including anti-inflammatory, anti-angiogenic and anti-mutagenic properties. However, there have been no reports on the effective isolation and purification of isoflavonoids from B. chinensis. To develop an efficient method for the preparative isolation and purification of isoflavones from B. chinensis by high-speed counter-current chromatography (HSCCC). A two-step HSCCC isolation method was developed using solvent system of n-hexane-ethyl acetate-2-propanol-methanol-water (5:6:2:3.5:6, v/v) and of ethyl acetate-methanol-water (10:2:9, v/v). FLASH purification system (45% methanol, isocratic) was also used for further purification. The purities and chemical structures of the isolated compounds were determined by high-performance liquid chromatography-photodiode array detection (HPLC-PDA), electrospray ionisation-mass spectrometry (ESI-MS), ¹H- and ¹³C-nuclear magnetic resonance spectrometry (NMR) and nuclear overhauser enhancement (NOE). HSCCC was successfully used for the preparative separation and purification of seven isoflavones, including tectoridin (145.4 mg, 97.5%), iridin (77.9 mg, 94.0%), irilin D (42.0 mg, 92.0%), tectorigenin (294.1 mg, 98.6%), iristectorigenin A (86.8 mg, 93.4%), irigenin (141.8 mg, 95.8%) and irisflorentin (73.4 mg, 94.7%) from the rhizomes of B. chinensis. Two isoflavone glycosides and five isoflavone derivatives were successfully isolated and purified from the crude methanol extract of dried rhizomes of the B. chinensis by HSCCC. Copyright © 2011 John Wiley & Sons, Ltd.

The coupling between hydrodynamic and purification efficiencies of ecological porous spur-dike in field drainage ditch

NASA Astrophysics Data System (ADS)

Rao, Lei; Wang, Pei-fang; Dai, Qing-song; Wang, Chao

2018-05-01

In this study, a series of ecological porous spur-dikes are arranged in an experiment channel to simulate a real field drainage ditch. The inside and outside flow fields of spur-dikes are determined by numerical simulations and experimental methods. An Ammonia-Nitrogen (NH3-N) degradation evaluation model is built to calculate the pollution removal rate by coupling with the inner flow field of the porous spur-dikes. The variations of the total pollutant removal rate in the channel are discussed in terms of different porosities and gap distances between spur-dikes and inlet flow velocities. It is indicated that a reasonable parameter matching of the porosity and the gap distance with the flow velocity of the ditch can bring about a satisfactory purification efficiency with a small delivery quantity of ecological porous materials.

A Novel Aqueous Micellar Two-Phase System Composed of Surfactant and Sorbitol for Purification of Pectinase Enzyme from Psidium guajava and Recycling Phase Components

PubMed Central

Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme. PMID:25756051

Porous graphene-based membranes for water purification from metal ions at low differential pressures.

PubMed

Park, Jaewoo; Bazylewski, Paul; Fanchini, Giovanni

2016-05-14

A new generation of membranes for water purification based on weakly oxidized and nanoporous few-layer graphene is here introduced. These membranes dramatically decrease the high energy requirements of water purification by reverse osmosis. They combine the advantages of porous and non-oxidized single-layer graphene, offering energy-efficient water filtration at relatively low differential pressures, and highly oxidized graphene oxide, exhibiting high performance in terms of impurity adsorption. In the reported fabrication process, leaks between juxtaposed few-layer graphene flakes are sealed by thermally annealed colloidal silica, in a treatment that precedes the opening of (sub)nanometre-size pores in graphene. This process, explored for the first time in this work, results in nanoporous graphene flakes that are water-tight at the edges without occluding the (sub)nanopores. With this method, removal of impurities from water occurs through a combination of size-based pore rejection and pore-edge adsorption. Thinness of graphene flakes allows these membranes to achieve water purification from metal ions in concentrations of few parts-per-million at differential pressures as low as 30 kPa, outperforming existing graphene or graphene oxide purification systems with comparable flow rates.

Porous graphene-based membranes for water purification from metal ions at low differential pressures

NASA Astrophysics Data System (ADS)

Park, Jaewoo; Bazylewski, Paul; Fanchini, Giovanni

2016-05-01

A new generation of membranes for water purification based on weakly oxidized and nanoporous few-layer graphene is here introduced. These membranes dramatically decrease the high energy requirements of water purification by reverse osmosis. They combine the advantages of porous and non-oxidized single-layer graphene, offering energy-efficient water filtration at relatively low differential pressures, and highly oxidized graphene oxide, exhibiting high performance in terms of impurity adsorption. In the reported fabrication process, leaks between juxtaposed few-layer graphene flakes are sealed by thermally annealed colloidal silica, in a treatment that precedes the opening of (sub)nanometre-size pores in graphene. This process, explored for the first time in this work, results in nanoporous graphene flakes that are water-tight at the edges without occluding the (sub)nanopores. With this method, removal of impurities from water occurs through a combination of size-based pore rejection and pore-edge adsorption. Thinness of graphene flakes allows these membranes to achieve water purification from metal ions in concentrations of few parts-per-million at differential pressures as low as 30 kPa, outperforming existing graphene or graphene oxide purification systems with comparable flow rates.

Eutrophic water purification efficiency using a combination of hydrodynamic cavitation and ozonation on a pilot scale.

PubMed

Li, Wei-Xin; Tang, Chuan-Dong; Wu, Zhi-Lin; Wang, Wei-Min; Zhang, Yu-Feng; Zhao, Yi; Cravotto, Giancarlo

2015-04-01

This paper presents the purification of eutrophic water using a combination of hydrodynamic cavitation (HC) and ozonation (O3) at a continuous flow of 0.8 m(3) h(-1) on a pilot scale. The maximum removal rate of chlorophyll a using O3 alone and the HC/O3 combination was 62.3 and 78.8%, respectively, under optimal conditions, where the ozone utilization efficiency was 64.5 and 94.8% and total energy consumption was 8.89 and 8.25 kWh m(-3), respectively. Thus, the removal rate of chlorophyll a and the ozone utilization efficiency were improved by 26.5% and 46.9%, respectively, by using the combined technique. Meanwhile, total energy consumption was reduced by 7.2%. Turbidity linearly decreased with chlorophyll a removal rate, but no linear relationship exists between the removal of COD or UV254 and chlorophyll a. As expected, the suction-cavitation-assisted O3 exhibited higher energy efficiency than the extrusion-cavitation-assisted O3 and O3 alone methods.

Optimization of extractants, purifying packings, and eluents for analytical extraction of organochlorine pesticides in Hydragric Acrisols.

PubMed

Zhao, Zhenhua; Xia, Liling; Wang, Fang; Jiang, Xin; Gao, Yanzheng

2012-08-01

In this study, we screened for an economic, rapid, and efficient hypotoxic pretreatment method for organochlorine pesticides in soil samples for gas chromatography (GC) analysis. The analytical extraction efficiencies of 11 different extractants, nine types of solid-phase purification (SPP) cartridge packings, and three types of eluents for 13 organochlorine pesticides (OCPs) in spiked and natural Chinese red soil (Hydragric Acrisols) were evaluated using an ultrasonic extraction and solid-phase purification method. High percent recoveries (85-106%) were obtained for the 13 organochlorine pesticides in soil when petroleum ether/acetone/water (10:5:2, v/v) was used an extractant. They were purified using celite SPP cartridge packing and eluted with 9 mL of dichloromethane/petroleum ether (1:9, v/v). The OCPs purification pretreatment of Hydragric Acrisols, using the above method, meets the GC analysis requirements. Compared with other traditional pretreatment methods for OCPs in soil samples, this method has several advantages, such as a short extraction time, reducing the amount of solvent, having no emulsion phenomenon, and hypotoxicity to the laboratory technicians. The concentrations of 1,1,1,-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl) ethane (DDTs; 3.42-8.08 ng g(-1)) in field soils were higher than the hexachlorocyclohexane concentration (2.94-6.12 ng g(-1)). The 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p'-DDE) + 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethane (p,p'-DDD)/p,p'-DDT ratio in this field soil was approximately 2.7, suggesting that no new DDT pollution source was introduced into the sampling site.

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages.

PubMed

Kramberger, Petra; Urbas, Lidija; Štrancar, Aleš

2015-01-01

Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production.

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

PubMed Central

Kramberger, Petra; Urbas, Lidija; Štrancar, Aleš

2015-01-01

Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production. PMID:25751122

Rapid one-step purification of single-cells encapsulated in alginate microcapsules from oil to aqueous phase using a hydrophobic filter paper: implications for single-cell experiments.

PubMed

Lee, Do-Hyun; Jang, Miran; Park, Je-Kyun

2014-10-01

By virtue of the biocompatibility and physical properties of hydrogel, picoliter-sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one-step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil-suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF-7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (~10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long-term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single-cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single-cell experiments. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel liquid/liquid extraction process composed of surfactant and acetonitrile for purification of polygalacturonase enzyme from Durio zibethinus.

PubMed

Amid, Mehrnoush; Manap, Yazid; Azmira, Farhana; Hussin, Muhaini; Sarker, Zaidul Islam

2015-07-01

Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme. Copyright © 2015 Elsevier B.V. All rights reserved.

Photocatalytic Cellulosic Electrospun Fibers for the Degradation of Potent Cyanobacteria Toxin Microcystin-LR

DTIC Science & Technology

2012-01-01

treatment applications using solar light as a renewable source of energy. Introduction The need for low cost and efficient water treatment strategies... photocatalysis with nanoparticles (such as titania, TiO2) show tremendous promise as a simple and energy efficient tech- nology for water purification and...which limits the amount of available sunlight that can be used for photocatalysis . To circumvent this issue, methods have been developed to extend

Concentration and Purification of Influenza Virus on Insoluble Polyelectrolytes

PubMed Central

Wallis, Craig; Homma, Akira; Melnick, Joseph L.

1972-01-01

A method for rapid concentration and purification of influenza virus by adsorption on and elution from an insoluble polyelectrolyte is described. To accomplish this task, influenza virus had to be rendered stable at pH 4 to 5, since viruses adsorb to the polyelectrolyte more efficiently at this pH range. A precipitate which forms in influenza harvests under acid conditions in the cold can be removed by ammonium sulfate at a concentration which traps the precipitate but not the virus. Thus, ammonium sulfate-treated influenza virus in allantoic fluid could be readily concentrated on the polyelectrolyte. Elution yielded a virus concentrate essentially free of nonviral proteins. PMID:4553141

Purification and Autoactivation Method for Recombinant Coagulation Factor VII.

PubMed

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.

Integrated process of distillation with side reactors for synthesis of organic acid esters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panchal, Chandrakant B; Prindle, John C; Kolah, Aspri

An integrated process and system for synthesis of organic-acid esters is provided. The method of synthesizing combines reaction and distillation where an organic acid and alcohol composition are passed through a distillation chamber having a plurality of zones. Side reactors are used for drawing off portions of the composition and then recycling them to the distillation column for further purification. Water is removed from a pre-reactor prior to insertion into the distillation column. An integrated heat integration system is contained within the distillation column for further purification and optimizing efficiency in the obtaining of the final product.

Purification of bacteriophage M13 by anion exchange chromatography.

PubMed

Monjezi, Razieh; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2010-07-01

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious. Copyright 2010 Elsevier B.V. All rights reserved.

Prospective technologies for using biohydrogen in power installations on the basis of fuel cells (a review)

NASA Astrophysics Data System (ADS)

Dunikov, D. O.; Borzenko, V. I.; Malyshenko, S. P.; Blinov, D. V.; Kazakov, A. N.

2013-03-01

The present state of technology for obtaining hydrogen by biological methods and for purifying it is reviewed from the viewpoint of its possible use in kilowatt-class power installations. Hybrid membranesorption biohydrogen purification methods combining membrane-based pretreatment and sorption-based final treatment, also with the use of metal hydrides, should be regarded as the most efficient ones.

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

PubMed

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

2014-01-01

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

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

PubMed

Genji, Takahisa; Nozawa, Akira; Tozawa, Yuzuru

2010-10-01

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield. Copyright © 2010 Elsevier Inc. All rights reserved.

Electro-purification of carbon nanotube networks without damaging the assembly structure and crystallinity

NASA Astrophysics Data System (ADS)

Yang, Xueqin; Yang, Ming; Zhang, Huichao; Zhao, Jingna; Zhang, Xiaohua; Li, Qingwen

2018-06-01

Fe-containing nanoparticles are of a high mass fraction in the as-grown carbon nanotube (CNT) network. By controlling the S-to-Fe atom ratio in the growth feedstock and introducing water as a weak oxidant, highly crystalline few-walled CNT network can be obtained, with a mass fraction of over 20 wt% for the Fe-containing nanoparticles. We report here an electron-oxidation-based purification method to efficiently remove the Fe-containing nanoparticles without inducing clear damage to either the assembly structure or the tube crystallinity. The purification could increase the ratio between Raman D and G peak intensities slightly from 0.08 to 0.12, decrease the specific conductivity from 0.31 to 0.24 S m2/g and the Fe content from >20 wt% to ≈1 wt%, and modify the capacitance just by about 13 F/g. All these indicate that the CNT network was well maintained by such gentle electro-oxidation-based purification. In addition, the purified CNT network can exhibit advantages in mechanical and electrical applications.

Silver nanocluster catalytic microreactors for water purification

NASA Astrophysics Data System (ADS)

Da Silva, B.; Habibi, M.; Ognier, S.; Schelcher, G.; Mostafavi-Amjad, J.; Khalesifard, H. R. M.; Tatoulian, M.; Bonn, D.

2016-07-01

A new method for the elaboration of a novel type of catalytic microsystem with a high specific area catalyst is developed. A silver nanocluster catalytic microreactor was elaborated by doping a soda-lime glass with a silver salt. By applying a high power laser beam to the glass, silver nanoclusters are obtained at one of the surfaces which were characterized by BET measurements and AFM. A microfluidic chip was obtained by sealing the silver coated glass with a NOA 81 microchannel. The catalytic activity of the silver nanoclusters was then tested for the efficiency of water purification by using catalytic ozonation to oxidize an organic pollutant. The silver nanoclusters were found to be very stable in the microreactor and efficiently oxidized the pollutant, in spite of the very short residence times in the microchannel. This opens the way to study catalytic reactions in microchannels without the need of introducing the catalyst as a powder or manufacturing complex packed bed microreactors.

Enzymatic production of single-molecule FISH and RNA capture probes.

PubMed

Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

2017-10-01

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Preparative isolation and purification of three stilbene glycosides from the tibetan medicinal plant Rheum tanguticum maxim. Ex Balf. by high-speed counter-current chromatography.

PubMed

Zhao, Xiao-Hui; Han, Fa; Li, Yu-Lin; Yue, Hui-Lan

2013-02-01

Stilbene glycosides are the primary constituents of Rheum tanguticum Maxim. ex Balf., to which different bioactivities has been attributed, including: anti-HIV, anti-oxidant, anti-tumour, anti-malarial, and anti-allergy activity. However, effective methods for the isolation and purification of stilbene glycosides, such as trans-rhapontin, cis-rhapontin and trans-desoxyrhaponticin, from this herb are not currently available. To develop an efficient method for the preparative isolation and purification of three stilbene glycosides from Rheum tanguticum Maxim. ex Balf. via high-speed counter-current chromatography (HSCCC). A solvent system composed of chloroform:n-butanol:methanol:water (4:1:3:2, v/v/v/v) was developed for the separation. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase. The flow rate was 1.8 mL/min. The apparatus was controlled at 800 rpm and 25 °C, and the effluent was monitored at 280 nm. Chemical constituents were analysed by high-performance liquid chromatography (HPLC), and their structures were identified by ¹H- and ¹³C-NMR. Under the optimised conditions, 25.5 mg trans-rhapontin, 16.0 mg cis-rhapontin and 20.5 mg trans-desoxyrhaponticin were separated from 80 mg crude sample; the isolates had purities of 99.6, 97.2 and 99.2%, respectively. A simple and efficient HSCCC method has been optimised for the preparative separation of stilbene glycosides from Rheum tanguticum Maxim. ex Balf. Copyright © 2012 John Wiley & Sons, Ltd.

A Method that Will Captivate U.

PubMed

Martin, Sophie; Coller, Jeff

2015-09-03

In an age of next-generation sequencing, the ability to purify RNA transcripts has become a critical issue. In this issue, Duffy et al. (2015) improve on a pre-existing technique of RNA labeling and purification by 4-thiouridine tagging. By increasing the efficiency of RNA capture, this method will enhance the ability to study RNA dynamics, especially for transcripts normally inefficiently captured by previous methods. Copyright © 2015 Elsevier Inc. All rights reserved.

Feasibility of continuous-variable quantum key distribution with noisy coherent states

DOE Office of Scientific and Technical Information (OSTI.GOV)

Usenko, Vladyslav C.; Department of Optics, Palacky University, CZ-772 07 Olomouc; Filip, Radim

2010-02-15

We address security of the quantum key distribution scheme based on the noisy modulation of coherent states and investigate how it is robust against noise in the modulation regardless of the particular technical implementation. As the trusted preparation noise is shown to be security breaking even for purely lossy channels, we reveal the essential difference between two types of trusted noise, namely sender-side preparation noise and receiver-side detection noise, the latter being security preserving. We consider the method of sender-side state purification to compensate the preparation noise and show its applicability in the realistic conditions of channel loss, untrusted channelmore » excess noise, and trusted detection noise. We show that purification makes the scheme robust to the preparation noise (i.e., even the arbitrary noisy coherent states can in principle be used for the purpose of quantum key distribution). We also take into account the effect of realistic reconciliation and show that the purification method is still efficient in this case up to a limited value of preparation noise.« less

Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

PubMed Central

Lim, Natalie Y. N.; Roco, Constance A.; Frostegård, Åsa

2016-01-01

Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of “representative samples” is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis. PMID:27803690

Size-amplified acoustofluidic separation of circulating tumor cells with removable microbeads

NASA Astrophysics Data System (ADS)

Liu, Huiqin; Ao, Zheng; Cai, Bo; Shu, Xi; Chen, Keke; Rao, Lang; Luo, Changliang; Wang, Fu-Bin; Liu, Wei; Bondesson, Maria; Guo, Shishang; Guo, Feng

2018-06-01

Isolation and analysis of rare circulating tumor cells (CTCs) is of great interest in cancer diagnosis, prognosis, and treatment efficacy evaluation. Acoustofluidic cell separation becomes an attractive method due to its contactless, noninvasive, simple, and versatile features. However, the indistinctive physical difference between CTCs and normal blood cells limits the purity of CTCs using current acoustic methods. Herein, we demonstrate a size-amplified acoustic separation and release of CTCs with removable microbeads. CTCs selectively bound to size-amplifiers (40 μm-diameter anti-EpCAM/gelatin-coated SiO2 microbeads) have significant physical differences (size and mechanics) compared to normal blood cells, resulting in an amplification of acoustic radiation force approximately a hundredfold over that of bare CTCs or normal blood cells. Therefore, CTCs can be efficiently sorted out with size-amplifiers in a traveling surface acoustic wave microfluidic device and released from size-amplifiers by enzymatic degradation for further purification or downstream analysis. We demonstrate a cell separation from blood samples with a total efficiency (E total) of ∼ 77%, purity (P) of ∼ 96%, and viability (V) of ∼83% after releasing cells from size-amplifiers. Our method substantially improves the emerging application of rare cell purification for translational medicine.

Near-Zero Emissions Oxy-Combustion Flue Gas Purification Task 2: SOx/Nox/Hg Removal for High Sulfur Coal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nick Degenstein; Minish Shah; Doughlas Louie

2012-05-01

The goal of this project is to develop a near-zero emissions flue gas purification technology for existing PC (pulverized coal) power plants that are retrofitted with oxy-combustion technology. The objective of Task 2 of this project was to evaluate an alternative method of SOx, NOx and Hg removal from flue gas produced by burning high sulfur coal in oxy-combustion power plants. The goal of the program was not only to investigate a new method of flue gas purification but also to produce useful acid byproduct streams as an alternative to using a traditional FGD and SCR for flue gas processing.more » During the project two main constraints were identified that limit the ability of the process to achieve project goals. 1) Due to boiler island corrosion issues >60% of the sulfur must be removed in the boiler island with the use of an FGD. 2) A suitable method could not be found to remove NOx from the concentrated sulfuric acid product, which limits sale-ability of the acid, as well as the NOx removal efficiency of the process. Given the complexity and safety issues inherent in the cycle it is concluded that the acid product would not be directly saleable and, in this case, other flue gas purification schemes are better suited for SOx/NOx/Hg control when burning high sulfur coal, e.g. this project's Task 3 process or a traditional FGD and SCR.« less

Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

PubMed

Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

2015-01-30

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

Experimental study and simulation of phosphorus purification effects of bioretention systems on urban surface runoff

PubMed Central

Liang, Zheng; Li, Yajiao; Li, Peng; Jiang, Chunbo

2018-01-01

Excessive phosphorus (P) contributes to eutrophication by degrading water quality and limiting human use of water resources. Identifying economic and convenient methods to control soluble reactive phosphorus (SRP) pollution in urban runoff is the key point of rainwater management strategies. Through three series of different tests involving influencing factors, continuous operation and intermittent operation, this study explored the purification effects of bioretention tanks under different experimental conditions, it included nine intermittent tests, single field continuous test with three groups of different fillers (Fly ash mixed with sand, Blast furnace slag, and Soil), and eight intermittent tests with single filler (Blast furnace slag mixed with sand). Among the three filler combinations studied, the filler with fly ash mixed with sand achieved the best pollution reduction efficiency. The setting of the submerged zone exerted minimal influence on the P removal of the three filler combinations. An extension of the dry period slightly promoted the P purification effect. The combination of fly ash mixed with sand demonstrated a positive purification effect on SRP during short- or long-term simulated rainfall duration. Blast furnace slag also presented a positive purification effect in the short term, although its continuous purification effect on SRP was poor in the long term. The purification abilities of soil in the short and long terms were weak. Under intermittent operations across different seasons, SRP removal was unstable, and effluent concentration processes were different. The purification effect of the bioretention system on SRP was predicted through partial least squares regression (PLS) modeling analysis. The event mean concentration removal of SRP was positively related to the adsorption capacity of filler and rainfall interval time and negatively related to submerged zones, influent concentration and volume. PMID:29742120

Effects of L-arginine on solubilization and purification of plant membrane proteins.

PubMed

Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

2011-11-01

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

Halophilic viruses with varying biochemical and biophysical properties are amenable to purification with asymmetrical flow field-flow fractionation.

PubMed

Eskelin, Katri; Lampi, Mirka; Meier, Florian; Moldenhauer, Evelin; Bamford, Dennis H; Oksanen, Hanna M

2017-11-01

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 µm, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.

PubMed

Moen, Aina E F; Tannæs, Tone M; Vatn, Simen; Ricanek, Petr; Vatn, Morten Harald; Jahnsen, Jørgen

2016-06-28

Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.

A new non-degradative method to purify glycogen.

PubMed

Tan, Xinle; Sullivan, Mitchell A; Gao, Fei; Li, Shihan; Schulz, Benjamin L; Gilbert, Robert G

2016-08-20

Liver glycogen, a complex branched glucose polymer containing a small amount of protein, is important for maintaining glucose homeostasis (blood-sugar control) in humans. It has recently been found that glycogen molecular structure is impaired in diabetes. Isolating the carbohydrate polymer and any intrinsically-attached protein(s) is an essential prerequisite for studying this structural impairment. This requires an effective, non-degradative and efficient purification method to exclude the many other proteins present in liver. Proteins and glycogen have different ranges of molecular sizes. Despite the plethora of proteins that might still be present in significant abundance after other isolation techniques, SEC (size exclusion chromatography, also known as GPC), which separates by molecular size, should separate those extraneous to glycogen from glycogen with any intrinsically associated protein(s). A novel purification method is developed for this, based on preparative SEC following sucrose gradient centrifugation. Proteomics is used to show that the new method compares favourably with current methods in the literature. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rapid screening, identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC-ESI-TOF-MS combined with semipreparative HPLC.

PubMed

Zhang, Minmin; Zhao, Hengqiang; Zhao, Zhiguo; Yan, Huijiao; Lv, Ruimin; Cui, Li; Yuan, Jinpeng; Wang, Daijie; Geng, Yanling; Liu, Daicheng; Wang, Xiao

2016-06-01

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti-influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti-influenza components from Zicao. Semipreparative high-performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high-performance liquid chromatography with the purity over 98% for all of them by high-performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β-dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti-influenza active ingredients from complex Chinese herbal medicines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hyperentanglement purification using imperfect spatial entanglement.

PubMed

Wang, Tie-Jun; Mi, Si-Chen; Wang, Chuan

2017-02-06

As the interaction between the photons and the environment which will make the entangled photon pairs in less entangled states or even in mixed states, the security and the efficiency of quantum communication will decrease. We present an efficient hyperentanglement purification protocol that distills nonlocal high-fidelity hyper-entangled Bell states in both polarization and spatial-mode degrees of freedom from ensembles of two-photon system in mixed states using linear optics. Here, we consider the influence of the photon loss in the channel which generally is ignored in the conventional entanglement purification and hyperentanglement purification (HEP) schemes. Compared with previous HEP schemes, our HEP scheme decreases the requirement for nonlocal resources by employing high-dimensional mode-check measurement, and leads to a higher fidelity, especially in the range where the conventional HEP schemes become invalid but our scheme still can work.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

PubMed

Brett, Sabine I; Lucien, Fabrice; Guo, Charles; Williams, Karla C; Kim, Yohan; Durfee, Paul N; Brinker, C J; Chin, Joseph I; Yang, Jun; Leong, Hon S

2017-05-01

The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches. © 2017 Wiley Periodicals, Inc.

Purification of crude glycerol from transesterification reaction of palm oil using direct method and multistep method

NASA Astrophysics Data System (ADS)

Nasir, N. F.; Mirus, M. F.; Ismail, M.

2017-09-01

Crude glycerol which produced from transesterification reaction has limited usage if it does not undergo purification process. It also contains excess methanol, catalyst and soap. Conventionally, purification method of the crude glycerol involves high cost and complex processes. This study aimed to determine the effects of using different purification methods which are direct method (comprises of ion exchange and methanol removal steps) and multistep method (comprises of neutralization, filtration, ion exchange and methanol removal steps). Two crude glycerol samples were investigated; the self-produced sample through the transesterification process of palm oil and the sample obtained from biodiesel plant. Samples were analysed using Fourier Transform Infrared Spectroscopy, Gas Chromatography and High Performance Liquid Chromatography. The results of this study for both samples after purification have showed that the pure glycerol was successfully produced and fatty acid salts were eliminated. Also, the results indicated the absence of methanol in both samples after purification process. In short, the combination of 4 purification steps has contributed to a higher quality of glycerol. Multistep purification method gave a better result compared to the direct method as neutralization and filtration steps helped in removing most excess salt, fatty acid and catalyst.

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification.

PubMed

Mayer, Daniel; Baginsky, Sacha; Schwemmle, Martin

2005-11-01

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

The bubble method of water purification

NASA Astrophysics Data System (ADS)

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.

Drawbacks of Dialysis Procedures for Removal of EDTA

PubMed Central

Mónico, Andreia; Martínez-Senra, Eva; Cañada, F. Javier; Zorrilla, Silvia

2017-01-01

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent commonly used in protein purification, both to eliminate contaminating divalent cations and to inhibit protease activity. For a number of subsequent applications EDTA needs to be exhaustively removed. Most purification methods rely in extensive dialysis and/or gel filtration in order to exchange or remove protein buffer components, including metal chelators. We report here that dialysis protocols, even as extensive as those typically employed for protein refolding, may not effectively remove EDTA, which is reduced only by approximately two-fold and it also persists after spin-column gel filtration, as determined by NMR and by colorimetric methods. Remarkably, the most efficient removal was achieved by ultrafiltration, after which EDTA became virtually undetectable. These results highlight a potentially widespread source of experimental variability affecting free divalent cation concentrations in protein applications. PMID:28099451

An effective purification method using large bottles for human pancreatic islet isolation

PubMed Central

Shimoda, Masayuki; Itoh, Takeshi; Iwahashi, Shuichi; Takita, Morihito; Sugimoto, Koji; Kanak, Mazhar A.; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F.; Grayburn, Paul A.; Matsumoto, Shinichi

2012-01-01

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification. PMID:23221740

A sparse matrix-vector multiplication based algorithm for accurate density matrix computations on systems of millions of atoms

NASA Astrophysics Data System (ADS)

Ghale, Purnima; Johnson, Harley T.

2018-06-01

We present an efficient sparse matrix-vector (SpMV) based method to compute the density matrix P from a given Hamiltonian in electronic structure computations. Our method is a hybrid approach based on Chebyshev-Jackson approximation theory and matrix purification methods like the second order spectral projection purification (SP2). Recent methods to compute the density matrix scale as O(N) in the number of floating point operations but are accompanied by large memory and communication overhead, and they are based on iterative use of the sparse matrix-matrix multiplication kernel (SpGEMM), which is known to be computationally irregular. In addition to irregularity in the sparse Hamiltonian H, the nonzero structure of intermediate estimates of P depends on products of H and evolves over the course of computation. On the other hand, an expansion of the density matrix P in terms of Chebyshev polynomials is straightforward and SpMV based; however, the resulting density matrix may not satisfy the required constraints exactly. In this paper, we analyze the strengths and weaknesses of the Chebyshev-Jackson polynomials and the second order spectral projection purification (SP2) method, and propose to combine them so that the accurate density matrix can be computed using the SpMV computational kernel only, and without having to store the density matrix P. Our method accomplishes these objectives by using the Chebyshev polynomial estimate as the initial guess for SP2, which is followed by using sparse matrix-vector multiplications (SpMVs) to replicate the behavior of the SP2 algorithm for purification. We demonstrate the method on a tight-binding model system of an oxide material containing more than 3 million atoms. In addition, we also present the predicted behavior of our method when applied to near-metallic Hamiltonians with a wide energy spectrum.

Recent progress in the applications of layer-by-layer assembly to the preparation of nanostructured ion-rejecting water purification membranes.

PubMed

Sanyal, Oishi; Lee, Ilsoon

2014-03-01

Reverse osmosis (RO) and nanofiltration (NF) are the two dominant membrane separation processes responsible for ion rejection. While RO is highly efficient in removal of ions it needs a high operating pressure and offers very low selectivity between ions. Nanofiltration on the other hand has a comparatively low operating pressure and most commercial membranes offer selectivity in terms of ion rejection. However in many nanofiltration operations rejection of monovalent ions is not appreciable. Therefore a high flux high rejection membrane is needed that can be applied to water purification systems. One such alternative is the usage of polyelectrolyte multilayer membranes that are prepared by the deposition of alternately charged polyelectrolytes via layer-by-layer (LbL) assembly method. LbL is one of the most common self-assembly techniques and finds application in various areas. It has a number of tunable parameters like deposition conditions, number of bilayers deposited etc. which can be manipulated as per the type of application. This technique can be applied to make a nanothin membrane skin which gives high rejection and at the same time allow a high water flux across it. Several research groups have applied this highly versatile technique to prepare membranes that can be employed for water purification. Some of these membranes have shown better performance than the commercial nanofiltration and reverse osmosis membranes. These membranes have the potential to be applied to various different aspects of water treatment like water softening, desalination and recovery of certain ions. Besides the conventional method of LbL technique other alternative methods have also been suggested that can make the technique fast, more efficient and thereby make it more commercially acceptable.

Californium purification and electrodeposition

DOE PAGES

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

2014-11-30

The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of themore » feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO 3 and HCl, and Eichrom TEVA resin in NH 4SCN. The TEVA NH 4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.« less

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

PubMed

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch; Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève; Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yieldedmore » 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic –activated cell sorting step.« less

Innovative Process for Comprehensive Treatment of Liquid Radioactive Waste - 12551

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penzin, R.A.; Sarychev, G.A.

This paper presents the results of research activities aimed at creation of a principally new LRW distilling treatment method. The new process is based on the instantaneous evaporation method widely used in distillation units. The main difference of the proposed process is that the vapor condensation is conducted without using heat exchangers in practically ideal mode by way of direct contacting in a vapor-liquid system. This process is conducted in a specially designed ejector unit in supersonic mode. Further recuperation of excess heat of vaporization is carried out in a standard heat exchanger. Such an arrangement of the process, togethermore » with use of the barometric height principle, allows to carry out LRW evaporation under low temperatures, which enables to use excess heat from NPS for heating initial LRW. Thermal calculations and model experiments have revealed that, in this case, the expenditure of energy for LRW treatment by distilling will not exceed 3 kilowatt-hour/m{sup 3}, which is comparable with the reverse-osmosis desalination method. Besides, the proposed devices are 4 to 5 times less metal-intensive than standard evaporation units. These devices are also characterized by versatility. Experiments have revealed that the new method can be used for evaporation of practically any types of LRW, including those containing a considerable amount of oil products. Owing to arrangement of the evaporation process at low temperatures, the new devices are not sensitive to 'scale formation'. This is why, they can be used for concentrating brines of up to 500-600 g/l. New types of such evaporating devices can be required both for LRW treatment processes at nuclear-power plants under design and for treating 'non-standard' LRW with complex physicochemical and radionuclide composition resulting from the disaster at the Fukushima I Nuclear Power Plant.) As a result of accidents at nuclear energy objects, as it has recently happened at NPP 'Fukushima-1', personnel faces the necessity to take emergency measures and to use marine water for cooling of reactor zone in contravention of the technological regulations. In these cases significant amount of liquid radioactive wastes of complex physicochemical composition is being generated, the purification of which by traditional methods is close to impossible. According to the practice of elimination of the accident after-effects at NPP 'Fukushima' there are still no technical means for the efficient purification of liquid radioactive wastes of complex composition like marine water from radionuclides. Therefore development of state-of-the-art highly efficient facilities capable of fast and safe purification of big amounts of liquid radioactive wastes of complex physicochemical composition from radionuclides turns to be utterly topical problem. Cesium radionuclides, being extremely dangerous for the environment, present over 90% of total radioactivity contained in liquid radioactive wastes left as a result of accidents at nuclear power objects. For the purpose of radiation accidents aftereffects liquidation VNIIHT proposes to create a plant for LRW reprocessing, consisting of 4 major technological modules: Module of LRW pretreatment to remove mechanical and organic impurities including oil products; Module of sorption purification of LWR by means of selective inorganic sorbents; Module of reverse osmotic purification and desalination; Module of deep evaporation of LRW concentrates. The first free modules are based on completed technological and designing concepts implemented by VNIIHT in the framework of LLRW Project in the period of 2000-2001 in Russia for comprehensive treatment of LWR of atomic fleet. These industrial plants proved to be highly efficient and secure during their long operation life. Module of deep evaporation is a new technological development. It will ensure conduction of evaporation and purification of LRW of different physicochemical composition, including those containing hardness salts, resulted in generation of LRW concentrate 300-600 g/l. The method is based on utilization of supersonic ejector for intensification of thermal physic processes and performance of evaporation in brine recycling mode. All proposed technological solutions are totally based on patented Russian developments. Proposed work will allow to construct modular plants, which will be totally prepared for efficient purification of any types of liquid radioactive wastes from radionuclides in case of force majeure. According to proposed scheme concentration level of cesium radionuclides in safe-for-storage form will make up not less than 5000. With respect to purification from cesium radionuclides of liquid radioactive wastes stored at NPP 'Fukushima' about 10 t of inorganic sorbents, loaded in 160 protective filter-containers, will be required for solving this problem. The amount of secondary wastes will be reduced approximately in 5 times in comparison with traditional schemes, applied in purification of secondary LRW of Fukushima-1 by Areva (France) and Kurion (USA) companies. All units of modular plants will be constructed and manufactured as totally automated, providing their twenty-four-hour safe operation. Modular design will ensure efficiency and let optimize the costs of secondary LRW treatment. In order to ensure off-line operation in emergency conditions the plant should be equipped with auxiliary modules: energy and ventilation ones. Under normal conditions these modules can be stored in 'mothballed' condition at special warehouses under the authority of federal bodies. It will be reasonable to choose required transport facilities, the most suitable for transportation of modules to target destination beforehand, using vessel classification list.« less

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

PubMed

Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

2015-01-01

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling.

PubMed

Mao, Shihong; Goodrich, Robert J; Hauser, Russ; Schrader, Steven M; Chen, Zhen; Krawetz, Stephen A

2013-10-01

Different semen storage and sperm purification methods may affect the integrity of isolated spermatozoal RNA. RNA-Seq was applied to determine whether semen storage methods (pelleted vs. liquefied) and somatic cell lysis buffer (SCLB) vs. PureSperm (PS) purification methods affect the quantity and quality of sperm RNA. The results indicate that the method of semen storage does not markedly impact RNA profiling whereas the choice of purification can yield significant differences. RNA-Seq showed that the majority of mitochondrial and mid-piece associated transcripts were lost after SCLB purification, which indicated that the mid-piece of spermatozoa may have been compromised. In addition, the number of stable transcript pairs from SCLB-samples was less than that from the PS samples. This study supports the view that PS purification better maintains the integrity of spermatozoal RNAs.

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

PubMed Central

Pua, Teen-Lee; Chan, Xiao Ying; Loh, Hwei-San; Omar, Abdul Rahman; Yusibov, Vidadi; Musiychuk, Konstantin; Hall, Alexandra C.; Coffin, Megan V.; Shoji, Yoko; Chichester, Jessica A.; Bi, Hong; Streatfield, Stephen J.

2017-01-01

ABSTRACT Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance. PMID:27929750

Online hyphenation of extraction, Sephadex LH-20 column chromatography, and high-speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step.

PubMed

Zhang, Ying-Qi; Wang, Shan-Shan; Han, Chao; Xu, Jin-Fang; Luo, Jian-Guang; Kong, Ling-Yi

2017-12-01

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH-20 column chromatography combined with high-speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH-20 column directly to cut the nontarget fractions followed by the second-dimensional high-speed countercurrent chromatography, hyphenated by a six-port valve equipped at the post-end of Sephadex LH-20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Combined Negative and Positive Enrichment Assay for Cancer Cells Isolation and Purification.

PubMed

Cheng, Boran; Wang, Shuyi; Chen, Yuanyuan; Fang, Yuan; Chen, Fangfang; Wang, Zhenmeng; Xiong, Bin

2016-02-01

Cancer cells that detach from solid tumor and circulate in the peripheral blood (CTCs) have been considered as a new "biomarker" for the detection and characterization of cancers. However, isolating and detecting cancer cells from the cancer patient peripheral blood have been technically challenging, owing to the small sub-population of CTCs (a few to hundreds per milliliter). Here we demonstrate a simple and efficient cancer cells isolation and purification method. A biocompatible and surface roughness controllable TiO2 nanofilm was deposited onto a glass slide to achieve enhanced topographic interactions with nanoscale cellular surface components, again, anti-CD45 (a leukocyte common antigen) and anti-EpCAM (epithelial cell adhesion molecule) were then coated onto the surface of the nanofilm for advance depletion of white blood cells (WBCs) and specific isolation of CTCs, respectively. Comparing to the conventional positive enrichment technology, this method exhibited excellent biocompatibility and equally high capture efficiency. Moreover, the maximum number of background cells (WBCs) was removed, and viable and functional cancer cells were isolated with high purity. Utilizing the horizontally packed TiO2 nanofilm improved pure CTC-capture through combining cell-capture-agent and cancer cell-preferred nanoscale topography, which represented a new method capable of obtaining biologically functional CTCs for subsequent molecular analysis. © The Author(s) 2014.

Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.

PubMed

Bright, G R; Spooner, B S

1983-06-01

The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

PubMed Central

Lezin, George; Kuehn, Michael R.; Brunelli, Luca

2011-01-01

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

Efficient and low-cost alternative of lipase concentration aiming at the application in the treatment of waste cooking oils.

PubMed

Preczeski, Karina P; Kamanski, Angela B; Scapini, Thamarys; Camargo, Aline F; Modkoski, Tatiani A; Rossetto, Vanusa; Venturin, Bruno; Mulinari, Jéssica; Golunski, Simone M; Mossi, Altemir J; Treichel, Helen

2018-06-01

In this study, we evaluated the concentration of lipases from Aspergillus niger using efficient and low-cost methods aiming at application in the treatment of waste cooking oils. The change in ionic strength of the medium by the addition of salt and precipitation with ethanol increased the specific activity from 2.90 to 28.50 U/mg, resulting in a purification factor of 9.82-fold. The use of acetone resulted in a specific activity of 33.63 U/mg, resulting in a purification factor of 11.60-fold. After that, the concentrated lipase was used in the hydrolysis of waste cooking oil and 753.07 and 421.60 µmol/mL of free fatty acids were obtained for the enzyme precipitated with ethanol and acetone, respectively. The hydrolysis of waste cooking oil catalyzed by homemade purified lipase in ultrasonic media can be considered a pretreatment of oil by converting a significant amount of triglycerides into free fatty acids.

In-tube electro-membrane extraction with a sub-microliter organic solvent consumption as an efficient technique for synthetic food dyes determination in foodstuff samples.

PubMed

Bazregar, Mohammad; Rajabi, Maryam; Yamini, Yadollah; Asghari, Alireza; Abdossalami asl, Yousef

2015-09-04

A simple and efficient extraction technique with a sub-microliter organic solvent consumption termed as in-tube electro-membrane extraction (IEME) is introduced. This method is based upon the electro-kinetic migration of ionized compounds by the application of an electrical potential difference. For this purpose, a thin polypropylene (PP) sheet placed inside a tube acts as a support for the membrane solvent, and 30μL of an aqueous acceptor solution is separated by this solvent from 1.2mL of an aqueous donor solution. This method yielded high extraction recoveries (63-81%), and the consumption of the organic solvent used was only 0.5μL. By performing this method, the purification is high, and the utilization of the organic solvent, used as a mediator, is very simple and repeatable. The proposed method was evaluated by extraction of four synthetic food dyes (Amaranth, Ponceau 4R, Allura Red, and Carmoisine) as the model analytes. Optimization of variables affecting the method was carried out in order to achieve the best extraction efficiency. These variables were the type of membrane solvent, applied extraction voltage, extraction time, pH range, and concentration of salt added. Under the optimized conditions, IEME-HPLC-UV provided a good linearity in the range of 1.00-800ngmL(-1), low limits of detection (0.3-1ngmL(-1)), and good extraction repeatabilities (RSDs below 5.2%, n=5). It seems that this design is a proper one for the automation of the method. Also the consumption of the organic solvent in a sub-microliter scale, and its simplicity, high efficiency, and high purification can help one getting closer to the objectives of the green chemistry. Copyright © 2015 Elsevier B.V. All rights reserved.

The modified swirl sedimentation tanks for water purification.

PubMed

Ochowiak, Marek; Matuszak, Magdalena; Włodarczak, Sylwia; Ancukiewicz, Małgorzata; Krupińska, Andżelika

2017-03-15

This paper discusses design, evaluation, and application for the use of swirl/vortex technologies as liquid purification system. A study was performed using modified swirl sedimentation tanks. The vortex separators (OW, OWK, OWR and OWKR) have been studied under laboratory conditions at liquid flow rate from 2.8⋅10 -5 to 5.1⋅10 -4 [m 3 /s]. The pressure drop and the efficiency of purification of liquid stream were analyzed. The suspended particles of different diameters were successfully removed from liquid with the application of swirl chambers of proposed constructions. It was found that damming of liquid in the tank increases alongside liquid stream at the inlet and depends on the tank construction. The efficiency of the sedimentation tanks increases alongside the diameters of solid particles and decrease in the liquid flow rate. The best construction proved to be the OWR sedimentation tank due to smallest liquid damming, even at high flow rates, and the highest efficiency of the purification liquid stream for solid particles of the smallest diameter. The proposed solution is an alternative to the classical constructions of sedimentation tanks. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation Of A Powder-Free DNA Extraction Method For Skeletal Remains.

PubMed

Harrel, Michelle; Mayes, Carrie; Gangitano, David; Hughes-Stamm, Sheree

2018-02-07

Bones are often recovered in forensic investigations, including missing persons and mass disasters. While traditional DNA extraction methods rely on grinding bone into powder prior to DNA purification, the TBone Ex buffer (DNA Chip Research Inc.) digests bone chips without powdering. In this study, six bones were extracted using the TBone Ex kit in conjunction with the PrepFiler ® BTA™ DNA extraction kit (Thermo Fisher Scientific) both manually and via an automated platform. Comparable amounts of DNA were recovered from a 50 mg bone chip using the TBone Ex kit and 50 mg of powdered bone with the PrepFiler ® BTA™ kit. However, automated DNA purification decreased DNA yield (p < 0.05). Nevertheless, short tandem repeat (STR) success was comparable across all methods tested. This study demonstrates that digestion of whole bone fragments is an efficient alternative to powdering bones for DNA extraction without compromising downstream STR profile quality. © 2018 American Academy of Forensic Sciences.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yacoby, I.; Tegler, L. T.; Pochekailov, S.

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,more » 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.« less

Optimized anion exchange column isolation of zirconium-89 ( 89 Zr) from yttrium cyclotron target: Method development and implementation on an automated fluidic platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

O’Hara, Matthew J.; Murray, Nathaniel J.; Carter, Jennifer C.

Zirconium-89 (89Zr), produced by the (p,n) reaction from naturally monoisotopic yttrium (natY), is a promising positron emitting isotope for immunoPET imaging. Its long half-life of 78.4 h is sufficient for evaluating slow physiological processes. A prototype automated fluidic system, coupled to on-line and in-line detectors, has been constructed to facilitate development of new 89Zr purification methodologies. The highly reproducible reagent delivery platform and near-real time monitoring of column effluents allows for efficient method optimization. The separation of Zr from dissolved Y metal targets was evaluated using several anion exchange resins. Each resin was evaluated against its ability to quantitatively capturemore » Zr from a load solution that is high in dissolved Y. The most appropriate anion exchange resin for this application was identified, and the separation method was optimized. The method is capable of a high Y decontamination factor (>105) and has been shown to separate Fe, an abundant contaminant in Y foils, from the 89Zr elution fraction. Finally, the performance of the method was evaluated using cyclotron bombarded Y foil targets. The separation method was shown to achieve >95% recovery of the 89Zr present in the foils. The 89Zr eluent, however, was in a chemical matrix not immediately conducive to labeling onto proteins. The main intent of this study was to develop a tandem column 89Zr purification process, wherein the anion exchange column method described here is the first separation in a dual-column purification process.« less

Study on a new water purification equipment with spiral lamellas

NASA Astrophysics Data System (ADS)

Feng, X. R.

2017-08-01

A new water purification equipment was introduced, especially the section of spiral lamellas. Utilization of spiral lamellas made the sedimentation space reach to 100%, not only improving sedimentation efficiency and reducing the cover space, but also saving investment. Production test results showed that the new water purification equipment with spiral lamellas had characteristics of excellent treatment efficiency and high shock resistant capacity. As the treatment water volume was 240 m3/d, when the turbidity, CODMn and UV254 were 203 NTU, 1.90 mg/L and 0.030 cm-1 in raw water, they were 0.32 NTU, 0.72mg/L and 0.011 cm-1 respectively in effluent water, which could fully meet the drinking water hygiene requirement.

Pesticide removal from waste spray-tank water by organoclay adsorption after field application: an approach for a formulation of cyprodinil containing antifoaming/defoaming agents.

PubMed

Suciu, Nicoleta A; Ferrari, Tommaso; Ferrari, Federico; Trevisan, Marco; Capri, Ettore

2012-05-01

Many reports on purification of water containing pesticides are based on studies using unformulated active ingredients. However, most commercial formulations contain additives/adjuvants or are manufactured using microencapsulation which may influence the purification process. Therefore, the main objective of this work was to develop and test a pilot scheme for decontaminating water containing pesticides formulated with antifoaming/defoaming agents. The Freundlich adsorption coefficients of formulation of cyprodinil, a new-generation fungicide, onto the organoclay Cloisite 20A have been determined in the laboratory in order to predict the efficiency of this organoclay in removing the fungicide from waste spray-tank water. Subsequently, the adsorption tests were repeated in the pilot system in order to test the practical operation of the purification scheme. The laboratory adsorption tests successfully predicted the efficiency of the pilot purification system, which removed more than 96% cyprodinil over a few hours. The passing of the organoclay-cyprodinil suspension through a layer of biomass gave 100% recovery of the organoclay at the surface of the biomass after 1 week. The organoclay was composted after the treatment to try to break down the fungicide so as to allow safe disposal of the waste, but cyprodinil was not significantly dissipated after 90 days. The purification scheme proved to be efficient for decontaminating water containing cyprodinil formulated with antifoaming/defoaming agents, but additional treatments for the adsorbed residues still appear to be necessary even for a moderately persistent pesticide such as cyprodinil. Furthermore, a significant conclusion of this study concerns the high influence of pesticide formulations on the process of purification of water containing these compounds, which should be taken into account when developing innovative decontamination schemes, especially for practical applications.

The MIMIC Method with Scale Purification for Detecting Differential Item Functioning

ERIC Educational Resources Information Center

Wang, Wen-Chung; Shih, Ching-Lin; Yang, Chih-Chien

2009-01-01

This study implements a scale purification procedure onto the standard MIMIC method for differential item functioning (DIF) detection and assesses its performance through a series of simulations. It is found that the MIMIC method with scale purification (denoted as M-SP) outperforms the standard MIMIC method (denoted as M-ST) in controlling…

Membrane-Based Technologies in the Pharmaceutical Industry and Continuous Production of Polymer-Coated Crystals/Particles.

PubMed

Chen, Dengyue; Sirkar, Kamalesh K; Jin, Chi; Singh, Dhananjay; Pfeffer, Robert

2017-01-01

Membrane technologies are of increasing importance in a variety of separation and purification applications involving liquid phases and gaseous mixtures. Although the most widely used applications at this time are in water treatment including desalination, there are many applications in chemical, food, healthcare, paper and petrochemical industries. This brief review is concerned with existing and emerging applications of various membrane technologies in the pharmaceutical and biopharmaceutical industry. The goal of this review article is to identify important membrane processes and techniques which are being used or proposed to be used in the pharmaceutical and biopharmaceutical operations. How novel membrane processes can be useful for delivery of crystalline/particulate drugs is also of interest. Membrane separation technologies are extensively used in downstream processes for bio-pharmaceutical separation and purification operations via microfiltration, ultrafiltration and diafiltration. Also the new technique of membrane chromatography allows efficient purification of monoclonal antibodies. Membrane filtration techniques of reverse osmosis and nanofiltration are being combined with bioreactors and advanced oxidation processes to treat wastewaters from pharmaceutical plants. Nanofiltration with organic solvent-stable membranes can implement solvent exchange and catalyst recovery during organic solvent-based drug synthesis of pharmaceutical compounds/intermediates. Membranes in the form of hollow fibers can be conveniently used to implement crystallization of pharmaceutical compounds. The novel crystallization methods of solid hollow fiber cooling crystallizer (SHFCC) and porous hollow fiber anti-solvent crystallization (PHFAC) are being developed to provide efficient methods for continuous production of polymer-coated drug crystals in the area of drug delivery. This brief review provides a general introduction to various applications of membrane technologies in the pharmaceutical/biopharmaceutical industry with special emphasis on novel membrane techniques for pharmaceutical applications. The method of coating a drug particle with a polymer using the SHFCC method is stable and ready for scale-up for operation over an extended period. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Application of alkaline solid residue of electric arc furnace dust for neutralization/purification of electroplating wastewaters.

PubMed

Elez, Loris; Orescanin, Visnja; Sofilic, Tahir; Mikulic, Nenad; Ruk, Damir

2008-10-01

The purpose of this work was development of an appropriate procedure for the neutralization/purification of electroplating wastewater (EWW) with alkaline solid residue (ASR) by-product of the alkaline extraction of zinc and lead from electric arc furnace dust (EAFD). Removal efficiency of ASR at optimum purification conditions (pH 8 and mixing time; 20 minutes) for the elements Pb, Cr (VI), Cr (III), Fe, Ni, Cu and Zn were 94.92%, 97.58%, 99.59%, 99.48%, 97.25% and 99.97%, respectively. The concentrations of all elements in the purified wastewater were significantly lower in relation to the upper permissible limit for wastewaters suitable for discharge into the environment. The remaining waste mud was regenerated in the strong alkaline medium and successfully applied once again for the neutralization/purification of EWW. Removal efficiencies of heavy metals accomplished with regenerated waste mud were comparable to these achieved by original ASR. Elemental concentrations in the leachates of the waste mud were in accordance with regulated values.

Oligomerization triggered by foldon: a simple method to enhance the catalytic efficiency of lichenase and xylanase.

PubMed

Wang, Xinzhe; Ge, Huihua; Zhang, Dandan; Wu, Shuyu; Zhang, Guangya

2017-07-03

Effective and simple methods that lead to higher enzymatic efficiencies are highly sough. Here we proposed a foldon-triggered trimerization of the target enzymes with significantly improved catalytic performances by fusing a foldon domain at the C-terminus of the enzymes via elastin-like polypeptides (ELPs). The foldon domain comprises 27 residues and can forms trimers with high stability. Lichenase and xylanase can hydrolyze lichenan and xylan to produce value added products and biofuels, and they have great potentials as biotechnological tools in various industrial applications. We took them as the examples and compared the kinetic parameters of the engineered trimeric enzymes to those of the monomeric and wild type ones. When compared with the monomeric ones, the catalytic efficiency (k cat /K m ) of the trimeric lichenase and xylanase increased 4.2- and 3.9- fold. The catalytic constant (k cat ) of the trimeric lichenase and xylanase increased 1.8- fold and 5.0- fold than their corresponding wild-type counterparts. Also, the specific activities of trimeric lichenase and xylanase increased by 149% and 94% than those of the monomeric ones. Besides, the recovery of the lichenase and xylanase activities increased by 12.4% and 6.1% during the purification process using ELPs as the non-chromatographic tag. The possible reason is the foldon domain can reduce the transition temperature of the ELPs. The trimeric lichenase and xylanase induced by foldon have advantages in the catalytic performances. Besides, they were easier to purify with increased purification fold and decreased the loss of activities compared to their corresponding monomeric ones. Trimerizing of the target enzymes triggered by the foldon domain could improve their activities and facilitate the purification, which represents a simple and effective enzyme-engineering tool. It should have exciting potentials both in industrial and laboratory scales.

Separation and purification of five alkaloids from Aconitum duclouxii by counter-current chromatography.

PubMed

Wang, Yarong; Cai, Shining; Chen, Yang; Deng, Liang; Zhou, Xumei; Liu, Jia; Xu, Xin; Xia, Qiang; Lin, Mao; Zhang, Jili; Huang, Weili; Wang, Wenjun; Xiang, Canhui; Cui, Guozhen; Du, Lianfeng; He, Huan; Qi, Baohui

2015-07-01

C19 -diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter-current chromatography. In the process of separation, the critical conditions of counter-current chromatography were optimized. The two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1, 24.1 mg of 2, 250.6 mg of 3, 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra-high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

PubMed Central

2010-01-01

Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research. PMID:20180960

Sensitivity of measurement-based purification processes to inner interactions

NASA Astrophysics Data System (ADS)

Militello, Benedetto; Napoli, Anna

2018-02-01

The sensitivity of a repeated measurement-based purification scheme to additional undesired couplings is analyzed, focusing on the very simple and archetypical system consisting of two two-level systems interacting with a repeatedly measured one. Several regimes are considered and in the strong coupling limit (i.e., when the coupling constant of the undesired interaction is very large) the occurrence of a quantum Zeno effect is proven to dramatically jeopardize the efficiency of the purification process.

Large-Scale Purification, Characterization, and Spore Outgrowth Inhibitory Effect of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361.

PubMed

Wang, Gaoyan; Manns, David C; Guron, Giselle K; Churey, John J; Worobo, Randy W

2014-06-01

Large-scale purification of the highly hydrophobic bacteriocin thurincin H was accomplished via a novel and simple two-step method: ammonia sulfate precipitation and C18 solid-phase extraction. The inhibition spectrum and stability of thurincin H as well as its antagonistic activity against Bacillus cereus F4552 spores were further characterized. In the purification method, secreted proteins contained in the supernatant of a 40 h incubated culture of B. thuringiensis SF361 were precipitated by 68 % ammonia sulfate and purified by reverse-phase chromatography, with a yield of 18.53 mg/l of pure thurincin H. Silver-stained SDS-PAGE, high-performance liquid chromatography, and liquid chromatography-mass spectrometry confirmed the high purity of the prepared sample. Thurincin H exhibited a broad antimicrobial activity against 22 tested bacterial strains among six different genera including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. There was no detectable activity against any of the selected yeast or fungi. The bacteriocin activity was stable for 30 min at 50 °C and decreased to undetectable levels within 10 min at temperatures above 80 °C. Thurincin H is also stable from pH 2-7 for at least 24 h at room temperature. Thurincin H is germicidal against B. cereus spores in brain heart infusion broth, but not in Tris-NaCl buffer. The efficient purification method enables the large-scale production of pure thurincin H. The broad inhibitory spectrum of this bacteriocin may be of interest as a potential natural biopreservative in the food industry, particularly in post-processed and ready-to-eat food.

Sodium purification apparatus and method

DOEpatents

Gould, Marc I. [Van Nuys, CA

1980-03-04

An apparatus for and method of collecting and storing oxide impurities contained in high-temperature liquid alkali metal. A method and apparatus are provided for nucleating and precipitating oxide impurities by cooling, wherein the nucleation and precipitation are enhanced by causing a substantial increase in pressure drop and corresponding change in the velocity head of the alkali metal. Thereafter the liquid alkali metal is introduced into a quiescent zone wherein the liquid velocity is maintained below a specific maximum whereby it is possible to obtain high oxide removal efficiencies without the necessity of a mesh or filter.

ELISA AND SOL-GEL BASED IMMUNOAFFINITY PURIFICATION OF THE PYRETHROID BIOALLETHRIN IN FOOD AND ENVIRONMENTAL SAMPLES

EPA Science Inventory

The peer-reviewed article describes the development of a new sol-gel based immunoaffinity purification procedure and an immunoassay for the pyrethroid bioallethrin. The immunoaffinity chromatography procedure was applied to food samples providing an efficient cleanup prior to im...

Magnetic purification of curcumin from Curcuma longa rhizome by novel naked maghemite nanoparticles.

PubMed

Magro, Massimiliano; Campos, Rene; Baratella, Davide; Ferreira, Maria Izabela; Bonaiuto, Emanuela; Corraducci, Vittorino; Uliana, Maíra Rodrigues; Lima, Giuseppina Pace Pereira; Santagata, Silvia; Sambo, Paolo; Vianello, Fabio

2015-01-28

Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids.

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli

PubMed Central

2013-01-01

Background Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. Results We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. Conclusion A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification. PMID:23638724

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli.

PubMed

Pandey, Neeraj; Sachan, Annapurna; Chen, Qi; Ruebling-Jass, Kristin; Bhalla, Ritu; Panguluri, Kiran Kumar; Rouviere, Pierre E; Cheng, Qiong

2013-05-02

Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

PubMed

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

TiO2-Based Advanced Oxidation Nanotechnologies For Water Purification And Reuse

EPA Science Inventory

TiO₂ photocatalysis, one of the UV-based advanced oxidation technologies (AOTs) and nanotechnologies (AONs), has attracted great attention for the development of efficient water treatment and purification systems due to the effectiveness of TiO₂ to generate ...

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

PubMed

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

2015-05-01

Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC. Copyright © 2014 John Wiley & Sons, Ltd.

Production, Purification and Preliminary X-ray Crystallographic Studies of Adeno-Associated Virus Serotype 9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, M.; Nam, H; Carter, A

2009-01-01

Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P32, with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetricmore » unit capsid have been determined by molecular-replacement methods and structure determination is in progress.« less

Bacterial Expression and Purification of the Amyloidogenic Peptide PAPf39 for Multidimensional NMR Spectroscopy

PubMed Central

Shanmuganathan, Aranganathan; Bishop, Anthony C.; French, Kinsley C.; McCallum, Scott A.; Makhatadze, George I.

2013-01-01

PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enable structural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression, as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus, and purification of uniformly labeled 13C- and 15N-labeled PAPf39 peptide. This allows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH. PMID:23314347

Design and function of biomimetic multilayer water purification membranes

PubMed Central

Ling, Shengjie; Qin, Zhao; Huang, Wenwen; Cao, Sufeng; Kaplan, David L.; Buehler, Markus J.

2017-01-01

Multilayer architectures in water purification membranes enable increased water throughput, high filter efficiency, and high molecular loading capacity. However, the preparation of membranes with well-organized multilayer structures, starting from the nanoscale to maximize filtration efficiency, remains a challenge. We report a complete strategy to fully realize a novel biomaterial-based multilayer nanoporous membrane via the integration of computational simulation and experimental fabrication. Our comparative computational simulations, based on coarse-grained models of protein nanofibrils and mineral plates, reveal that the multilayer structure can only form with weak interactions between nanofibrils and mineral plates. We demonstrate experimentally that silk nanofibril (SNF) and hydroxyapatite (HAP) can be used to fabricate highly ordered multilayer membranes with nanoporous features by combining protein self-assembly and in situ biomineralization. The production is optimized to be a simple and highly repeatable process that does not require sophisticated equipment and is suitable for scaled production of low-cost water purification membranes. These membranes not only show ultrafast water penetration but also exhibit broad utility and high efficiency of removal and even reuse (in some cases) of contaminants, including heavy metal ions, dyes, proteins, and other nanoparticles in water. Our biomimetic design and synthesis of these functional SNF/HAP materials have established a paradigm that could lead to the large-scale, low-cost production of multilayer materials with broad spectrum and efficiency for water purification, with applications in wastewater treatment, biomedicine, food industry, and the life sciences. PMID:28435877

Design and function of biomimetic multilayer water purification membranes.

PubMed

Ling, Shengjie; Qin, Zhao; Huang, Wenwen; Cao, Sufeng; Kaplan, David L; Buehler, Markus J

2017-04-01

Multilayer architectures in water purification membranes enable increased water throughput, high filter efficiency, and high molecular loading capacity. However, the preparation of membranes with well-organized multilayer structures, starting from the nanoscale to maximize filtration efficiency, remains a challenge. We report a complete strategy to fully realize a novel biomaterial-based multilayer nanoporous membrane via the integration of computational simulation and experimental fabrication. Our comparative computational simulations, based on coarse-grained models of protein nanofibrils and mineral plates, reveal that the multilayer structure can only form with weak interactions between nanofibrils and mineral plates. We demonstrate experimentally that silk nanofibril (SNF) and hydroxyapatite (HAP) can be used to fabricate highly ordered multilayer membranes with nanoporous features by combining protein self-assembly and in situ biomineralization. The production is optimized to be a simple and highly repeatable process that does not require sophisticated equipment and is suitable for scaled production of low-cost water purification membranes. These membranes not only show ultrafast water penetration but also exhibit broad utility and high efficiency of removal and even reuse (in some cases) of contaminants, including heavy metal ions, dyes, proteins, and other nanoparticles in water. Our biomimetic design and synthesis of these functional SNF/HAP materials have established a paradigm that could lead to the large-scale, low-cost production of multilayer materials with broad spectrum and efficiency for water purification, with applications in wastewater treatment, biomedicine, food industry, and the life sciences.

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

PubMed

Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

2009-01-01

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

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

PubMed

Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou

2006-09-01

An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.

Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae

PubMed Central

2011-01-01

Background There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in E. coli, yeast and baculovirus grown in insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV). Findings Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in insect larvae. Recognition of G21-465 by ß-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (Oncorhynchus mykiss) was demonstrated. Conclusions Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies. PMID:21693048

Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae.

PubMed

Encinas, Paloma; Gomez-Sebastian, Silvia; Nunez, Maria Carmen; Gomez-Casado, Eduardo; Escribano, Jose M; Estepa, Amparo; Coll, Julio

2011-06-21

There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in E. coli, yeast and baculovirus grown in insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV). Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in insect larvae. Recognition of G21-465 by ß-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (Oncorhynchus mykiss) was demonstrated. Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.

Expression and affinity purification of recombinant proteins from plants

NASA Technical Reports Server (NTRS)

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

2002-01-01

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

Modeling Xenon Purification Systems in a Laser Inertial Fusion Engine

NASA Astrophysics Data System (ADS)

Hopkins, Ann; Gentile, Charles

2011-10-01

A Laser Inertial Fusion Engine (LIFE) is a proposed method to employ fusion energy to produce electricity for consumers. However, before it can be built and used as such, each aspect of a LIFE power plant must first be meticulously planned. We are in the process of developing and perfecting models for an exhaust processing and fuel recovery system. Such a system is especially essential because it must be able to recapture and purify expensive materials involved in the reaction so they may be reused. One such material is xenon, which is to be used as an intervention gas in the target chamber. Using Aspen HYSYS, we have modeled several subsystems for exhaust processing, including a subsystem for xenon recovery and purification. After removing hydrogen isotopes using lithium bubblers, we propose to use cryogenic distillation to purify the xenon from remaining contaminants. Aspen HYSYS allows us to analyze predicted flow rates, temperatures, pressures, and compositions within almost all areas of the xenon purification system. Through use of Aspen models, we hope to establish that we can use xenon in LIFE efficiently and in a practical manner.

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY.

PubMed

Edupuganti, Soujanya Ratna; Edupuganti, Om Prakash; O'Kennedy, Richard; Defrancq, Eric; Boullanger, Stéphanie

2013-04-01

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology.

PubMed

Schmedes, Sarah; Marshall, Pamela; King, Jonathan L; Budowle, Bruce

2013-07-01

Various types of biological samples present challenges for extraction of DNA suitable for subsequent molecular analyses. Commonly used extraction methods, such as silica membrane columns and phenol-chloroform, while highly successful may still fail to provide a sufficiently pure DNA extract with some samples. Synchronous coefficient of drag alteration (SCODA), implemented in Boreal Genomics' Aurora Nucleic Acid Extraction System (Boreal Genomics, Vancouver, BC), is a new technology that offers the potential to remove inhibitors effectively while simultaneously concentrating DNA. In this initial study, SCODA was tested for its ability to remove various concentrations of forensically and medically relevant polymerase chain reaction (PCR) inhibitors naturally found in tissue, hair, blood, plant, and soil samples. SCODA was used to purify and concentrate DNA from intentionally contaminated DNA samples containing known concentrations of hematin, humic acid, melanin, and tannic acid. The internal positive control (IPC) provided in the Quantifiler™ Human DNA Quantification Kit (Life Technologies, Foster City, CA) and short tandem repeat (STR) profiling (AmpFℓSTR® Identifiler® Plus PCR Amplification Kit; Life Technologies, Foster City, CA) were used to measure inhibition effects and hence purification. SCODA methodology yielded overall higher efficiency of purification of highly contaminated samples compared with the QIAquick® PCR Purification Kit (Qiagen, Valencia, CA). SCODA-purified DNA yielded no cycle shift of the IPC for each sample and yielded greater allele percentage recovery and relative fluorescence unit values compared with the QIAquick® purification method. The Aurora provided an automated, minimal-step approach to successfully remove inhibitors and concentrate DNA from challenged samples.

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

PubMed

Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W; Clark, Chad R; Bharucha, Adil; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong-Heon

2017-12-21

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

PubMed Central

Vassylyeva, Marina N.; Klyuyev, Sergiy; Vassylyev, Alexey D.; Wesson, Hunter; Zhang, Zhuo; Renfrow, Matthew B.; Wang, Hengbin; Higgins, N. Patrick; Chow, Louise T.; Vassylyev, Dmitry G.

2017-01-01

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10−14–10−17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays. PMID:28607052

High-throughput protein concentration and buffer exchange: comparison of ultrafiltration and ammonium sulfate precipitation.

PubMed

Moore, Priscilla A; Kery, Vladimir

2009-01-01

High-throughput protein purification is a complex, multi-step process. There are several technical challenges in the course of this process that are not experienced when purifying a single protein. Among the most challenging are the high-throughput protein concentration and buffer exchange, which are not only labor-intensive but can also result in significant losses of purified proteins. We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration.

An efficient dye-sensitized BiOCl photocatalyst for air and water purification under visible light irradiation.

PubMed

Li, Guisheng; Jiang, Bo; Xiao, Shuning; Lian, Zichao; Zhang, Dieqing; Yu, Jimmy C; Li, Hexing

2014-08-01

A photosensitized BiOCl catalyst was found to be effective for photocatalytic water purification and air remediation under visible light irradiation (λ > 420 nm). Prepared by a solvothermal method, the BiOCl crystals possessed a 3D hierarchical spherical structure with the highly active facets exposed. When sensitized by Rhodamine B (RhB), the photocatalyst system was more active than N-doped TiO2 for breaking down 4-chlorophenol (4-CP, 200 ppm) and nitric monoxide (NO, 500 ppb). The high activity could be attributed to the hierarchical structure (supplying feasible reaction tunnels for adsorption and transition of reactants or products) and the efficient exposure of the {001} facets. The former provides an enriched oxygen atom density that promotes adsorption of cationic dye RhB, and creates an oxygen vacancy state. The HO˙ and ˙O2(-) radicals produced from the injected electrons from the excited dye molecule (RhB*) into the conduction band of BiOCl were responsible for the excellent photocatalytic performance of the RhB-BiOCl system.

[ASSESSMENT OF POTENTIAL RISK FOR CONTAMINATION OF SURFACE WATER RESERVOIRS BY PATHOGENS OF HUMAN PARASITIC DISEASES].

PubMed

Khromenkova, E P; Dimidova, L L; Dumbadze, O S; Aidinov, G T; Shendo, G L; Agirov, A Kh; Batchaev, Kh Kh

2015-01-01

Sanitary and parasitological studies of the waste effluents and surface reservoir waters were conducted in the south of Russia. The efficiency of purification of waste effluents from the pathogens of parasitic diseases was investigated in the region's sewage-purification facilities. The water of the surface water reservoirs was found to contain helminthic eggs and larvae and intestinal protozoan cysts because of the poor purification and disinfection of service fecal sewage waters. The poor purification and disinvasion of waste effluents in the region determine the potential risk of contamination of the surface water reservoirs and infection of the population with the pathogens of human parasitic diseases.

A facile method to prepare dual-functional membrane for efficient oil removal and in situ reversible mercury ions adsorption from wastewater

NASA Astrophysics Data System (ADS)

Zhang, Qingdong; Liu, Na; Cao, Yingze; Zhang, Weifeng; Wei, Yen; Feng, Lin; Jiang, Lei

2018-03-01

In this work, a novel thiol covered polyamide (nylon 66) microfiltration membrane was fabricated by combining mussel-inspired chemistry and coupling reaction, which owns excellent dual-function that can simultaneously remove oil from water efficiently and adsorb the mercury ions contained in the wastewater reversibly. Such membrane exhibited high oil/water separation efficiency, outstanding mercury adsorption ability, and good stability. Moreover, it can be regenerated in nitric acid solution, and maintain its good adsorption performance. The as-prepared membrane showed great potentials for water purification to reduce the heavy metal ion pollution and complicated industrial oily wastewater and living wastewater.

Functional magnetic mesoporous nanoparticles for efficient purification of laccase from fermentation broth in magnetically stabilized fluidized bed.

PubMed

Wang, Feng; Guo, Chen; Liu, Chun-Zhao

2013-12-01

A magnetically stabilized fluidized bed (MSFB) with the Cu(2+)-chelated magnetic mesoporous silica nanoparticles (MMSNPs-Cu(2+)) was established to purify laccase directly from the fermentation broth of Trametes versicolor. The MMSNPs-Cu(2+) particles in the MSFB maintained a stable bed expansion of two to threefold at a flow rate of 120-180 cm/h. At the optimal magnetic field intensity of 120 Gs, both the maximal Bodenstein number and the smallest axial dispersion coefficient were achieved, which resulted in a stable fluidization stage. The dynamic binding capacity of laccase in the MSFB decreased from 192.5 to144.3 mg/g when the flow velocity through the bed increased from 44.2 to 69.8 cm/h. The MSFB with MMSNPs-Cu(2+) achieved efficient laccase purification from the fermentation broth with 62.4-fold purification of laccase and 108.9 % activity yield. These results provided an excellent platform for the application of these magnetic mesoporous nanoparticles integrated with the MSFB in developing novel protein purification process.

High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris.

PubMed

Song, Dongmin; Gao, Zhendong; Zhao, Liqiang; Wang, Xiangxiang; Xu, Haijin; Bai, Yanling; Zhang, Xiuming; Linder, Markus B; Feng, Hui; Qiao, Mingqiang

2016-12-01

Hydrophobins are proteins produced by filamentous fungi with high natural-surfactant activities and that can self-assemble in interfaces of air-water or solid-water to form amphiphilic membranes. Here, we reported a high-yield fermentation method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris, attaining production of 300 mg/L by keeping the dissolved oxygen level at 15%-25% by turning the methanol-feeding speed. We also developed a novel HGFI-purification method enabling large-scare purification of HGFI, with >90% recovery. Additionally, we observed that hydrophobin HGFI in fermentation broth precipitated at pH < 7.0 and temperatures >90 °C. We also identified the structure and properties of proteins purified by this method through atomic force microscopy, circular dichroism, X-ray photoelectron spectroscopy, and water-contact angle measurement, which is similar to protein purification by ultrafiltration without heating treatment that enables our method to maintain native HGFI structure and properties. Furthermore, the purification method presented here can be applied to large-scale purification of other type I hydrophobins. Copyright © 2016. Published by Elsevier Inc.

Design and optimization of a recombinant system for large-scale production of the MPT64 antigen from Mycobacterium tuberculosis.

PubMed

Geisbrecht, Brian V; Nikonenko, Boris; Samala, Rowena; Nakamura, Reiko; Nacy, Carol A; Sacksteder, Katherine A

2006-03-01

Early clinical trials of a potential new tuberculosis (TB) diagnostic, the Patch Test for Active TB (PTAT), used MPB64 protein that was purified from the spent medium of Bacillus Calmette-Guérin (BCG) Tokyo 172 vaccine production. The yield was poor, 0.05 mg/L, and the process for purification of the protein was complex, requiring four chromatographic steps. The combination of yield and purification complexity compromised the ability to produce the PTAT diagnostic in quantities sufficient for larger clinical trials and commercialization. We report here a highly efficient method for the overexpression and purification of recombinant MPT64 from Escherichia coli (rMPT64) based upon a mild insolubility of rMPT64 following induction, and scalable anion-exchange and gel filtration chromatographies. Yields of protein were improved substantially to approximately 250 mg/L, and resulted in a preparation greater than 98% pure. Quantitative release assays were developed and used with MALDI-TOF mass spectrometry to confirm the identity of rMPT64. Using a guinea pig model of active TB, we found that rMPT64 elicited a specific immune response indistinguishable from that of MPB64 purified from BCG Tokyo culture filtrates. These results describe the first efficient and scalable protocol for production of rMPT64, demonstrate its activity in an animal model of active TB, and lay the foundation of ongoing and future use of the PTAT in clinical settings.

A method suitable for DNA extraction from humus-rich soil.

PubMed

Miao, Tianjin; Gao, Song; Jiang, Shengwei; Kan, Guoshi; Liu, Pengju; Wu, Xianming; An, Yingfeng; Yao, Shuo

2014-11-01

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

Analysis of gas membrane ultra-high purification of small quantities of mono-isotopic silane

DOE PAGES

de Almeida, Valmor F.; Hart, Kevin J.

2017-01-03

A small quantity of high-value, crude, mono-isotopic silane is a prospective gas for a small-scale, high-recovery, ultra-high membrane purification process. This is an unusual application of gas membrane separation for which we provide a comprehensive analysis of a simple purification model. The goal is to develop direct analytic expressions for estimating the feasibility and efficiency of the method, and guide process design; this is only possible for binary mixtures of silane in the dilute limit which is a somewhat realistic case. In addition, analytic solutions are invaluable to verify numerical solutions obtained from computer-aided methods. Hence, in this paper wemore » provide new analytic solutions for the purification loops proposed. Among the common impurities in crude silane, methane poses a special membrane separation challenge since it is chemically similar to silane. Other potential problematic compounds are: ethylene, diborane and ethane (in this order). Nevertheless, we demonstrate, theoretically, that a carefully designed membrane system may be able to purify mono-isotopic, crude silane to electronics-grade level in a reasonable amount of time and expenses. We advocate a combination of membrane materials that preferentially reject heavy impurities based on mobility selectivity, and light impurities based on solubility selectivity. We provide estimates for the purification of significant contaminants of interest. In this study, we suggest cellulose acetate and polydimethylsiloxane as examples of membrane materials on the basis of limited permeability data found in the open literature. We provide estimates on the membrane area needed and priming volume of the cell enclosure for fabrication purposes when using the suggested membrane materials. These estimates are largely theoretical in view of the absence of reliable experimental data for the permeability of silane. And finally, future extension of this work to the non-dilute limit may apply to the recovery of silane from rejected streams of natural silicon semi-conductor processes.« less

Analysis of gas membrane ultra-high purification of small quantities of mono-isotopic silane

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Almeida, Valmor F.; Hart, Kevin J.

A small quantity of high-value, crude, mono-isotopic silane is a prospective gas for a small-scale, high-recovery, ultra-high membrane purification process. This is an unusual application of gas membrane separation for which we provide a comprehensive analysis of a simple purification model. The goal is to develop direct analytic expressions for estimating the feasibility and efficiency of the method, and guide process design; this is only possible for binary mixtures of silane in the dilute limit which is a somewhat realistic case. In addition, analytic solutions are invaluable to verify numerical solutions obtained from computer-aided methods. Hence, in this paper wemore » provide new analytic solutions for the purification loops proposed. Among the common impurities in crude silane, methane poses a special membrane separation challenge since it is chemically similar to silane. Other potential problematic compounds are: ethylene, diborane and ethane (in this order). Nevertheless, we demonstrate, theoretically, that a carefully designed membrane system may be able to purify mono-isotopic, crude silane to electronics-grade level in a reasonable amount of time and expenses. We advocate a combination of membrane materials that preferentially reject heavy impurities based on mobility selectivity, and light impurities based on solubility selectivity. We provide estimates for the purification of significant contaminants of interest. In this study, we suggest cellulose acetate and polydimethylsiloxane as examples of membrane materials on the basis of limited permeability data found in the open literature. We provide estimates on the membrane area needed and priming volume of the cell enclosure for fabrication purposes when using the suggested membrane materials. These estimates are largely theoretical in view of the absence of reliable experimental data for the permeability of silane. And finally, future extension of this work to the non-dilute limit may apply to the recovery of silane from rejected streams of natural silicon semi-conductor processes.« less

Sodium purification apparatus and method

DOEpatents

Gould, M.I.

1980-03-04

An apparatus for and method of collecting and storing oxide impurities contained in high-temperature liquid alkali metal are disclosed. A method and apparatus are provided for nucleating and precipitating oxide impurities by cooling, wherein the nucleation and precipitation are enhanced by causing a substantial increase in pressure drop and corresponding change in the velocity head of the alkali metal. Thereafter the liquid alkali metal is introduced into a quiescent zone wherein the liquid velocity is maintained below a specific maximum whereby it is possible to obtain high oxide removal efficiencies without the necessity of a mesh or filter. 1 fig.

Histamine monolith versatility to purify supercoiled plasmid deoxyribonucleic acid from Escherichia coli lysate.

PubMed

Sousa, A; Almeida, A M; Černigoj, U; Sousa, F; Queiroz, J A

2014-08-15

Preparation of high quantities of supercoiled plasmid DNA of pharmaceutical grade purity is a research area where intensive investigation is being performed. From this standpoint, several downstream methods have been proposed, among them the monolithic chromatographic strategies owing to excellent mass transfer properties of monolithic supports and their high binding capacity for large biomolecules. The present study explores the physicochemical properties of histamine ligand in a supercoiled plasmid DNA purification process from an Escherichia coli clarified lysate, where the emphasis is given to the elution strategy that allows higher selectivity and efficient removal of other impurities besides the open circular isoform. The combination of high NaCl concentration and acidic pH allowed the elimination of 89% of RNA during the preparative loading of the lysate sample. The results of the purification strategy with ascending sodium chloride gradient revealed that 97% of supercoiled plasmid DNA was recovered with a purity degree of 99%. In addition, using a combined purification strategy with ascending sodium chloride (capture step) and then descending ammonium sulfate (polishing step) gradient, it was achieved a lower supercoiled plasmid DNA recovery yield of 79% with a purity degree of 92%, although the dynamic binding capacity under these conditions was higher than in the previous strategy. A significant reduction of host contents, such as proteins, RNA and genomic DNA, was obtained in both purification strategies. Accordingly, histamine is a useful and versatile ligand that allows the desirable supercoiled plasmid purification with high yield and purity level. Copyright © 2014. Published by Elsevier B.V.

Synthesis of the human insulin gene. Part II. Further improvements in the modified phosphotriester method and the synthesis of seventeen deoxyribooligonucleotide fragments constituting human insulin chains B and mini-CDNA.

PubMed Central

Sung, W L; Hsiung, H M; Brousseau, R; Michniewicz, J; Wu, R; Narang, S A

1979-01-01

The purification of protected deoxyribooligonucleotides containing phosphotriester internucleotidic linkages has been improved by developing a deactivated silica gel chromatographic technique. The efficiency of this technique as applied in the modified phosphotriester approach has been demonstrated in the rapid synthesis of seventeen pure fragments constituting the sequence of human insulin B and mini-C DNA. The sequence of each oligomer was confirmed by the two-dimensional mobility shift method of fingerprinting. Images PMID:230464

Necessity of purification during bacterial DNA extraction with environmental soils

PubMed Central

Choi, Jung-Hyun

2017-01-01

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. PMID:28793754

Bismuth Oxysulfide and Its Polymer Nanocomposites for Efficient Purification

PubMed Central

Luo, Yidong; Qiao, Lina; Wang, Huanchun; Lan, Shun; Shen, Yang; Lin, Yuanhua; Nan, Cewen

2018-01-01

The danger of toxic organic pollutants in both aquatic and air environments calls for high-efficiency purification material. Herein, layered bismuth copper oxychalcogenides, BiCuSO, nanosheets of high photocatalytic activity were introduced to the PVDF (Polyvinylidene Fluoride). The fibrous membranes provide an easy, efficient, and recyclable way to purify organic pollutant. The physical and photophysical properties of the BiCuSO and its polymer composite were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), ultraviolet-visible diffuse reflection spectroscopy (DRS), X-ray photoelectron spectroscopy (XPS), electron spin resonance (EPR). Photocatalysis of Congo Red reveals that the BiCuSO/PVDF shows a superior photocatalytic activity of a 55% degradation rate in 70 min at visible light. The high photocatalytic activity is attributed to the exposed active {101} facets and the triple vacant associates VBi‴VO••VBi‴. By engineering the intrinsic defects on the surface of bismuth oxysulfide, high solar-driven photocatalytic activity can be approached. The successful fabrication of the bismuth oxysulfide and its polymer nanocomposites provides an easy and general approach for high-performance purification materials for various applications. PMID:29562701

Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale

PubMed Central

Zang, Yuguo; Kammerer, Bernd; Eisenkolb, Maike; Lohr, Katrin; Kiefer, Hans

2011-01-01

Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step. PMID:21966480

Improvements to direct quantitative analysis of multiple microRNAs facilitating faster analysis.

PubMed

Ghasemi, Farhad; Wegman, David W; Kanoatov, Mirzo; Yang, Burton B; Liu, Stanley K; Yousef, George M; Krylov, Sergey N

2013-11-05

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a "miRNA fingerprint"-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting.

A simple chromatographic method for purification of egg lecithin.

PubMed

Nielsen, J R

1980-06-01

Egg lecithin was purified from the CdCl2-lecithin complex by column chromatography on Alumina. The yield from 5 eggs was 2.8 g. The purified lecithin had correct chemical values for pure lecithin and a fatty acid composition similar to lecithin prepared by other methods. The method probably can be adapted for purification of other lipids containing the phosphocholine moiety and for purification of synthetic lecithin.

A methodological approach for production and purification of polyclonal antibody against dog IgG.

PubMed

Sadeghi, Somayeh; Aghebati-Maleki, Leili; Nozari, Samira; Majidi, Jafar

2018-01-01

Antibodies are a class of biomolecules that has an important role in the immune system and lots of applications in biotechnological methods and in pharmaceutics. Production and purification of antibodies in laboratory animals is one of the first ways to manufacture of these prominent tools. The obtained antibodies from these process could be used in various types of bioassay techniques such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay, etc. Also, antibodies employed in diagnostics applications in humans and other animals in order to detect specific antigens. In this study, we aimed to produce and purify anti-dog IgG via immunizing rabbits with dog IgG in combination with Freund's adjuvant. Polyclonal IgG were purified by ion exchange chromatography and then the purified antibody was labeled with horse radish peroxidase (HPR). Direct ELISA was used to determine the optimum titer and cross-reactivity of HRP conjugated IgG. The purity of various IgG preparations and the optimum dilution of prepared HRP conjugated IgG, respectively, was about 95.00% and 1:8000. This study showed that efficiency ion-exchange chromatography could be an appropriate method for purification of IgG antibodies. This antibody could be a useful tool for future dog immune diagnosis tests. This product characterization shown here sets the foundations for future work on dog IgGs.

Hydrophilic interaction liquid chromatography for the separation, purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz.

PubMed

Liang, Tu; Fu, Qing; Li, Fangbing; Zhou, Wei; Xin, Huaxia; Wang, Hui; Jin, Yu; Liang, Xinmiao

2015-08-01

A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid-phase extraction followed by hydrophilic interaction liquid chromatography at semi-preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Method for Rapid Purification of Class IIa Bacteriocins and Comparison of Their Activities

PubMed Central

Guyonnet, D.; Fremaux, C.; Cenatiempo, Y.; Berjeaud, J. M.

2000-01-01

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared. PMID:10742275

Waste water biological purification plants of dairy products industry and energy management

NASA Astrophysics Data System (ADS)

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.

Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

PubMed Central

Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

2011-01-01

Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process. PMID:21589929

A cell culture-derived whole virus influenza A vaccine based on magnetic sulfated cellulose particles confers protection in mice against lethal influenza A virus infection.

PubMed

Pieler, Michael M; Frentzel, Sarah; Bruder, Dunja; Wolff, Michael W; Reichl, Udo

2016-12-07

Downstream processing and formulation of viral vaccines employs a large number of different unit operations to achieve the desired product qualities. The complexity of individual process steps involved, the need for time consuming studies towards the optimization of virus yields, and very high requirements regarding potency and safety of vaccines results typically in long lead times for the establishment of new processes. To overcome such obstacles, to enable fast screening of potential vaccine candidates, and to explore options for production of low cost veterinary vaccines a new platform for whole virus particle purification and formulation based on magnetic particles has been established. Proof of concept was carried out with influenza A virus particles produced in suspension Madin Darby canine kidney (MDCK) cells. The clarified, inactivated, concentrated, and diafiltered virus particles were bound to magnetic sulfated cellulose particles (MSCP), and directly injected into mice for immunization including positive and negative controls. We show here, that in contrast to the mock-immunized group, vaccination of mice with antigen-loaded MSCP (aMSCP) resulted in high anti-influenza A antibody responses and full protection against a lethal challenge with replication competent influenza A virus. Antiviral protection correlated with a 400-fold reduced number of influenza nucleoprotein gene copies in the lungs of aMSCP immunized mice compared to mock-treated animals, indicating the efficient induction of antiviral immunity by this novel approach. Thus, our data proved the use of MSCP for purification and formulation of the influenza vaccine to be fast and efficient, and to confer protection of mice against influenza A virus infection. Furthermore, the method proposed has the potential for fast purification of virus particles directly from bioreactor harvests with a minimum number of process steps towards formulation of low-cost veterinary vaccines, and for screening studies requiring fast purification protocols. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns.

PubMed

Liu, Keyang; Wen, Zhanbo; Li, Na; Yang, Wenhui; Hu, Lingfei; Wang, Jie; Yin, Zhe; Dong, Xiaokai; Li, Jinsong

2012-12-01

Bacteriophages are used widely in many fields, and phages with high purity and infectivity are required. Convective interaction media (CIM) methacrylate monoliths were used for the purification of mycobacteriophage D29. The lytic phages D29 from bacterial lysate were purified primarily by polyethylene glycol 8000 or ammonium sulphate, and then the resulting phages were passed through the CIM monolithic columns for further purification. After the whole purification process, more than 99% of the total proteins were removed irrespective which primary purification method was used. The total recovery rates of viable phages were around 10-30%. Comparable results were obtained when the purification method was scaled-up from a 0.34 mL CIM DEAE (diethylamine) monolithic disk to an 8 mL CIM DEAE monolithic column. Copyright © 2012 Elsevier B.V. All rights reserved.

Magnetic techniques for the isolation and purification of proteins and peptides

PubMed Central

Safarik, Ivo; Safarikova, Mirka

2004-01-01

Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. PMID:15566570

How much DNA is lost? Measuring DNA loss of short-tandem-repeat length fragments targeted by the PowerPlex 16® system using the Qiagen MinElute Purification Kit.

PubMed

Kemp, Brian M; Winters, Misa; Monroe, Cara; Barta, Jodi Lynn

2014-01-01

The success in recovering genetic profiles from aged and degraded biological samples is diminished by fundamental aspects of DNA extraction, as well as its long-term preservation, that are not well understood. While numerous studies have been conducted to determine whether one extraction method was superior to others, nearly all of them were initiated with no knowledge of the actual starting DNA quantity in the samples prior to extraction, so they ultimately compared the outcome of all methods relative to the best. Using quantitative PCR to estimate the copy count of synthetic standards before (i.e., "copies in") and after (i.e., "copies out") purification by the Qiagen MinElute PCR Purification Kit, we documented DNA loss within a pool of 16 different-sized fragments ranging from 106 to 409 bp in length, corresponding to those targeted by the PowerPlex 16 System (Promega, Madison, WI). Across all standards from 10(4) to 10(7) copies/μL, loss averaged between 21.75% and 60.56% (mean, 39.03%), which is not congruent with Qiagen's claim that 80% of 70 bp to 4 kb fragments are retained using this product (i.e., 20% loss). Our study also found no clear relationship either between DNA strand length and retention or between starting copy number and retention. This suggests that there is no molecule bias across the MinElute column membrane and highlights the need for manufacturers to clearly and accurately describe on what their claims are based, and should also encourage researchers to document DNA retention efficiencies of their own methods and protocols. Understanding how and where to reduce loss of molecules during extraction and purification will serve to generate clearer and more accurate data, which will enhance the utility of ancient and low-copy-number DNA as a tool for closing forensic cases or in reconstructing the evolutionary history of humans and other organisms.

A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components.

PubMed

Amid, Mehrnoush; Manap, Mohd Yazid; Hussin, Muhaini; Mustafa, Shuhaimi

2015-06-17

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

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

PubMed

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

2014-09-07

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

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

PubMed

Serpa, Gisele; Augusto, Elisabeth Fátima Pires; Tamashiro, Wirla Maria Silva Cunha; Ribeiro, Mariana Borçoe; Miranda, Everson Alves; Bueno, Sônia Maria Alves

2005-02-25

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2002-06-01

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

PubMed

Branavan, Manoharanehru; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Ahern, Jeremy C; Sivanesan, Tulasi; Hudson, Chris; Stead, Thomas; Kremer, Jessica; Garg, Neha; Baker, Mark; Sadiq, Syed T; Balachandran, Wamadeva

2016-08-01

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

[Determination of patulin in fruits and jam by solid phase extraction-ultra performance liquid chromatography].

PubMed

Lü, Weichao; Shen, Shuchang; Wang, Chao

2017-11-08

With magnesium silicate, silica gel, diatomite and calcium sulfate as raw materials, a new solid phase extraction column was prepared through a series of processes of grinding to ethanol homogenate, drying and packing into polypropylene tube. The sample was hydrolyzed by pectinase, extracted by acetonitrile and purified by solid phase extraction. The target compounds were separated on a C18 column (100 mm×2.1 mm, 1.8 μm), using 0.8% (v/v) tetrahydrofuran solution as mobile phase with a flow rate of 0.5 mL/min. The detection wavelength was 276 nm. The effect of pectinase on extraction yield and purification effect of solid-phase extraction column were investigated. The optimum chromatographic conditions were selected. There was a good linear relationship between the peak heights and the mass concentrations of patulin in the range of 0.1 to 10 mg/L with the correlation coefficient ( R 2 ) of 1. The limit of detection for this method was 10.22 μg/kg. The spiked recoveries of samples were 86.58%-94.84% with the relative standard deviations (RSDs) of 1.45%-2.28%. The results indicated that the self-made solid phase extraction column had a good purification efficiency, and the UPLC had a high separation efficiency. The method is simple, accurate and of great significance for the quality and safety control of fruit products.

Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes.

PubMed

Li, Bin; Yang, Hui; Wang, Xiaochen; Zhan, Yongkun; Sheng, Wei; Cai, Huanhuan; Xin, Haoyang; Liang, Qianqian; Zhou, Ping; Lu, Chao; Qian, Ruizhe; Chen, Sifeng; Yang, Pengyuan; Zhang, Jianyi; Shou, Weinian; Huang, Guoying; Liang, Ping; Sun, Ning

2017-09-29

Most infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle. Although current pluripotent stem cells (PSCs) and directed cardiac differentiation techniques are able to generate fetal-like human cardiomyocytes, isolation of pure ventricular cardiomyocytes has been challenging. For repairing ventricular damage, we aimed to establish a highly efficient purification system to obtain homogeneous ventricular cardiomyocytes and prepare engineered human ventricular heart muscles in a dish. The purification system used TALEN-mediated genomic editing techniques to insert the neomycin or EGFP selection marker directly after the myosin light chain 2 (MYL2) locus in human pluripotent stem cells. Purified early ventricular cardiomyocytes were estimated by immunofluorescence, fluorescence-activated cell sorting, quantitative PCR, microelectrode array, and patch clamp. In subsequent experiments, the mixture of mature MYL2-positive ventricular cardiomyocytes and mesenchymal cells were cocultured with decellularized natural heart matrix. Histological and electrophysiology analyses of the formed tissues were performed 2 weeks later. Human ventricular cardiomyocytes were efficiently isolated based on the purification system using G418 or flow cytometry selection. When combined with the decellularized natural heart matrix as the scaffold, functional human ventricular heart muscles were prepared in a dish. These engineered human ventricular muscles can be great tools for regenerative therapy of human ventricular damage as well as drug screening and ventricular-specific disease modeling in the future.

A rapid Orthopoxvirus purification protocol suitable for high-containment laboratories.

PubMed

Hughes, Laura; Wilkins, Kimberly; Goldsmith, Cynthia S; Smith, Scott; Hudson, Paul; Patel, Nishi; Karem, Kevin; Damon, Inger; Li, Yu; Olson, Victoria A; Satheshkumar, P S

2017-05-01

Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron ® . Genetron ® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield. Published by Elsevier B.V.

Symmetry-conserving purification of quantum states within the density matrix renormalization group

DOE PAGES

Nocera, Alberto; Alvarez, Gonzalo

2016-01-28

The density matrix renormalization group (DMRG) algorithm was originally designed to efficiently compute the zero-temperature or ground-state properties of one-dimensional strongly correlated quantum systems. The development of the algorithm at finite temperature has been a topic of much interest, because of the usefulness of thermodynamics quantities in understanding the physics of condensed matter systems, and because of the increased complexity associated with efficiently computing temperature-dependent properties. The ancilla method is a DMRG technique that enables the computation of these thermodynamic quantities. In this paper, we review the ancilla method, and improve its performance by working on reduced Hilbert spaces andmore » using canonical approaches. Furthermore we explore its applicability beyond spins systems to t-J and Hubbard models.« less

High throughput photo-oxidations in a packed bed reactor system.

PubMed

Kong, Caleb J; Fisher, Daniel; Desai, Bimbisar K; Yang, Yuan; Ahmad, Saeed; Belecki, Katherine; Gupton, B Frank

2017-12-01

The efficiency gains produced by continuous-flow systems in conducting photochemical transformations have been extensively demonstrated. Recently, these systems have been used in developing safe and efficient methods for photo-oxidations using singlet oxygen generated by photosensitizers. Much of the previous work has focused on the use of homogeneous photocatalysts. The development of a unique, packed-bed photoreactor system using immobilized rose bengal expands these capabilities as this robust photocatalyst allows access to and elaboration from these highly useful building blocks without the need for further purification. With this platform we were able to demonstrate a wide scope of singlet oxygen ene, [4+2] cycloadditions and heteroatom oxidations. Furthermore, we applied this method as a strategic element in the synthesis of the high-volume antimalarial artemisinin. Copyright © 2017. Published by Elsevier Ltd.

The effect of column purification on cDNA indirect labelling for microarrays

PubMed Central

Molas, M Lia; Kiss, John Z

2007-01-01

Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays. PMID:17597522

The effect of column purification on cDNA indirect labelling for microarrays.

PubMed

Molas, M Lia; Kiss, John Z

2007-06-27

The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays.

Recent Advances in Marine Algae Polysaccharides: Isolation, Structure, and Activities.

PubMed

Xu, Shu-Ying; Huang, Xuesong; Cheong, Kit-Leong

2017-12-13

Marine algae have attracted a great deal of interest as excellent sources of nutrients. Polysaccharides are the main components in marine algae, hence a great deal of attention has been directed at isolation and characterization of marine algae polysaccharides because of their numerous health benefits. In this review, extraction and purification approaches and chemico-physical properties of marine algae polysaccharides (MAPs) are summarized. The biological activities, which include immunomodulatory, antitumor, antiviral, antioxidant, and hypolipidemic, are also discussed. Additionally, structure-function relationships are analyzed and summarized. MAPs' biological activities are closely correlated with their monosaccharide composition, molecular weights, linkage types, and chain conformation. In order to promote further exploitation and utilization of polysaccharides from marine algae for functional food and pharmaceutical areas, high efficiency, and low-cost polysaccharide extraction and purification methods, quality control, structure-function activity relationships, and specific mechanisms of MAPs activation need to be extensively investigated.

Isolation and purification of lutein from the microalga Chlorella vulgaris by extraction after saponification.

PubMed

Li, Hua-Bin; Jiang, Yue; Chen, Feng

2002-02-27

A simple and efficient method for the isolation and purification of lutein from the microalga Chlorella vulgaris was developed. Crude lutein was obtained by extraction with dichloromethane from the microalga after saponification. Partition values of lutein in the two-phase system of ethanol-water-dichloromethane at different ratios were measured by HPLC so as to assist the determination of an appropriate condition for washing water-soluble impurities in the crude lutein. Partition values of lutein in another two-phase system of ethanol-water-hexane at different ratios were also measured by HPLC for determining the condition for removing fat-soluble impurities. The water-soluble impurities in the crude lutein were removed by washing with 30% aqueous ethanol, and the fat-soluble impurities were removed by extraction with hexane. The final purity of lutein obtained was 90-98%, and the yield was 85-91%.

Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

PubMed

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

An improved 96-well turbidity assay for T4 lysozyme activity.

PubMed

Toro, Tasha B; Nguyen, Thao P; Watt, Terry J

2015-01-01

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

An improved 96-well turbidity assay for T4 lysozyme activity

PubMed Central

Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

2015-01-01

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data.

PubMed

Eddhif, Balkis; Guignard, Nadia; Batonneau, Yann; Clarhaut, Jonathan; Papot, Sébastien; Geffroy-Rodier, Claude; Poinot, Pauline

2018-04-01

The data presented here are related to the research paper entitled "Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses" (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.

Analysis and evaluation in the production process and equipment area of the low-cost solar array project

NASA Technical Reports Server (NTRS)

Goldman, H.; Wolf, M.

1979-01-01

Analyses of slicing processes and junction formation processes are presented. A simple method for evaluation of the relative economic merits of competing process options with respect to the cost of energy produced by the system is described. An energy consumption analysis was developed and applied to determine the energy consumption in the solar module fabrication process sequence, from the mining of the SiO2 to shipping. The analysis shows that, in current technology practice, inordinate energy use in the purification step, and large wastage of the invested energy through losses, particularly poor conversion in slicing, as well as inadequate yields throughout. The cell process energy expenditures already show a downward trend based on increased throughput rates. The large improvement, however, depends on the introduction of a more efficient purification process and of acceptable ribbon growing techniques.

Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.

PubMed

Weiss, Agnes; Jérôme, Valérie; Freitag, Ruth

2007-06-15

The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis.

PubMed

Kanai, Masatake; Mano, Shoji; Nishimura, Mikio

2017-01-11

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

[Comparison of MPure-12 Automatic Nucleic Acid Purification and Chelex-100 Method].

PubMed

Shen, X; Li, M; Wang, Y L; Chen, Y L; Lin, Y; Zhao, Z M; Que, T Z

2017-04-01

To explore the forensic application value of MPure-12 automatic nucleic acid purification （MPure-12 Method） for DNA extraction by extracting and typing DNA from bloodstains and various kinds of biological samples with different DNA contents. Nine types of biological samples, such as bloodstains, semen stains, and saliva were collected. DNA were extracted using MPure-12 method and Chelex-100 method, followed by PCR amplification and electrophoresis for obtaining STR-profiles. The samples such as hair root, chutty, butt, muscular tissue, saliva stain, bloodstain and semen stain were typed successfully by MPure-12 method. Partial alleles were lacked in the samples of saliva, and the genotyping of contact swabs was unsatisfactory. Additional, all of the bloodstains （20 μL, 15 μL, 10 μL, 5 μL, 1 μL） showed good typing results using Chelex-100 method. But the loss of alleles occurred in 1 μL blood volume by MPure-12 method. MPure-12 method is suitable for DNA extraction of a certain concentration blood samples．Chelex-100 method may be better for the extraction of trace blood samples．This instrument used in nucleic acid extraction has the advantages of simplicity of operator, rapidity, high extraction efficiency, high rate of reportable STR-profiles and lower man-made pollution. Copyright© by the Editorial Department of Journal of Forensic Medicine

Investigation of Free-Standing Plasmonic Mesoporous Ag/CMK-8-Nafion Composite Membrane for the Removal of Organic Pollutants with 254-nm UV Irradiation

NASA Astrophysics Data System (ADS)

Tseng, Chuan Ming; Chen, Hsin Liang; Lai, Sz Nian; Chen, Ming Shiung; Peng, Chien Jung; Li, Chia Jui; Hung, Wei Hsuan

2017-05-01

"Carbon-based material" has demonstrated a great potential on water purification due to its strong physical adsorption to organic pollutants in the water. Three-dimensional cubic ordered mesoporous carbon (CMK-8), one of the well-known ordered mesoporous carbons, was prepared by using nanocasting method with mesoporous silica (KIT-6) as the template. In this study, CMK-8 blended with Nafion polymer to form a free-standing mesoporous CMK-8-Nafion composite membrane. The synthesis of high crystallinity CMK-8 was characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). More than 80% methyl orange (MO) removal efficiency was observed under 254-nm UV irradiation after 120 min. Ninety-two percent recycling performance was remained after four recycling tests, which indicated a reliable servicing lifetime for the water purification. Furthermore, an additional layer of plasmonic silver nanoparticles (Ag NPs) was integrated into this CMK-8-Nafion membrane for higher pollutant removal efficiency, attributing from the generation of plasmon-resonance hot electrons from Ag NPs. A 4-in. CMK-8-Nafion composite membrane was also fabricated for the demonstration of potential large-scale utilization.

Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica.

PubMed

Bulani, Siyavuya Ishmael; Moleleki, Lucy; Albertyn, Jacobus; Moleleki, Ntsane

2012-05-20

In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control cells indicating that mCherry was displayed on the cells. Expression of mCherry on cells of Y. lipolytica was confirmed by both fluorescent microscopy and flow cytometry. Furthermore, SDS-PAGE analysis and matrix-assisted laser desorption/ionization (MALDI)-time-of (TOF)-mass spectrometry (MS) peptide mass fingerprinting (PMF) confirmed that the protein cleaved from the yeast cells using enterokinase was mCherry. Efficient cleavage of mCherry reported in this work offers an alternative purification method for displayed heterologous proteins on Y. lipolytica cells using the plasmid constructed in this study. The developed displaying system offers great potential for industrial production and purification of heterologous proteins at low cost.

Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor.

PubMed

Berger, Bryan W; García, Roxana Y; Lenhoff, Abraham M; Kaler, Eric W; Robinson, Clifford R

2005-07-01

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.

Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen.

PubMed

Ng, Michelle Y T; Tan, Wen Siang; Abdullah, Norhafizah; Ling, Tau Chuan; Tey, Beng Ti

2006-10-01

Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 degrees C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 degrees C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.

Robust and Efficient Spin Purification for Determinantal Configuration Interaction.

PubMed

Fales, B Scott; Hohenstein, Edward G; Levine, Benjamin G

2017-09-12

The limited precision of floating point arithmetic can lead to the qualitative and even catastrophic failure of quantum chemical algorithms, especially when high accuracy solutions are sought. For example, numerical errors accumulated while solving for determinantal configuration interaction wave functions via Davidson diagonalization may lead to spin contamination in the trial subspace. This spin contamination may cause the procedure to converge to roots with undesired ⟨Ŝ 2 ⟩, wasting computer time in the best case and leading to incorrect conclusions in the worst. In hopes of finding a suitable remedy, we investigate five purification schemes for ensuring that the eigenvectors have the desired ⟨Ŝ 2 ⟩. These schemes are based on projection, penalty, and iterative approaches. All of these schemes rely on a direct, graphics processing unit-accelerated algorithm for calculating the S 2 c matrix-vector product. We assess the computational cost and convergence behavior of these methods by application to several benchmark systems and find that the first-order spin penalty method is the optimal choice, though first-order and Löwdin projection approaches also provide fast convergence to the desired spin state. Finally, to demonstrate the utility of these approaches, we computed the lowest several excited states of an open-shell silver cluster (Ag 19 ) using the state-averaged complete active space self-consistent field method, where spin purification was required to ensure spin stability of the CI vector coefficients. Several low-lying states with significant multiply excited character are predicted, suggesting the value of a multireference approach for modeling plasmonic nanomaterials.

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

PubMed Central

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 should be employed for future, more exhaustive optimization experiments and protein purification runs 4. The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach. PMID:21968976

Economic Methods of Ginger Protease'sextraction and Purification

NASA Astrophysics Data System (ADS)

Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

Efficient counter-current chromatographic isolation and structural identification of two new cinnamic acids from Echinacea purpurea.

PubMed

Lu, Ying; Li, JiaYin; Li, MiLu; Hu, Xia; Tan, Jun; Liu, Zhong Hua

2012-10-01

Two new cinnamic acids, 2-O-caffeoyl-3-O-isoferuloyltartaric (3), and 2, 3-di-O-isoferuloyltartaric acid (5), along with three known caffeic acids, cichoric acid (1), 2-O-caffeoyl-3-O-feruloyltartaric acid (2) and 2-O-caffeoyl-3-O-p-coumaroyltartaric acid (4), have been successfully isolated and purified from Echinacea purpurea. In this study, we investigated an efficient method for the preparative isolation and purification of cinnamic acids from E. purpurea by high-speed counter-current chromatography (HSCCC). The separation was performed using a two-phase solvent composed of n-hexane-ethyl-acetate-methanol-0.5% aqueous acetic acid (1:3:1:4, v/v). The upper phase was used as the stationary phase and the lower phase as the mobile phase, with a flow rate of 1.6 mL/min. From 250 mg of crude extracts, 65.1 mg of 1, 8.3 mg of 2, 4.0 mg of 3, 4.5 mg of 4, and 4.3 mg of 5 were isolated in one-step, with purities of 98.5%, 97.7%, 94.6%, 94.3%, and 98.6%, respectively, as evaluated by HPLC-DAD. The chemical structures were identified by electro spray ionization mass spectrometry (ESI-MS) and one- and two-dimensional NMR spectra. HSCCC was very efficient for the separation and purification of the cinnamic acids from

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branch, Darren W.; Vreeland, Erika C.; McClain, Jamie L.

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP andmore » the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.« less

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

DOE PAGES

Branch, Darren W.; Vreeland, Erika C.; McClain, Jamie L.; ...

2017-07-21

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP andmore » the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.« less

Two-step purification method of vitellogenin from three teleost fish species: rainbow trout (Oncorhynchus mykiss), gudgeon (Gobio gobio) and chub (Leuciscus cephalus).

PubMed

Brion, F; Rogerieux, F; Noury, P; Migeon, B; Flammarion, P; Thybaud, E; Porcher, J M

2000-01-14

A two-step purification protocol was developed to purify rainbow trout (Oncorhynchus mykiss) vitellogenin (Vtg) and was successfully applied to Vtg of chub (Leuciscus cephalus) and gudgeon (Gobio gobio). Capture and intermediate purification were performed by anion-exchange chromatography on a Resource Q column and a polishing step was performed by gel permeation chromatography on Superdex 200 column. This method is a rapid two-step purification procedure that gave a pure solution of Vtg as assessed by silver staining electrophoresis and immunochemical characterisation.

Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

PubMed

Shi, Changhua; Han, Tzu-Chiang; Wood, David W

2017-01-01

Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

Challenges and opportunities in the purification of recombinant tagged proteins.

PubMed

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

2014-01-01

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. Copyright © 2013 Elsevier Inc. All rights reserved.

Nanocellulose-Based Materials for Water Purification

PubMed Central

Voisin, Hugo; Bergström, Lennart; Liu, Peng; Mathew, Aji P.

2017-01-01

Nanocellulose is a renewable material that combines a high surface area with high strength, chemical inertness, and versatile surface chemistry. In this review, we will briefly describe how nanocellulose is produced, and present—in particular, how nanocellulose and its surface modified versions affects the adsorption behavior of important water pollutants, e.g., heavy metal species, dyes, microbes, and organic molecules. The processing of nanocellulose-based membranes and filters for water purification will be described in detail, and the uptake capacity, selectivity, and removal efficiency will also be discussed. The processing and performance of nanocellulose-based membranes, which combine a high removal efficiency with anti-fouling properties, will be highlighted. PMID:28336891

Optimized anion exchange column isolation of zirconium-89 ( 89Zr) from yttrium cyclotron target: Method development and implementation on an automated fluidic platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

O’Hara, Matthew J.; Murray, Nathaniel J.; Carter, Jennifer C.

Zirconium-89 ( 89Zr), produced by the (p, n) reaction from naturally monoisotopic yttrium ( natY), is a promising positron emitting isotope for immunoPET imaging. Its long half-life of 78.4 h is sufficient for evaluating slow physiological processes. A prototype automated fluidic system, coupled to on-line and in-line detectors, has been constructed to facilitate development of new 89Zr purification methodologies. The highly reproducible reagent delivery platform and near-real time monitoring of column effluents allows for efficient method optimization. The separation of Zr from dissolved Y metal targets was evaluated using several anion exchange resins. Each resin was evaluated against its abilitymore » to quantitatively capture Zr from a load solution high in dissolved Y. The most appropriate anion exchange resin for this application was identified, and the separation method was optimized. The method is capable of a high Y decontamination factor (>10 5) and has been shown to remove Fe, an abundant contaminant in Y foils, from the 89Zr elution fraction. Finally, the method was evaluated using cyclotron bombarded Y foil targets; the method was shown to achieve >95% recovery of the 89Zr present in the foils. The anion exchange column method described here is intended to be the first 89Zr isolation stage in a dual-column purification process.« less

Optimized anion exchange column isolation of zirconium-89 ( 89Zr) from yttrium cyclotron target: Method development and implementation on an automated fluidic platform

DOE PAGES

O’Hara, Matthew J.; Murray, Nathaniel J.; Carter, Jennifer C.; ...

2018-02-24

Zirconium-89 ( 89Zr), produced by the (p, n) reaction from naturally monoisotopic yttrium ( natY), is a promising positron emitting isotope for immunoPET imaging. Its long half-life of 78.4 h is sufficient for evaluating slow physiological processes. A prototype automated fluidic system, coupled to on-line and in-line detectors, has been constructed to facilitate development of new 89Zr purification methodologies. The highly reproducible reagent delivery platform and near-real time monitoring of column effluents allows for efficient method optimization. The separation of Zr from dissolved Y metal targets was evaluated using several anion exchange resins. Each resin was evaluated against its abilitymore » to quantitatively capture Zr from a load solution high in dissolved Y. The most appropriate anion exchange resin for this application was identified, and the separation method was optimized. The method is capable of a high Y decontamination factor (>10 5) and has been shown to remove Fe, an abundant contaminant in Y foils, from the 89Zr elution fraction. Finally, the method was evaluated using cyclotron bombarded Y foil targets; the method was shown to achieve >95% recovery of the 89Zr present in the foils. The anion exchange column method described here is intended to be the first 89Zr isolation stage in a dual-column purification process.« less

Separation and purification of thymopentin with molecular imprinting membrane by solid phase extraction disks.

PubMed

Wang, Chaoli; Hu, Xiaoling; Guan, Ping; Wu, Danfeng; Qian, Liwei; Li, Ji; Song, Renyuan

2015-01-01

The synthesis and performance of molecularly imprinted membranes (MIMs) as a solid phase extraction packing materials for the separation and purification of thymopentin from crude samples was described. In order to increase structural selectivity and imprinting efficiency, surface-initiated ATRP and ionic liquid (1-vinyl-3-ethyl acetate imidazolium chloride) were used to prepare molecularly imprinting membranes. The results demonstrated that solid phase extraction disks stuffed by MIMs with ionic liquids as functional monomer demonstrated high isolation and purification of performance to the thymopentin. The molecular recognition of thymopentin was analyzed by using molecular modeling software. Copyright © 2014 Elsevier B.V. All rights reserved.

Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lane, Michael Douglas; Nam, Hyun-Joo; Padron, Eric

2005-06-01

The production, purification, crystallization and preliminary X-ray crystallographic analysis of adeno-associated virus serotype 8 is reported. Adeno-associated viruses (AAVs) are actively being developed for clinical gene-therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of their viral capsid structures and the structural determinants of their tissue-transduction interactions. AAV8 is ∼80% identical to the more widely studied AAV2, but its liver-transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X-ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X-rays to 3.0 Åmore » resolution using synchrotron radiation and belong to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = 257.5, c = 443.5 Å. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit.« less

Preparation of a Sepia Melanin and Poly(ethylene-alt-maleic Anhydride) Hybrid Material as an Adsorbent for Water Purification.

PubMed

Panzarasa, Guido; Osypova, Alina; Consolati, Giovanni; Quasso, Fiorenza; Soliveri, Guido; Ribera, Javier; Schwarze, Francis W M R

2018-01-23

Meeting the increasing demand of clean water requires the development of novel efficient adsorbent materials for the removal of organic pollutants. In this context the use of natural, renewable sources is of special relevance and sepia melanin, thanks to its ability to bind a variety of organic and inorganic species, has already attracted interest for water purification. Here we describe the synthesis of a material obtained by the combination of sepia melanin and poly(ethylene- alt -maleic anhydride) (P(E- alt -MA)). Compared to sepia melanin, the resulting hybrid displays a high and fast adsorption efficiency towards methylene blue (a common industrial dye) for a wide pH range (from pH 2 to 12) and under high ionic strength conditions. It is easily recovered after use and can be reused up to three times. Given the wide availability of sepia melanin and P(E- alt -MA), the synthesis of our hybrid is simple and affordable, making it suitable for industrial water purification purposes.

Catalyst-Free Difunctionalization of Activated Alkenes in Water: Efficient Synthesis of β-Keto Sulfides and Sulfones.

PubMed

Wang, Huamin; Wang, Guangyu; Lu, Qingquan; Chiang, Chien-Wei; Peng, Pan; Zhou, Jiufu; Lei, Aiwen

2016-10-04

Difunctionalization of activated alkenes, a powerful strategy in chemical synthesis, has been accomplished for direct synthesis of a series of β-keto sulfides and β-keto sulfones. The transformation, mediated by O2 , proceeds smoothly in water and without any catalyst. Prominent advantages of this method include mild reaction conditions, purification simplicity, and gram-scale synthesis, underlining the practical utility of this methodology. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review.

PubMed

Meyer, Jeremy; Gonelle-Gispert, Carmen; Morel, Philippe; Bühler, Léo

2016-01-01

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30-141.9 million cells for 85-98% purities; for mice 9-9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5-100 million cells for 73.7-95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5-9 million cells with 90-98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver.

Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review

PubMed Central

Meyer, Jeremy; Gonelle-Gispert, Carmen; Morel, Philippe; Bühler, Léo

2016-01-01

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30–141.9 million cells for 85–98% purities; for mice 9–9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5–100 million cells for 73.7–95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5–9 million cells with 90–98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver. PMID:26992171

Homogeneous purification and characterization of LePGT1--a membrane-bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon.

PubMed

Ohara, Kazuaki; Mito, Koji; Yazaki, Kazufumi

2013-06-01

Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family. © 2013 The Authors Journal compilation © 2013 FEBS.

One-step separation and purification of three lignans and one flavonol from Sinopodophyllum emodi by medium-pressure liquid chromatography and high-speed counter-current chromatography.

PubMed

Wang, Ping; Liu, Yongling; Chen, Tao; Xu, Wenhua; You, Jinmao; Liu, Yongjun; Li, Yulin

2013-01-01

Lignans and flavonols are the primary constituents of Sinopodophyllum emodi and have been used as cathartic, anthelmintic, chemotherapeutic and anti-hypertensive compounds. Although these compounds have been isolated, there have been no reports on the separation of 4'-demethyl podophyllotoxin, podophyllotoxin, deoxypodophyllotoxin and kaempferol in one step by medium-pressure liquid chromatography (MPLC) and high-speed counter-current chromatography (HSCCC). Development of an efficient method for the preparative separation and purification of three lignans and one flavonol from S. emodi. The precipitate of crude extracts was first separated by MPLC into four parts, numbered GJ-1, GJ-2, GJ-3 and GJ-4. GJ-1 was separated and purified by HSCCC using a solvent system composed of n-hexane:ethyl acetate:methanol:water (1.75:1.5:1:0.75, v/v/v/v). The purities of the target compounds were assessed using high-performance liquid chromatography (HPLC) and chemical structures were identified by (1) H-NMR and (13) C-NMR. The HSCCC and MPLC methods were successfully used for the preparative separation and purification of 4'-demethyl podophyllotoxin (8.5 mg, 92.4%), podophyllotoxin (40.1 mg, 92.1%), deoxypodophyllotoxin (4.6 mg, 98.1%), and kaempferol (1.6 mg, 96.7%) from a 100 mg sample. Three lignans (4'-demethyl podophyllotoxin, podophyllotoxin, deoxypodophyllotoxin) and one flavonol (kaempferol) were successfully isolated by HSCCC and MPLC in one step. Copyright © 2013 John Wiley & Sons, Ltd.

Preparation and purification of organic samples for selenium isotope studies.

PubMed

Banning, Helena; Stelling, Monika; König, Stephan; Schoenberg, Ronny; Neumann, Thomas

2018-01-01

Selenium (Se) is an important micronutrient but also a strong toxin with a narrow tolerance range for many organisms. As such, a globally heterogeneous Se distribution in soils is responsible for various disease patterns (i.e. Se excess and deficiency) and environmental problems, whereby plants play a key role for the Se entrance into the biosphere. Selenium isotope variations were proved to be a powerful tracer for redox processes and are therefore promising for the exploration of the species dependent Se metabolism in plants and the Se cycling within the Critical Zone. Plant cultivation setups enable systematic controlled investigations, but samples derived from them-plant tissue and phytoagar-are particularly challenging and require specific preparation and purification steps to ensure precise and valid Se isotope analytics performed with HG-MC-ICP-MS. In this study, different methods for the entire process from solid tissue preparation to Se isotope measurements were tested, optimized and validated. A particular microwave digestion procedure for plant tissue and a vacuum filtration method for phytoagar led to full Se recoveries, whereby unfavorable organic residues were reduced to a minimum. Three purification methods predominantly described in the literature were systematically tested with pure Se solution, high concentrated multi-element standard solution as well as plant and phytoagar as target matrices. All these methods efficiently remove critical matrix elements, but differ in Se recovery and organic residues. Validation tests doping Se-free plant material and phytoagar with a reference material of known Se isotope composition revealed the high impact of organic residues on the accuracy of MC-ICP-MS measurements. Only the purification method with no detectable organic residues, hydride generation and trapping, results in valid mass bias correction for plant samples with an average deviation to true δ82/76Se values of 0.2 ‰ and a reproducibility (2 SD) of ± 0.2 ‰. For phytoagar this test yields a higher deviation of 1.1 ‰ from the true value and a 2 SD of ± 0.1 ‰. The application of the developed methods to cultivated plants shows sufficient accuracy and precision and is a promising approach to resolve plant internal Se isotope fractionations, for which respective δ82/76Se values of +2.3 to +3.5 ‰ for selenate and +1.2 to +1.9 ‰ for selenite were obtained.

Preparation and purification of organic samples for selenium isotope studies

PubMed Central

Stelling, Monika; König, Stephan; Schoenberg, Ronny; Neumann, Thomas

2018-01-01

Selenium (Se) is an important micronutrient but also a strong toxin with a narrow tolerance range for many organisms. As such, a globally heterogeneous Se distribution in soils is responsible for various disease patterns (i.e. Se excess and deficiency) and environmental problems, whereby plants play a key role for the Se entrance into the biosphere. Selenium isotope variations were proved to be a powerful tracer for redox processes and are therefore promising for the exploration of the species dependent Se metabolism in plants and the Se cycling within the Critical Zone. Plant cultivation setups enable systematic controlled investigations, but samples derived from them–plant tissue and phytoagar–are particularly challenging and require specific preparation and purification steps to ensure precise and valid Se isotope analytics performed with HG-MC-ICP-MS. In this study, different methods for the entire process from solid tissue preparation to Se isotope measurements were tested, optimized and validated. A particular microwave digestion procedure for plant tissue and a vacuum filtration method for phytoagar led to full Se recoveries, whereby unfavorable organic residues were reduced to a minimum. Three purification methods predominantly described in the literature were systematically tested with pure Se solution, high concentrated multi-element standard solution as well as plant and phytoagar as target matrices. All these methods efficiently remove critical matrix elements, but differ in Se recovery and organic residues. Validation tests doping Se-free plant material and phytoagar with a reference material of known Se isotope composition revealed the high impact of organic residues on the accuracy of MC-ICP-MS measurements. Only the purification method with no detectable organic residues, hydride generation and trapping, results in valid mass bias correction for plant samples with an average deviation to true δ82/76Se values of 0.2 ‰ and a reproducibility (2 SD) of ± 0.2 ‰. For phytoagar this test yields a higher deviation of 1.1 ‰ from the true value and a 2 SD of ± 0.1 ‰. The application of the developed methods to cultivated plants shows sufficient accuracy and precision and is a promising approach to resolve plant internal Se isotope fractionations, for which respective δ82/76Se values of +2.3 to +3.5 ‰ for selenate and +1.2 to +1.9 ‰ for selenite were obtained. PMID:29509798

Automated high throughput microscale antibody purification workflows for accelerating antibody discovery

PubMed Central

Luan, Peng; Lee, Sophia; Paluch, Maciej; Kansopon, Joe; Viajar, Sharon; Begum, Zahira; Chiang, Nancy; Nakamura, Gerald; Hass, Philip E.; Wong, Athena W.; Lazar, Greg A.

2018-01-01

ABSTRACT To rapidly find “best-in-class” antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification. PMID:29494273

Purification of functionalized DNA origami nanostructures.

PubMed

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.

Biocompatible water softening system using cationic protein from moringa oleifera extract

NASA Astrophysics Data System (ADS)

Nisha, R. R.; Jegathambal, P.; Parameswari, K.; Kirupa, K.

2017-10-01

In developing countries like India, the deciding factors for the selection of the specific water purification system are the flow rate, cost of implementation and maintenance, availability of materials for fabrication or assembling, technical manpower, energy requirement and reliability. But most of them are energy and cost intensive which necessitate the development of cost-effective water purification system. In this study, the feasibility of development of an efficient and cost-effective water purifier using Moringa oleifera cationic protein coated sand column to treat drinking water is presented. Moringa oleifera seeds contain cationic antimicrobial protein which acts as biocoagulant in the removal of turbidity and also aids in water softening. The main disadvantage of using Moringa seeds in water purification is that the dissolved organic matter (DOM) which is left over in the water contributes to growth of any pathogens that come into contact with the stored water. To overcome this limitation, the Moringa oleifera cationic protein coated sand (MOCP c-sand) is prepared in which the flocculant and antimicrobial properties of the MOCP are maintained and the DOM to be rinsed away. The efficiency of MOCP c-sand in removing suspended particles and reducing total hardness (TH), chloride, total dissolved solids (TDS), electrical conductivity (EC) was also studied. Also, it is shown that the functionalized sand showed the same treatment efficiency even after being stored dry and in dehydrated condition for 3 months. This confirms MOCP c-sand's potential as a locally sustainable water treatment option for developing countries since other chemicals used in water purification are expensive.

Automated high-throughput purification of genomic DNA from plant leaf or seed using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2004-06-01

Three different methods of automated high throughput purification of genomic DNA from plant materials processed in 96 well plates are described. One method uses MagneSil paramagnetic particles to purify DNA present in single leaf punch samples or small seed samples, using 320ul capacity 96 well plates which minimizes reagent and plate costs. A second method uses 2.2 ml and 1.2 ml capacity plates and allows the purification of larger amounts of DNA from 5-6 punches of materials or larger amounts of seeds. The third method uses the MagneSil ONE purification system to purify a fixed amount of DNA, thus simplifying the processing of downstream applications by normalizing the amounts of DNA so they do not require quantitation. Protocols for the purification of a fixed yield of DNA, e.g. 1 ug, from plant leaf or seed samples using MagneSil paramagnetic particles and a Beckman-Coulter BioMek FX robot are described. DNA from all three methods is suitable for applications such as PCR, RAPD, STR, READIT SNP analysis, and multiplexed PCR systems. The MagneSil ONE system is also suitable for use with SNP detection systems such as Third Wave Technology"s Invader methods.

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

PubMed

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

2016-02-01

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

Photoelectric characterization of fabricated dye-sensitized solar cell using dye extracted from red Siahkooti fruit as natural sensitizer

NASA Astrophysics Data System (ADS)

Mozaffari, Sayed Ahmad; Saeidi, Mahsa; Rahmanian, Reza

2015-05-01

Natural dye extracted from Siahkooti fruit with/without purification by solid phase extraction (SPE) technique was used in the fabrication of DSSC as natural sensitizer. The UV-Vis absorption spectroscopy and Fourier transform infrared (FTIR) were employed to indicate the presence of anthocyanins in the fruit of red Siahkooti. The photoelectrochemical performance and the efficiency of assembled DSSC using Siahkooti fruit dye extract were evaluated and efficiency enhancement was obtained by a preliminary purification of extracted dye. The efficiency and fill factor of the DSSC using purified Siahkooti fruit dye were 0.32% and 0.73%, respectively. The results successfully showed that the DSSC, using Siahkooti fruit extract as a dye sensitizer, is useful for the preparation of environmentally friendly, low-cost, renewable and clean sources of energy.

Single-step affinity purification for fungal proteomics.

PubMed

Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

2010-05-01

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

Purification process for vertically aligned carbon nanofibers

NASA Technical Reports Server (NTRS)

Nguyen, Cattien V.; Delziet, Lance; Matthews, Kristopher; Chen, Bin; Meyyappan, M.

2003-01-01

Individual, free-standing, vertically aligned multiwall carbon nanotubes or nanofibers are ideal for sensor and electrode applications. Our plasma-enhanced chemical vapor deposition techniques for producing free-standing and vertically aligned carbon nanofibers use catalyst particles at the tip of the fiber. Here we present a simple purification process for the removal of iron catalyst particles at the tip of vertically aligned carbon nanofibers derived by plasma-enhanced chemical vapor deposition. The first step involves thermal oxidation in air, at temperatures of 200-400 degrees C, resulting in the physical swelling of the iron particles from the formation of iron oxide. Subsequently, the complete removal of the iron oxide particles is achieved with diluted acid (12% HCl). The purification process appears to be very efficient at removing all of the iron catalyst particles. Electron microscopy images and Raman spectroscopy data indicate that the purification process does not damage the graphitic structure of the nanotubes.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification.

PubMed

Shin, Min Jae; Tan, Lihan; Jeong, Min Ho; Kim, Ji-Heung; Choe, Woo-Seok

2011-08-05

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification. Copyright © 2011 Elsevier B.V. All rights reserved.

The SNO+ Scintillator Purification Plant and Projected Sensitivity to Solar Neutrinos in the Pure Scintillator Phase

NASA Astrophysics Data System (ADS)

Pershing, Teal; SNO+ Collaboration

2016-03-01

The SNO+ detector is a neutrino and neutrinoless double-beta decay experiment utilizing the renovated SNO detector. In the second phase of operation, the SNO+ detector will contain 780 tons of organic liquid scintillator composed of 2 g/L 2,5-diphenyloxazole (PPO) in linear alkylbenzene (LAB). In this phase, SNO+ will strive to detect solar neutrinos in the sub-MeV range, including CNO production neutrinos and pp production neutrinos. To achieve the necessary detector sensitivity, a four-part scintillator purification plant has been constructed in SNOLAB for the removal of ionic and radioactive impurities. We present an overview of the SNO+ scintillator purification plant stages, including distillation, water extraction, gas stripping, and metal scavenger columns. We also give the projected SNO+ sensitivities to various solar-produced neutrinos based on the scintillator plant's projected purification efficiency.

Generation of Envelope-Modified Baculoviruses for Gene Delivery into Mammalian Cells.

PubMed

Hofmann, Christian

2016-01-01

Genetically modified baculoviruses can efficiently deliver and express genes in mammalian cells. The major prerequisite for the expression of a gene transferred by baculovirus is its control by a promoter that is active in mammalian cells. This chapter describes methods for producing second generation baculovirus vectors through modification of their envelope. Envelope modified baculoviruses offer additional new applications of the system, such as their use in in vivo gene delivery, targeting, and vaccination. Methods of generating a recombinant baculovirus vector with a modified envelope and its amplification and purification, including technical scale production, are discussed. A variety of notes give clues regarding specific technical procedures. Finally, methods to analyze the virus and transduction procedures are presented.

Advanced Method for Isolation of Mouse Hepatocytes, Liver Sinusoidal Endothelial Cells, and Kupffer Cells.

PubMed

Liu, Jia; Huang, Xuan; Werner, Melanie; Broering, Ruth; Yang, Dongliang; Lu, Mengji

2017-01-01

Separation of pure cell populations from the liver is a prerequisite to study the role of hepatic parenchymal and non-parenchymal cells in liver physiology, pathophysiology, and immunology. Traditional methods for hepatic cell separation usually purify only single cell types from liver specimens. Here, we describe an efficient method that can simultaneously purify populations of hepatocytes (HCs), liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs) from a single mouse liver specimen. A liberase-based perfusion technique in combination with a low-speed centrifugation and magnetic-activated cell sorting (MACS) led to the isolation and purification of HCs, KCs, and LSECs with high yields and purity.

Fermentation and purification strategies for the production of betulinic acid and its lupane-type precursors in Saccharomyces cerevisiae.

PubMed

Czarnotta, Eik; Dianat, Mariam; Korf, Marcel; Granica, Fabian; Merz, Juliane; Maury, Jérôme; Baallal Jacobsen, Simo A; Förster, Jochen; Ebert, Birgitta E; Blank, Lars M

2017-11-01

Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L, and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering. © 2017 Wiley Periodicals, Inc.

Enzymatic Purification of Microplastics in Environmental Samples.

PubMed

Löder, Martin G J; Imhof, Hannes K; Ladehoff, Maike; Löschel, Lena A; Lorenz, Claudia; Mintenig, Svenja; Piehl, Sarah; Primpke, Sebastian; Schrank, Isabella; Laforsch, Christian; Gerdts, Gunnar

2017-12-19

Micro-Fourier transform infrared (micro-FTIR) spectroscopy and Raman spectroscopy enable the reliable identification and quantification of microplastics (MPs) in the lower micron range. Since concentrations of MPs in the environment are usually low, the large sample volumes required for these techniques lead to an excess of coenriched organic or inorganic materials. While inorganic materials can be separated from MPs using density separation, the organic fraction impedes the ability to conduct reliable analyses. Hence, the purification of MPs from organic materials is crucial prior to conducting an identification via spectroscopic techniques. Strong acidic or alkaline treatments bear the danger of degrading sensitive synthetic polymers. We suggest an alternative method, which uses a series of technical grade enzymes for purifying MPs in environmental samples. A basic enzymatic purification protocol (BEPP) proved to be efficient while reducing 98.3 ± 0.1% of the sample matrix in surface water samples. After showing a high recovery rate (84.5 ± 3.3%), the BEPP was successfully applied to environmental samples from the North Sea where numbers of MPs range from 0.05 to 4.42 items m -3 . Experiences with different environmental sample matrices were considered in an improved and universally applicable version of the BEPP, which is suitable for focal plane array detector (FPA)-based micro-FTIR analyses of water, wastewater, sediment, biota, and food samples.

High performance of a unique mesoporous polystyrene-based adsorbent for blood purification

PubMed Central

Chen, Jian; Han, Wenyan; Chen, Jie; Zong, Wenhui; Wang, Weichao; Wang, Yue; Cheng, Guanghui; Li, Chunran; Ou, Lailiang; Yu, Yaoting

2017-01-01

A multi-functional polystyrene based adsorbent (NKU-9) with a unique mesoporous and a high surface area was prepared by suspension polymerization for removal of therapeutic toxins in blood purification. The adsorbent produced had an almost equal amount of mesopore distribution in the range from 2 to 50 nm. The adsorption of serum toxins with different molecular weights were examined by in vitro adsorption assays and compared with some clinical currently used adsorbents such as HA-330, Cytosorb and BL-300 which are produced by China, America and Japan, respectively. Test results indicated that the adsorption rate for pentobarbital by NKU-9 was 81.24% which is nearly as high as HA-330 (81.44%). The latter adsorbent is currently used for acute detoxification treatment in China. To reach adsorption equilibrium, NKU-9 was faster than HA-330, which implies short treatment time. For the removal of middle molecular toxins such as β2-microglobulin (98.88%), NKU-9 performed better adsorptive selectivity than Cytosorb (92.80%). In addition, NKU-9 showed high performance for the removal of albumin-bound toxins (e.g., bilirubin), and its adsorption rate for total bilirubin (80.79%) in plasma was 8.4% higher than that of anion exchange resin BL-300 which is currently used to eliminate bilirubin in clinic. Therefore, our results indicate that the newly developed adsorbent with a wide distribution and almost equal amount of mesopores is a multifunctional adsorbent for high efficient removal of serum toxins with different molecular weights which might be an excellent blood purification adsorbent especially to treat diseases that conventional medical methods are low or not efficient. PMID:28149527

High performance of a unique mesoporous polystyrene-based adsorbent for blood purification.

PubMed

Chen, Jian; Han, Wenyan; Chen, Jie; Zong, Wenhui; Wang, Weichao; Wang, Yue; Cheng, Guanghui; Li, Chunran; Ou, Lailiang; Yu, Yaoting

2017-02-01

A multi-functional polystyrene based adsorbent (NKU-9) with a unique mesoporous and a high surface area was prepared by suspension polymerization for removal of therapeutic toxins in blood purification. The adsorbent produced had an almost equal amount of mesopore distribution in the range from 2 to 50 nm. The adsorption of serum toxins with different molecular weights were examined by in vitro adsorption assays and compared with some clinical currently used adsorbents such as HA-330, Cytosorb and BL-300 which are produced by China, America and Japan, respectively. Test results indicated that the adsorption rate for pentobarbital by NKU-9 was 81.24% which is nearly as high as HA-330 (81.44%). The latter adsorbent is currently used for acute detoxification treatment in China. To reach adsorption equilibrium, NKU-9 was faster than HA-330, which implies short treatment time. For the removal of middle molecular toxins such as β2-microglobulin (98.88%), NKU-9 performed better adsorptive selectivity than Cytosorb (92.80%). In addition, NKU-9 showed high performance for the removal of albumin-bound toxins (e.g., bilirubin), and its adsorption rate for total bilirubin (80.79%) in plasma was 8.4% higher than that of anion exchange resin BL-300 which is currently used to eliminate bilirubin in clinic. Therefore, our results indicate that the newly developed adsorbent with a wide distribution and almost equal amount of mesopores is a multifunctional adsorbent for high efficient removal of serum toxins with different molecular weights which might be an excellent blood purification adsorbent especially to treat diseases that conventional medical methods are low or not efficient.

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni.

PubMed

Gencay, Yilmaz Emre; Birk, Tina; Sørensen, Martine Camilla Holst; Brøndsted, Lone

2017-01-01

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Purification of swine haptoglobin by affinity chromatography.

PubMed Central

Eurell, T E; Hall, W F; Bane, D P

1990-01-01

A globin-agarose affinity chromatography technique was used to purify swine haptoglobin. This technique provides a highly specific, single-step purification method without the contamination of extraneous serum proteins reported by previous studies. Complex formation between the haptoglobin isolate and swine hemoglobin confirmed that biological activity was maintained during the purification process. Immunoelectrophoretic and Ouchterlony immunodiffusion methods revealed that the swine haptoglobin isolate cross-reacted with polyvalent antisera against human haptoglobin. Images Fig. 2. Fig. 3. PMID:2123414

Comparison of two methods for purification of enterocin B, a bacteriocin produced by Enterococcus faecium W3.

PubMed

Dündar, Halil; Atakay, Mehmet; Çelikbıçak, Ömür; Salih, Bekir; Bozoğlu, Faruk

2015-01-01

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

Enrichment of Undifferentiated Type A Spermatogonia from Goat Testis Using Discontinuous Percoll Density Gradient and Differential Plating

PubMed Central

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-01-01

Background The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Methods Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). Results The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Conclusion Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes. PMID:24834311

Purification of liquid metal systems with sodium coolant from oxygen using getters

NASA Astrophysics Data System (ADS)

Kozlov, F. A.; Konovalov, M. A.; Sorokin, A. P.

2016-05-01

For increasing the safety and economic parameters of nuclear power stations (NPSs) with sodium coolant, it was decided to install all systems contacting radioactive sodium, including purification systems of circuit I, in the reactor vessel. The performance and capacity of cold traps (CTs) (conventional element of coolant purification systems) in these conditions are limited by their volume. It was proposed to use hot traps (HTs) in circuit I for coolant purification from oxygen. It was demonstrated that, at rated parameters of the installation when the temperature of the coolant streamlining the getter (gas absorber) is equal to 550°C, the hot trap can provide the required coolant purity. In shutdown modes at 250-300°C, the performance of the hot trap is reduced by four orders of magnitude. Possible HT operation regimes for shutdown modes and while reaching rated parameters were proposed and analyzed. Basic attention was paid to purification modes at power rise after commissioning and accidental contamination of the coolant when the initial oxygen concentration in it reached 25 mln-1. It was demonstrated that the efficiency of purification systems can be increased using HTs with the getter in the form of a foil or granules. The possibility of implementing the "fast purification" mode in which the coolant is purified simultaneously with passing over from the shutdown mode to the rated parameters was substantiated.

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Very large scale monoclonal antibody purification: the case for conventional unit operations.

PubMed

Kelley, Brian

2007-01-01

Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using today's commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the world's largest single-plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non-conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.

Graphene-Based Standalone Solar Energy Converter for Water Desalination and Purification.

PubMed

Yang, Yang; Zhao, Ruiqi; Zhang, Tengfei; Zhao, Kai; Xiao, Peishuang; Ma, Yanfeng; Ajayan, Pulickel M; Shi, Gaoquan; Chen, Yongsheng

2018-01-23

Harvesting solar energy for desalination and sewage treatment has been considered as a promising solution to produce clean water. However, state-of-the-art technologies often require optical concentrators and complicated systems with multiple components, leading to poor efficiency and high cost. Here, we demonstrate an extremely simple and standalone solar energy converter consisting of only an as-prepared 3D cross-linked honeycomb graphene foam material without any other supporting components. This simple all-in-one material can act as an ideal solar thermal converter capable of capturing and converting sunlight into heat, which in turn can distill water from various water sources into steam and produce purified water under ambient conditions and low solar flux with very high efficiency. High specific water production rate of 2.6 kg h -1 m -2 g -1 was achieved with near ∼87% under 1 sun intensity and >80% efficiency even under ambient sunlight (<1 sun). This scalable sheet-like material was used to obtain pure drinkable water from both seawater and sewage water under ambient conditions. Our results demonstrate a competent monolithic material platform providing a paradigm change in water purification by using a simple, point of use, reusable, and low-cost solar thermal water purification system for a variety of environmental conditions.

Microplate-Based Method for High-Throughput Screening (HTS) of Chromatographic Conditions Studies for Recombinant Protein Purification.

PubMed

Carvalho, Rimenys J; Cruz, Thayana A

2018-01-01

High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation of several conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification. Here, we propose a method for studying different purification conditions that can be used for any recombinant protein, including complex and glycosylated proteins, using low binding filter microplates.

Application of dielectric constant measurement in microwave sludge disintegration and wastewater purification processes.

PubMed

Kovács, Petra Veszelovszki; Lemmer, Balázs; Keszthelyi-Szabó, Gábor; Hodúr, Cecilia; Beszédes, Sándor

2018-05-01

It has been numerously verified that microwave radiation could be advantageous as a pre-treatment for enhanced disintegration of sludge. Very few data related to the dielectric parameters of wastewater of different origins are available; therefore, the objective of our work was to measure the dielectric constant of municipal and meat industrial wastewater during a continuous flow operating microwave process. Determination of the dielectric constant and its change during wastewater and sludge processing make it possible to decide on the applicability of dielectric measurements for detecting the organic matter removal efficiency of wastewater purification process or disintegration degree of sludge. With the measurement of dielectric constant as a function of temperature, total solids (TS) content and microwave specific process parameters regression models were developed. Our results verified that in the case of municipal wastewater sludge, the TS content has a significant effect on the dielectric constant and disintegration degree (DD), as does the temperature. The dielectric constant has a decreasing tendency with increasing temperature for wastewater sludge of low TS content, but an adverse effect was found for samples with high TS and organic matter contents. DD of meat processing wastewater sludge was influenced significantly by the volumetric flow rate and power level, as process parameters of continuously flow microwave pre-treatments. It can be concluded that the disintegration process of food industry sludge can be detected by dielectric constant measurements. From technical purposes the applicability of dielectric measurements was tested in the purification process of municipal wastewater, as well. Determination of dielectric behaviour was a sensitive method to detect the purification degree of municipal wastewater.

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

PubMed

Mackin, Robert B

2014-01-01

The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin

PubMed Central

Mackin, Robert B.

2014-01-01

The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix. PMID:26150942

Synthesis of amino-rich silica-coated magnetic nanoparticles for the efficient capture of DNA for PCR.

PubMed

Bai, Yalong; Cui, Yan; Paoli, George C; Shi, Chunlei; Wang, Dapeng; Zhou, Min; Zhang, Lida; Shi, Xianming

2016-09-01

Magnetic separation has great advantages over traditional bio-separation methods and has become popular in the development of methods for the detection of bacterial pathogens, viruses, and transgenic crops. Functionalization of magnetic nanoparticles is a key factor for efficient capture of the target analytes. In this paper, we report the synthesis of amino-rich silica-coated magnetic nanoparticles using a one-pot method. This type of magnetic nanoparticle has a rough surface and a higher density of amino groups than the nanoparticles prepared by a post-modification method. Furthermore, the results of hydrochloric acid treatment indicated that the magnetic nanoparticles were stably coated. The developed amino-rich silica-coated magnetic nanoparticles were used to directly adsorb DNA. After magnetic separation and blocking, the magnetic nanoparticles and DNA complexes were used directly for the polymerase chain reaction (PCR), without onerous and time-consuming purification and elution steps. The results of real-time quantitative PCR showed that the nanoparticles with higher amino group density resulted in improved DNA capture efficiency. The results suggest that amino-rich silica-coated magnetic nanoparticles are of great potential for efficient bio-separation of DNA prior to detection by PCR. Copyright © 2016. Published by Elsevier B.V.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications.

PubMed

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-06-26

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

PubMed Central

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-01-01

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications. PMID:26113394

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

NASA Astrophysics Data System (ADS)

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-06-01

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

A simple microfluidic platform for rapid and efficient production of the radiotracer [18F]fallypride.

PubMed

Zhang, Xin; Liu, Fei; Knapp, Karla-Anne; Nickels, Michael L; Manning, H Charles; Bellan, Leon M

2018-05-01

Herein, we report the development of a simple, high-throughput and efficient microfluidic system for synthesizing radioactive [18F]fallypride, a PET imaging radiotracer widely used in medical research. The microfluidic chip contains all essential modules required for the synthesis and purification of radioactive fallypride. The radiochemical yield of the tracer is sufficient for multiple animal injections for preclinical imaging studies. To produce the on-chip concentration and purification columns, we employ a simple "trapping" mechanism by inserting rows of square pillars with predefined gaps near the outlet of microchannel. Microspheres with appropriate functionality are suspended in solution and loaded into the microchannels to form columns for radioactivity concentration and product purification. Instead of relying on complicated flow control elements (e.g., micromechanical valves requiring complex external pneumatic actuation), external valves are utilized to control transfer of the reagents between different modules. The on-chip ion exchange column can efficiently capture [18F]fluoride with negligible loss (∼98% trapping efficiency), and subsequently release a burst of concentrated [18F]fluoride to the reaction cavity. A thin layer of PDMS with a small hole in the center facilitates rapid and reliable water evaporation (with the aid of azeotropic distillation and nitrogen flow) while reducing fluoride loss. During the solvent exchange and fluorination reaction, the entire chip is uniformly heated to the desired temperature using a hot plate. All aspects of the [18F]fallypride synthesis were monitored by high-performance liquid chromatography (HPLC) analysis, resulting in labelling efficiency in fluorination reaction ranging from 67-87% (n = 5). Moreover, after isolating unreacted [18F]fluoride, remaining fallypride precursor, and various by-products via an on-chip purification column, the eluted [18F]fallypride is radiochemically pure and of a sufficient quantity to allow for PET imaging (∼5 mCi). Finally, a positron emission tomography (PET) image of a rat brain injected with ∼300 μCi [18F]fallypride produced by our microfluidic chip is provided, demonstrating the utility of the product produced by the microfluidic reactor. With a short synthesis time (∼60 min) and a highly integrated on-chip modular configuration that allows for concentration, reaction, and product purification, our microfluidic chip offers numerous exciting advantages with the potential for applications in radiochemical research and clinical production. Moreover, due to its simplicity and potential for automation, we anticipate it may be easily integrated into a clinical environment.

Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein.

PubMed

Ding, Xueqin; Wang, Yuzhi; Zeng, Qun; Chen, Jing; Huang, Yanhua; Xu, Kaijia

2014-03-07

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by (1)H nuclear magnetic resonance ((1)H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV-vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV-vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Optimized anion exchange column isolation of zirconium-89 (89Zr) from yttrium cyclotron target: Method development and implementation on an automated fluidic platform.

PubMed

O'Hara, Matthew J; Murray, Nathaniel J; Carter, Jennifer C; Morrison, Samuel S

2018-04-13

Zirconium-89 ( 89 Zr), produced by the (p, n) reaction from naturally monoisotopic yttrium ( nat Y), is a promising positron emitting isotope for immunoPET imaging. Its long half-life of 78.4 h is sufficient for evaluating slow physiological processes. A prototype automated fluidic system, coupled to on-line and in-line detectors, has been constructed to facilitate development of new 89 Zr purification methodologies. The highly reproducible reagent delivery platform and near-real time monitoring of column effluents allows for efficient method optimization. The separation of Zr from dissolved Y metal targets was evaluated using several anion exchange resins. Each resin was evaluated against its ability to quantitatively capture Zr from a load solution high in dissolved Y. The most appropriate anion exchange resin for this application was identified, and the separation method was optimized. The method is capable of a high Y decontamination factor (>10 5 ) and has been shown to remove Fe, an abundant contaminant in Y foils, from the 89 Zr elution fraction. Finally, the method was evaluated using cyclotron bombarded Y foil targets; the method was shown to achieve >95% recovery of the 89 Zr present in the foils. The anion exchange column method described here is intended to be the first 89 Zr isolation stage in a dual-column purification process. Copyright © 2018 Elsevier B.V. All rights reserved.

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ugai, Hideyo; Yamasaki, Takahito; Hirose, Megumi

2005-06-17

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 deg C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-formingmore » units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35 x 10{sup 10} pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.« less

Removal of Pb, Cd, and Cr in a water purification system using modified mineral waste materials and activated carbon derived from waste materials

NASA Astrophysics Data System (ADS)

Lu, H. R.; Su, L. C.; Ruan, H. D.

2016-08-01

This study attempts to find out and optimize the removal efficiency of heavy metals in a water purification unit using a low-cost waste material and modified mineral waste materials (MMWM) accompanied with activated carbon (AC) derived from waste materials. The factors of the inner diameter of the purification unit (2.6-5cm), the height of the packing materials (5-20cm), the size of AC (200-20mesh), the size of MMWM (1-0.045mm), and the ratio between AC and MMWM in the packing materials (1:0 - 0:1) were examined based on a L18 (5) 3 orthogonal array design. In order to achieve an optimally maximum removal efficiency, the factors of the inner diameter of the purification unit (2.6-7.5cm), the height of the packing materials (10-30cm), and the ratio between AC and MMWM in the packing materials (1:4-4:1) were examined based on a L16 (4) 3 orthogonal array design. A height of 25cm, inner diameter of 5cm, ratio between AC and MMWM of 3:2 with size of 60-40mesh and 0.075-0.045mm, respectively, were the best conditions determined by the ICP-OES analysis to perform the adsorption of heavy metals in this study.

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

PubMed Central

McGrath, M; Witte, O; Pincus, T; Weissman, I L

1978-01-01

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques. Images PMID:205680

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.

PubMed

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-04-01

The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.

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

PubMed

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

2017-03-01

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

Purification of a Recombinant Glutathione Transferase from the Causative Agent of Hydatidosis, "Echinococcus granulosus"

ERIC Educational Resources Information Center

Fleitas, Andrea L.; Randall, Lía M.; Möller, Matías N.; Denicola, Ana

2016-01-01

This practical class activity was designed to introduce students to recombinant protein expression and purification. The principal goal is to shed light on basic aspects concerning recombinant protein production, in particular protein expression, chromatography methods for protein purification, and enzyme activity as a tool to evaluate purity and…

A Versatile and Inexpensive Enzyme Purification Experiment for Undergraduate Biochemistry Labs.

ERIC Educational Resources Information Center

Farrell, Shawn O.; Choo, Darryl

1989-01-01

Develops an experiment that could be done in two- to three-hour blocks and does not rely on cold room procedures for most of the purification. Describes the materials, methods, and results of the purification of bovine heart lactate dehydrogenase using ammonium sulfate fractionation, dialysis, and separation using affinity chromatography and…

Photoelectric characterization of fabricated dye-sensitized solar cell using dye extracted from red Siahkooti fruit as natural sensitizer.

PubMed

Mozaffari, Sayed Ahmad; Saeidi, Mahsa; Rahmanian, Reza

2015-05-05

Natural dye extracted from Siahkooti fruit with/without purification by solid phase extraction (SPE) technique was used in the fabrication of DSSC as natural sensitizer. The UV-Vis absorption spectroscopy and Fourier transform infrared (FTIR) were employed to indicate the presence of anthocyanins in the fruit of red Siahkooti. The photoelectrochemical performance and the efficiency of assembled DSSC using Siahkooti fruit dye extract were evaluated and efficiency enhancement was obtained by a preliminary purification of extracted dye. The efficiency and fill factor of the DSSC using purified Siahkooti fruit dye were 0.32% and 0.73%, respectively. The results successfully showed that the DSSC, using Siahkooti fruit extract as a dye sensitizer, is useful for the preparation of environmentally friendly, low-cost, renewable and clean sources of energy. Copyright © 2015 Elsevier B.V. All rights reserved.

A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves

PubMed Central

Andersen, Natalia D.; Srinivas, Shruthi; Piñero, Gonzalo; Monje, Paula V.

2016-01-01

We herein developed a protocol for the rapid procurement of adult nerve-derived Schwann cells (SCs) that was optimized to implement an immediate enzymatic dissociation of fresh nerve tissue while maintaining high cell viability, improving yields and minimizing fibroblast and myelin contamination. This protocol introduces: (1) an efficient method for enzymatic cell release immediately after removal of the epineurium and extensive teasing of the nerve fibers; (2) an adaptable drop-plating method for selective cell attachment, removal of myelin debris, and expansion of the initial SC population in chemically defined medium; (3) a magnetic-activated cell sorting purification protocol for rapid and effective fibroblast elimination; and (4) an optional step of cryopreservation for the storage of the excess of cells. Highly proliferative SC cultures devoid of myelin and fibroblast growth were obtained within three days of nerve processing. Characterization of the initial, expanded, and cryopreserved cell products confirmed maintenance of SC identity, viability and growth rates throughout the process. Most importantly, SCs retained their sensitivity to mitogens and potential for differentiation even after cryopreservation. To conclude, this easy-to-implement and clinically relevant protocol allows for the preparation of expandable homogeneous SC cultures while minimizing time, manipulation of the cells, and exposure to culture variables. PMID:27549422

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

PubMed

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

2014-06-01

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation of a Sepia Melanin and Poly(ethylene-alt-maleic Anhydride) Hybrid Material as an Adsorbent for Water Purification

PubMed Central

Osypova, Alina; Quasso, Fiorenza; Soliveri, Guido; Ribera, Javier; Schwarze, Francis W. M. R.

2018-01-01

Meeting the increasing demand of clean water requires the development of novel efficient adsorbent materials for the removal of organic pollutants. In this context the use of natural, renewable sources is of special relevance and sepia melanin, thanks to its ability to bind a variety of organic and inorganic species, has already attracted interest for water purification. Here we describe the synthesis of a material obtained by the combination of sepia melanin and poly(ethylene-alt-maleic anhydride) (P(E-alt-MA)). Compared to sepia melanin, the resulting hybrid displays a high and fast adsorption efficiency towards methylene blue (a common industrial dye) for a wide pH range (from pH 2 to 12) and under high ionic strength conditions. It is easily recovered after use and can be reused up to three times. Given the wide availability of sepia melanin and P(E-alt-MA), the synthesis of our hybrid is simple and affordable, making it suitable for industrial water purification purposes. PMID:29360734

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

PubMed

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Separation and purification of fructooligosaccharides on a zeolite fixed-bed column.

PubMed

Kuhn, Raquel Cristine; Mazutti, Marcio Antonio; Maugeri Filho, Francisco

2014-04-01

Fructooligosaccharides (FOS), a well-known prebiotic product, are obtained by enzymatic synthesis and consist of a mixture of mono- and disaccharides. In this work, a methodology for their separation and purification was developed using a zeolite fixed-bed column. The effects of column temperature (40-60°C), eluent flow rate (0.10-0.14 mL/min), injected to bed volume percent ratio (2.6-5.1%), and ethanol concentration in the eluent (40-60%, v/v) were investigated using a fractionary factorial design (2(4-1)), having the separation efficiency and purity as target responses. Additional experiments were performed as well, where the temperature and ethanol concentration were studied in a central composite design (2(2)). In this work, the zeolite fixed-bed column was shown to be a good alternative for FOS purification, allowing a FOS purity of 90% and separation efficiency of 6.86 between FOS and glucose, using an eluent at 45°C with 60% ethanol concentration. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation and purification of all-trans diadinoxanthin and all-trans diatoxanthin from diatom Phaeodactylum tricornutum.

PubMed

Kuczynska, Paulina; Jemiola-Rzeminska, Malgorzata

2017-01-01

Two diatom-specific carotenoids are engaged in the diadinoxanthin cycle, an important mechanism which protects these organisms against photoinhibition caused by absorption of excessive light energy. A high-performance and economical procedure of isolation and purification of diadinoxanthin and diatoxanthin from the marine diatom Phaeodactylum tricornutum using a four-step procedure has been developed. It is based on the use of commonly available materials and does not require advanced technology. Extraction of pigments, saponification, separation by partition and then open column chromatography, which comprise the complete experimental procedure, can be performed within 2 days. This method allows HPLC grade diadinoxanthin and diatoxanthin of a purity of 99 % or more to be obtained, and the efficiency was estimated to be 63 % for diadinoxanthin and 73 % for diatoxanthin. Carefully selected diatom culture conditions as well as analytical ones ensure highly reproducible performance. A protocol can be used to isolate and purify the diadinoxanthin cycle pigments both on analytical and preparative scale.

Removal of arsenic, phosphates and ammonia from well water using electrochemical/chemical methods and advanced oxidation: a pilot plant approach.

PubMed

Orescanin, Visnja; Kollar, Robert; Nad, Karlo; Halkijevic, Ivan; Kuspilic, Marin; Findri Gustek, Stefica

2014-01-01

The purpose of this work was to develop a pilot plant purification system and apply it to groundwater used for human consumption, containing high concentrations of arsenic and increased levels of phosphates, ammonia, mercury and color. The groundwater used was obtained from the production well in the Vinkovci County (Eastern Croatia). Due to a complex composition of the treated water, the purification system involved a combined electrochemical treatment, using iron and aluminum electrode plates with simultaneous ozonation, followed by a post-treatment with UV, ozone and hydrogen peroxide. The removal of the contaminant with the waste sludge collected during the electrochemical treatment was also tested. The combined electrochemical and advanced oxidation treatment resulted in the complete removal of arsenic, phosphates, color, turbidity, suspended solids and ammonia, while the removal of other contaminants of interest was up to 96.7%. Comparable removal efficiencies were obtained by using waste sludge as a coagulant.

Comparing cooling systems for the COBE 2991 cell separator used in the purification of human pancreatic islets of Langerhans.

PubMed

Adewola, A; Mage, R; Hansen, M; Barbaro, B; Mendoza-Elias, J; Harvat, T; Morel, P H; Oberholzer, J; Wang, Y

2010-01-01

Two different approaches of controlled cooling of the COBE 2991 cell-separator for islet purification were evaluated. The first method is the new Geneva COBE cooling system (GCCS), which consists of an electronically controlled liquid nitrogen injection system. The second is the University of Illinois at Chicago cooling system (UICCS), which consists of a specially designed "Cold Room" maintained at 1-8 C. For the GCCS, the mean temperatures of the gradient solutions were measured at the beginning and end of centrifugation were found to be 7 +/-0.7 C and 6.8 +/-0.6 C respectively. For the UICCS, the mean temperature of the gradients at the beginning and end of centrifugation were 4.7 +/-0.53 C and 7.03 C+/-0.91 C respectively. The presented COBE cooling systems can easily be adapted to a COBE 2991 cell-separator and are efficient in maintaining gradient solutions at a defined low temperature during centrifugation.

ELECTROMAGNETIC STIRRING IN ZONE REFINING

DOE Office of Scientific and Technical Information (OSTI.GOV)

Braun, I.; Frank, F.C.; Marshall, S.

1958-02-01

The efficiency of the zone refining process can obviously be increased by stirring the molten zone to disperse the impurity-rich layer at the solid- liquid surface. Induction heating is sometimes preferred to radiant heat because it produces more convection, but no marked improvement has been reported. Pfann and Dorsi(1967) have described a method of stirring the melt by passing an electric current through the ingot and compressing a magnetic field across the molten zone. Preliminary results obtained by using a rotating magnetic field us the stirring agent during the purification of aluminum are described. (A.C.)

Process for purification of solids

NASA Technical Reports Server (NTRS)

Herzer, H.; Rath, H. J.; Schmidt, D.

1981-01-01

A process for purifying solids, especially silicon, by melting and subsequent resolidification, is described. Silicon used in solar cell manufacturing is processed more efficiently and cost effectively.

Towards the Ultimate Membranes: Two-dimensional Nanoporous Materials and Films.

PubMed

Agrawal, Kumar Varoon

2018-05-30

The energy-efficient separation of molecules has been a popular topic in chemistry and chemical engineering as a consequence of the large energy-footprint of separation processes in the chemical industry. The Laboratory of Advanced Separations (LAS) at EPFL, led by Prof. Kumar Varoon Agrawal, is focused to develop next-generation, high-performance membranes that can improve the energy efficiency of hydrogen purification, carbon capture, hydrocarbon and water purification. For this, LAS is seeking to develop the ultimate nanoporous membranes, those with a thickness of 1 nm and possessing an array of size-selective nanopores. In this article, the research activities at LAS, especially in the bottom-up and top-down synthesis of chemically and thermally stable, nanoporous two-dimensional materials and membranes are discussed.

TlBr purification and single crystal growth for the detector applications

NASA Astrophysics Data System (ADS)

Kozlov, Vasilij; Heikkilä, Mikko; Kostamo, Pasi; Lipsanen, Harri; Leskelä, Markku

2011-05-01

The combination of distillation, Bridgman-Stockbarger, hydrothermal recrystallisation and travelling molten zone (TMZ) methods were used for TlBr purification. Grown crystals were characterised by XRD rocking curve and FTIR spectroscopy methods, and by electrical measurements made from 200 to 300 K.

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

PubMed

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.

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

Application of visual basic in high-throughput mass spectrometry-directed purification of combinatorial libraries.

PubMed

Li, B; Chan, E C Y

2003-01-01

We present an approach to customize the sample submission process for high-throughput purification (HTP) of combinatorial parallel libraries using preparative liquid chromatography electrospray ionization mass spectrometry. In this study, Visual Basic and Visual Basic for Applications programs were developed using Microsoft Visual Basic 6 and Microsoft Excel 2000, respectively. These programs are subsequently applied for the seamless electronic submission and handling of data for HTP. Functions were incorporated into these programs where medicinal chemists can perform on-line verification of the purification status and on-line retrieval of postpurification data. The application of these user friendly and cost effective programs in our HTP technology has greatly increased our work efficiency by reducing paper work and manual manipulation of data.

Experimental Methods for Protein Interaction Identification and Characterization

NASA Astrophysics Data System (ADS)

Uetz, Peter; Titz, Björn; Cagney, Gerard

There are dozens of methods for the detection of protein-protein interactions but they fall into a few broad categories. Fragment complementation assays such as the yeast two-hybrid (Y2H) system are based on split proteins that are functionally reconstituted by fusions of interacting proteins. Biophysical methods include structure determination and mass spectrometric (MS) identification of proteins in complexes. Biochemical methods include methods such as far western blotting and peptide arrays. Only the Y2H and protein complex purification combined with MS have been used on a larger scale. Due to the lack of data it is still difficult to compare these methods with respect to their efficiency and error rates. Current data does not favor any particular method and thus multiple experimental approaches are necessary to maximally cover the interactome of any target cell or organism.

A novel method for simultaneous production of two ribosome-inactivating proteins, α-MMC and MAP30, from Momordica charantia L.

PubMed

Meng, Yao; Lin, Sen; Liu, Shuangfeng; Fan, Xiang; Li, Gangrui; Meng, Yanfa

2014-01-01

Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) from Momordica charantia L. have been confirmed to possess anti-tumor and anti-virus activities. Traditional purification methods of these two ribosome-inactivating proteins (RIPs) were separate which was time consuming and cost effective as well as low efficient. In order to obtain sufficient samples for researches, a strategy combining ion-exchange and gel filtration chromatography was developed and optimized in this study. Using this novel purification method, averagely 1162 mg of α-MMC and 535 mg of MAP30 were obtained from 400 g of Momordica charantia L seeds. The homogeneities of them were assessed by electrophoresis analysis. Determination of molecular weights of α-MMC and MAP30 were 28.585 kDa and 29.094 kDa by MALDI-TOF/TOF and pI were 9.02 and 9.12, respectively. The single glycoproteins were identified by Periodate-Schiff's base (PAS) and the saccharide content was tested to be 1.25% and 1.1% by anthrone-sulfuric acid method. Biological activities were evidenced by their ability to inhibit proliferation of lung adenocarcinoma A549 cell and to convert supercoiled plasmid pUC18 into relaxed forms. Finally, we also found that both two RIPs exhibited no superoxide dismutase (SOD) activity.

Solid-phase extraction method for the isolation of plant thionins from European mistletoe, wheat and barley using zirconium silicate embedded in poly(styrene-co-divinylbenzene) hollow-monoliths.

PubMed

Hussain, Shah; Güzel, Yüksel; Schönbichler, Stefan A; Rainer, Matthias; Huck, Christian W; Bonn, Günther K

2013-09-01

Thionins are cysteine-rich, biologically active small (∼5 kDa) and basic proteins occurring ubiquitously in the plant kingdom. This study describes an efficient solid-phase extraction (SPE) method for the selective isolation of these pharmacologically active proteins. Hollow-monolithic extraction tips based on poly(styrene-co-divinylbenzene) with embedded zirconium silicate nano-powder were designed, which showed an excellent selectivity for sulphur-rich proteins owing to strong co-ordination between zirconium and the sulphur atoms from the thiol-group of cysteine. The sorbent provides a combination of strong hydrophobic and electrostatic interactions which may help in targeted separation of certain classes of proteins in a complex mixture based upon the binding strength of different proteins. European mistletoe, wheat and barley samples were used for selective isolation of viscotoxins, purothionins and hordothionins, respectively. The enriched fractions were subjected to analysis by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometer to prove the selectivity of the SPE method towards thionins. For peptide mass-fingerprint analysis, tryptic digests of SPE eluates were examined. Reversed-phase high-performance liquid chromatography hyphenated to diode-array detection was employed for the purification of individual isoforms. The developed method was found to be highly specific for the isolation and purification of thionins.

Entanglement of purification: from spin chains to holography

NASA Astrophysics Data System (ADS)

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tykvart, J.; Sacha, P.; Barinka, C.

2012-02-07

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo.more » We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.« less

Item Purification Does Not Always Improve DIF Detection: A Counterexample with Angoff's Delta Plot

ERIC Educational Resources Information Center

Magis, David; Facon, Bruno

2013-01-01

Item purification is an iterative process that is often advocated as improving the identification of items affected by differential item functioning (DIF). With test-score-based DIF detection methods, item purification iteratively removes the items currently flagged as DIF from the test scores to get purified sets of items, unaffected by DIF. The…

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

PubMed

Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

2015-11-19

Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

Isotopic composition analysis and age dating of uranium samples by high resolution gamma ray spectrometry

NASA Astrophysics Data System (ADS)

Apostol, A. I.; Pantelica, A.; Sima, O.; Fugaru, V.

2016-09-01

Non-destructive methods were applied to determine the isotopic composition and the time elapsed since last chemical purification of nine uranium samples. The applied methods are based on measuring gamma and X radiations of uranium samples by high resolution low energy gamma spectrometric system with planar high purity germanium detector and low background gamma spectrometric system with coaxial high purity germanium detector. The ;Multigroup γ-ray Analysis Method for Uranium; (MGAU) code was used for the precise determination of samples' isotopic composition. The age of the samples was determined from the isotopic ratio 214Bi/234U. This ratio was calculated from the analyzed spectra of each uranium sample, using relative detection efficiency. Special attention is paid to the coincidence summing corrections that have to be taken into account when performing this type of analysis. In addition, an alternative approach for the age determination using full energy peak efficiencies obtained by Monte Carlo simulations with the GESPECOR code is described.

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.

PubMed

Zheng, Hewen; Korendovych, Ivan V; Luk, Yan-Yeung

2016-01-01

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa. Copyright © 2015 Elsevier Inc. All rights reserved.

Electrophoretic cell separation using microspheres. [purification of lymphocytes

NASA Technical Reports Server (NTRS)

Smolka, A.; Sachs, G.

1980-01-01

Methods of cell separation based on the electrokinetic properties of the cell membrane offer a degree of discrimination among cell populations which is not available with methods based on cell size or density alone. Studies aimed at extending red cell separations using microspheres to purification of lymphocytes.

Development of Ultrafiltration Membrane-Separation Technology for Energy-Efficient Water Treatment and Desalination Process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yim, Woosoon; Bae, Chulsung

The growing scarcity of fresh water is a major political and economic challenge in the 21st century. Compared to thermal-based distillation technique of water production, pressure driven membrane-based water purification process, such as ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO), can offer more energy-efficient and environmentally friendly solution to clean water production. Potential applications also include removal of hazardous chemicals (i.e., arsenic, pesticides, organics) from water. Although those membrane-separation technologies have been used to produce drinking water from seawater (desalination) and non-traditional water (i.e., municipal wastewater and brackish groundwater) over the last decades, they still have problems in ordermore » to be applied in large-scale operations. Currently, a major huddle of membrane-based water purification technology for large-scale commercialization is membrane fouling and its resulting increases in pressure and energy cost of filtration process. Membrane cleaning methods, which can restore the membrane properties to some degree, usually cause irreversible damage to the membranes. Considering that electricity for creating of pressure constitutes a majority of cost (~50%) in membrane-based water purification process, the development of new nano-porous membranes that are more resistant to degradation and less subject to fouling is highly desired. Styrene-ethylene/butylene-styrene (SEBS) block copolymer is one of the best known block copolymers that induces well defined morphologies. Due to the polarity difference of aromatic styrene unit and saturated ethylene/butylene unit, these two polymer chains self-assemble each other and form different phase-separated morphologies depending on the ratios of two polymer chain lengths. Because the surface of SEBS is hydrophobic which easily causes fouling of membrane, incorporation of ionic group (e,g, sulfonate) to the polymer is necessary to reduces fouling. Recently, sulfonated SEBS became commercially available and has been extensively explored for membrane-mediated water purification technology. The sulfonated block copolymer creates a well developed nano-sale phase-separated morphologies composed of hydrophilic domains (sulfonated polystyrene) and hydrophobic domains (polyethylene/polybutylene). The hydrophilic domains determines transport properties (water transport, salt and/or ion rejection, etc) and the hydrophobic domains provides mechanical stability of the membrane. Unfortunately, a high degree of sulfonation of SEBS induces excessive swelling and deterioration of mechanical stability of the membrane. In an effort to develop robust polymeric membrane materials for water purification technology, phosphonic acid-functionalized SEBS membranes are investigated during this report period. In compare to sulfonated polymers, the corresponding phosphonated polymers are known to swell less because of the formation of extensive hydrogen bonding networks between phosphonates. In addition to the expected better mechanical stability, phosphonated polymers has another advantage over sulfonated polymers for the use water purification membrane; each phosphonate can accommodate two ions while each sulfonate accommodates only one ion. Membrane properties (ion type, ionic density, etc) of new membranes will be studied and their separation performance will be evaluated in water purification and desalination process. Through systematic study of the relationship of chemical structure–surface property–membrane performance, we aim to better understand the nature of membrane fouling and develop more fouling-resistant water purification membranes. The basic understanding of this relationship will lead to the development of advanced membrane materials which can offer a solution to environmentally sustainable production of fresh water.« less

LOW COST, EFFICIENT MICROCHANNEL PLASMA OZONE GENERATOR FOR POINT OF USE WATER TREATMENT - PHASE I

EPA Science Inventory

A team of EP Purification and the University of Illinois engineers and chemists is pursuing the commercialization of low-cost microchannel plasma modules capable of efficiently producing ozone for...

High temperature metal purification using a compact portable rf heating and levitation system on the wake shield

NASA Technical Reports Server (NTRS)

Hahs, C. A.

1990-01-01

The Wake Shield Facility (WSF) can provide an ideal vacuum environment for the purification of high temperature metals in space. The Modular Electromagnetic Levitator (MEL), will provide the opportunity to study undercooling of metals in space and allow to determine material properties in space. The battery powered rf levitation and heating system developed for the MEL demonstrated efficiency of 36 percent. This system is being considered to purify metals at temperatures below 3000 C.

Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin.

PubMed

Sanz, J M; Lopez, R; Garcia, J L

1988-05-23

Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form). Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.

Recovery and purification process development for monoclonal antibody production

PubMed Central

Ma, Junfen; Winter, Charles; Bayer, Robert

2010-01-01

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

Synthesis of capped RNA using a DMT group as a purification handle.

PubMed

Veliath, Elizabeth; Gaffney, Barbara L; Jones, Roger A

2014-01-01

We report a new method for synthesis of capped RNA or 2'-OMe RNA that uses a N(2-)4,4'-dimethoxytrityl (DMT) group as a lipophilic purification handle to allow convenient isolation and purification of the capped RNA. The DMT group is easily removed under mild conditions without degradation of the cap. We have used this approach to prepare capped 10- and 20-mers. This method is compatible with the many condensation reactions that have been reported for preparation of capped RNA or cap analogues.

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

PubMed Central

Wysoczynski, Christina L.; Roemer, Sarah C.; Dostal, Vishantie; Barkley, Robert M.; Churchill, Mair E. A.; Malarkey, Christopher S.

2013-01-01

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications. PMID:24013567

Photocatalytic degradation properties of V-doped TiO2 to automobile exhaust.

PubMed

Wang, Tong; Shen, Dongya; Xu, Tao; Jiang, Ruiling

2017-05-15

To improve the photocatalytic degradation properties of titanium dioxide (TiO 2 ) used as raw materials for purifying automobile exhaust (AE), the vanadium (V)-doped TiO 2 samples were prepared. The photocatalytic degradation efficiencies of V-doped TiO 2 to each component in AE were evaluated under ultraviolet (UV) and visible light irradiation, respectively. Results indicated that the photocatalytic activity of V-doped TiO 2 to AE was higher than that of pure TiO 2 , and the optimal V dopant content of TiO 2 was 1.0% under UV light irradiation. The degradation efficiencies of V-doped TiO 2 to NOx and HC were higher than those to CO 2 and CO in AE because of the reversible reaction between CO 2 and CO. In addition, it was found that the photocatalytic degradation efficiencies of V-doped TiO 2 to each component in AE were also increased under visible light irradiation. The V-doped TiO 2 also showed higher degradation efficiencies to NOx and HC than those to CO 2 and CO under visible light irradiation. The V doped TiO 2 presented higher photocatalytic activity to CO 2 than that to CO, but the reversible reaction between CO and CO 2 was not found under visible light irradiation. The photocatalytic reactions of pure and V-doped TiO 2 samples to each component in AE followed the first order kinetic pathway under the two light irradiations. It is concluded that the V doping is a feasible method to improve the photocatalytic degradation properties of TiO 2 to AE for air purification, developing a sustainable environmental purification technology based on TiO 2 materials. Copyright © 2017 Elsevier B.V. All rights reserved.

Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

PubMed

Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

2015-02-01

Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium. © 2014 Wiley Periodicals, Inc.

Comparison of Influenza Virus Particle Purification Using Magnetic Sulfated Cellulose Particles with an Established Centrifugation Method for Analytics.

PubMed

Serve, Anja; Pieler, Michael Martin; Benndorf, Dirk; Rapp, Erdmann; Wolff, Michael Werner; Reichl, Udo

2015-11-03

A method for the purification of influenza virus particles using novel magnetic sulfated cellulose particles is presented and compared to an established centrifugation method for analytics. Therefore, purified influenza A virus particles from adherent and suspension MDCK host cell lines were characterized on the protein level with mass spectrometry to compare the viral and residual host cell proteins. Both methods allowed one to identify all 10 influenza A virus proteins, including low-abundance proteins like the matrix protein 2 and nonstructural protein 1, with a similar impurity level of host cell proteins. Compared to the centrifugation method, use of the novel magnetic sulfated cellulose particles reduced the influenza A virus particle purification time from 3.5 h to 30 min before mass spectrometry analysis.

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

PubMed

Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

2015-12-01

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A two-step electrodialysis method for DNA purification from polluted metallic environmental samples.

PubMed

Rodríguez-Mejía, José Luis; Martínez-Anaya, Claudia; Folch-Mallol, Jorge Luis; Dantán-González, Edgar

2008-08-01

Extracting DNA from samples of polluted environments using standard methods often results in low yields of poor-quality material unsuited to subsequent manipulation and analysis by molecular biological techniques. Here, we report a novel two-step electrodialysis-based method for the extraction of DNA from environmental samples. This technique permits the rapid and efficient isolation of high-quality DNA based on its acidic nature, and without the requirement for phenol-chloroform-isoamyl alcohol cleanup and ethanol precipitation steps. Subsequent PCR, endonuclease restriction, and cloning reactions were successfully performed utilizing DNA obtained by electrodialysis, whereas some or all of these techniques failed using DNA extracted with two alternative methods. We also show that his technique is applicable to purify DNA from a range of polluted and nonpolluted samples.

Arrays of probes for positional sequencing by hybridization

DOEpatents

Cantor, Charles R [Boston, MA; Prezetakiewiczr, Marek [East Boston, MA; Smith, Cassandra L [Boston, MA; Sano, Takeshi [Waltham, MA

2008-01-15

This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins ☆

PubMed Central

Donnelly, Mark I.; Zhou, Min; Millard, Cynthia Sanville; Clancy, Shonda; Stols, Lucy; Eschenfeldt, William H.; Collart, Frank R.; Joachimiak, Andrzej

2009-01-01

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his6-tag–maltose-binding protein (MBP), intended to facilitate purification and enhance proteins’ solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his6-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his6-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his6-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his6-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his6-tag. PMID:16497515

Quantitative investigation of non-hydrolytic disruptive activity on crystalline cellulose and application to recombinant swollenin.

PubMed

Wang, Yuguo; Tang, Rentao; Tao, Jin; Gao, Gui; Wang, Xiaonan; Mu, Ying; Feng, Yan

2011-09-01

For the efficient degradation and bioconversion of cellulosic biomass, it is important to efficiently disrupt and convert crystalline regions of cellulose into easily hydrolyzable regions than to simply hydrolyze cellulose. Expansin-like proteins such as swollenins have disruptive functions on lignocellulose, including crystalline cellulose, via non-hydrolytic mechanisms. In this work, we produced the swollenin from Trichoderma asperellum in Escherichia coli. The recombinant protein was then refolded into the bioactive form with simultaneous purification via a novel cellulose-assisted process. We devised a novel, simple, and efficient method to quantitatively determine the non-hydrolytic disruptive activity of swollenin on crystalline cellulose. This method is based on the synergism of the swollenin and the endoglucanase FnCel5A from Fervidobacterium nodosum. The change from crystalline regions into easily hydrolyzable forms, due to non-hydrolytic disruption, might be slight and not easily be observed. However, disrupted regions of cellulose could be hydrolyzed by FnCel5A, and reducing sugars were formed by the synergism. The disruptive function of the swollenin was quantitatively characterized by measuring the release of reducing sugars. These methods and processes will be useful for further research on non-hydrolytic disruptive bioactivities and provide novel approaches for the efficient and economical bioconversion of cellulosic biomass.

Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

PubMed

Acero-Navarro, Kevin E; Jiménez-Ramírez, Mariella; Villalobos, Miguel A; Vargas-Martínez, Rocío; Perales-Vela, Hugo V; Velasco-García, Roberto

2018-02-01

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl 2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

PubMed Central

Penna, Vessoni Thereza Christina; Martins, Silva Alzira Maria; Mazzola, Priscila Gava

2002-01-01

Background A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. Methodology Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. Results The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. Conclusions We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments. PMID:12182763

Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays.

PubMed

Tchou, Isabelle; Sabido, Odile; Lambert, Claude; Misery, Laurent; Garraud, Olivier; Genin, Christian

2003-03-03

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

[Progress in isolation and purification of porcine islets].

PubMed

Zhu, Haitao; Yu, Liang; Wang, Bo

2012-08-01

To review the common methods of isolation and purification of porcine islets and research progress. Domestic and abroad literature concerning the isolation and purification of porcine islets was reviewed and analyzed thoroughly. The efficacy of the isolation and purification depends on the selection of donor, the procurement and cryopreservation of high-quality donor pancreas, and the selection and improvement of the operation. The shortage of transplanted islets could be resolved by the establishment of standardized and optimal process, which may also promote the development of porcine islet xenograft.

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

PubMed

Saito, Maiko; Kurosawa, Yae; Okuyama, Tsuneo

2012-02-01

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.

Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host.

PubMed

Santos, João H P M; Costa, Iris M; Molino, João V D; Leite, Mariana S M; Pimenta, Marcela V; Coutinho, João A P; Pessoa, Adalberto; Ventura, Sónia P M; Lopes, André M; Monteiro, Gisele

2017-03-01

l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His) 6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni 2+ -charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg -1 ) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017. © 2016 American Institute of Chemical Engineers.

Development of a passive micromixer based on repeated fluid twisting and flattening, and its application to DNA purification.

PubMed

Lee, Nae Yoon; Yamada, Masumi; Seki, Minoru

2005-11-01

We have developed a three-dimensional passive micromixer based on new mixing principles, fluid twisting and flattening. This micromixer is constructed by repeating two microchannel segments, a "main channel" and a "flattened channel", which are very different in size and are arranged perpendicularly. At the intersection of these segments the fluid inside the micromixer is twisted and then, in the flattened channel, the diffusion length is greatly reduced, achieving high mixing efficiency. Several types of micromixer were fabricated and the effect of microchannel geometry on mixing performance was evaluated. We also integrated this micromixer with a miniaturized DNA purification device, in which the concentration of the buffer solution could be rapidly changed, to perform DNA purification based on solid-phase extraction.

Genetic selection for a highly functional cysteine-less membrane protein using site-saturation mutagenesis

PubMed Central

Arendt, Cassandra S.; Ri, Keirei; Yates, Phillip A.; Ullman, Buddy

2007-01-01

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiol-specific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. PMID:17481563

One-step purification of nisin A by immunoaffinity chromatography.

PubMed

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-12-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.

Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

NASA Astrophysics Data System (ADS)

Liu, Fang

Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

PubMed

Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana

2011-08-01

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. Copyright © 2011 Elsevier B.V. All rights reserved.

Affinity chromatography: A versatile technique for antibody purification.

PubMed

Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

2017-03-01

Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

The Blood Compatibilities of Blood Purification Membranes and Other Materials Developed in Japan

PubMed Central

Abe, Takaya; Kato, Karen; Fujioka, Tomoaki; Akizawa, Tadao

2011-01-01

The biocompatibilities in blood purification therapy are defined as “a concept to stipulate safety of blood purification therapy by an index based on interaction in the body arising from blood purification therapy itself.” The biocompatibilities are associated with not only materials to be used but also many factors such as sterilization method and eluted substance. It is often evaluated based on impacts on cellular pathways and on humoral pathways. Since the biocompatibilities of blood purification therapy in particular hemodialysis are not just a prognostic factor for dialysis patients but a contributory factor for long-term complications, it should be considered with adequate attention. It is important that blood purification therapy should be performed by consistently evaluating not only risks associated with these biocompatibilities but also the other advantages obtained from treatments. In this paper, the biocompatibilities of membrane and adsorption material based on Japanese original which are used for blood purification therapy are described. PMID:21969830

Overview of the Purification of Recombinant Proteins

PubMed Central

Wingfield, Paul T.

2015-01-01

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

Overview of the purification of recombinant proteins.

PubMed

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.

Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry.

PubMed

Papaioannou, A; Louis, M; Dhital, B; Ho, H P; Chang, E J; Boutis, G S

2015-05-01

Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel Self-driven Microbial Nutrient Recovery Cell with Simultaneous Wastewater Purification

PubMed Central

Chen, Xi; Sun, Dongya; Zhang, Xiaoyuan; Liang, Peng; Huang, Xia

2015-01-01

Conventional wastewater purification technologies consume large amounts of energy, while the abundant chemical energy and nutrient resources contained in sewage are wasted in such treatment processes. A microbial nutrient recovery cell (MNRC) has been developed to take advantage of the energy contained in wastewater, in order to simultaneously purify wastewater and recover nutrient ions. When wastewater was circulated between the anode and cathode chambers of the MNRC, the organics (COD) were removed by bacteria while ammonium and phosphate (NH4+-N and PO43−-P) were recovered by the electrical field that was produced using in situ energy in the wastewater without additional energy input. The removal efficiencies from wastewater were >82% for COD, >96% for NH4+-N, and >64% for PO43−-P in all the operational cycles. Simultaneously, the concentrations of NH4+ and PO43− in the recovery chamber increased to more than 1.5 and 2.2 times, respectively, compared with the initial concentrations in wastewater. The MNRC provides proof-of-concept as a sustainable, self-driven approach to efficient wastewater purification and nutrient recovery in a comprehensive bioelectrochemical system. PMID:26503712

Advanced model-based control strategies for the intensification of upstream and downstream processing in mAb production.

PubMed

Papathanasiou, Maria M; Quiroga-Campano, Ana L; Steinebach, Fabian; Elviro, Montaña; Mantalaris, Athanasios; Pistikopoulos, Efstratios N

2017-07-01

Current industrial trends encourage the development of sustainable, environmentally friendly processes with minimal energy and material consumption. In particular, the increasing market demand in biopharmaceutical industry and the tight regulations in product quality necessitate efficient operating procedures that guarantee products of high purity. In this direction, process intensification via continuous operation paves the way for the development of novel, eco-friendly processes, characterized by higher productivity and lower production costs. This work focuses on the development of advanced control strategies for (i) a cell culture system in a bioreactor and (ii) a semicontinuous purification process. More specifically, we consider a fed-batch culture of GS-NS0 cells and the semicontinuous Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) for the purification process. The controllers are designed following the PAROC framework/software platform and their capabilities are assessed in silico, against the process models. It is demonstrated that the proposed controllers efficiently manage to increase the system productivity, returning strategies that can lead to continuous, stable process operation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:966-988, 2017. © 2017 American Institute of Chemical Engineers.

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

PubMed

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.

Purification of polymorphic components of complex genomes

DOEpatents

Stodolsky, Marvin

1991-01-01

A method is disclosed for processing related subject and reference macromolecule populations composed of complementary strands into their respective subject and reference populations of representative fragments and effectuating purification of unique polymorphic subject fragments.

Purification of polymorphic components of complex genomes

DOEpatents

Stodolsky, M.

1988-01-21

A method for processing related subject and reference macromolecule composed of complementary strand into their respective subject and reference populations of representative fragments and effectuating purification of unique polymorphic subject fragments. 1 fig.

Purification of Carbon Nanotubes: Alternative Methods

NASA Technical Reports Server (NTRS)

Files, Bradley; Scott, Carl; Gorelik, Olga; Nikolaev, Pasha; Hulse, Lou; Arepalli, Sivaram

2000-01-01

Traditional carbon nanotube purification process involves nitric acid refluxing and cross flow filtration using surfactant TritonX. This is believed to result in damage to nanotubes and surfactant residue on nanotube surface. Alternative purification procedures involving solvent extraction, thermal zone refining and nitric acid refiuxing are used in the current study. The effect of duration and type of solvent to dissolve impurities including fullerenes and P ACs (polyaromatic compounds) are monitored by nuclear magnetic reasonance, high performance liquid chromatography, and thermogravimetric analysis. Thermal zone refining yielded sample areas rich in nanotubes as seen by scanning electric microscopy. Refluxing in boiling nitric acid seem to improve the nanotube content. Different procedural steps are needed to purify samples produced by laser process compared to arc process. These alternative methods of nanotube purification will be presented along with results from supporting analytical techniques.

Studies on separation and purification of fission (99)Mo from neutron activated uranium aluminum alloy.

PubMed

Rao, Ankita; Kumar Sharma, Abhishek; Kumar, Pradeep; Charyulu, M M; Tomar, B S; Ramakumar, K L

2014-07-01

A new method has been developed for separation and purification of fission (99)Mo from neutron activated uranium-aluminum alloy. Alkali dissolution of the irradiated target (100mg) results in aluminum along with (99)Mo and a few fission products passing into solution, while most of the fission products, activation products and uranium remain undissolved. Subsequent purification steps involve precipitation of aluminum as Al(OH)3, iodine as AgI/AgIO3 and molybdenum as Mo-α-benzoin oxime. Ruthenium is separated by volatilization as RuO4 and final purification of (99)Mo was carried out using anion exchange method. The radiochemical yield of fission (99)Mo was found to be >80% and the purity of the product was in conformity with the international pharmacopoeia standards. Copyright © 2014 Elsevier Ltd. All rights reserved.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

PubMed

Battle, Katrina N; Jackson, Joshua M; Witek, Małgorzata A; Hupert, Mateusz L; Hunsucker, Sally A; Armistead, Paul M; Soper, Steven A

2014-03-21

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

Virus purification by CsCl density gradient using general centrifugation.

PubMed

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.

A versatile bio-based material for efficiently removing toxic dyes, heavy metal ions and emulsified oil droplets from water simultaneously.

PubMed

Li, Daikun; Li, Qing; Mao, Daoyong; Bai, Ningning; Dong, Hongzhou

2017-12-01

Developing versatile materials for effective water purification is significant for environment and water source protection. Herein, a versatile bio-based material (CH-PAA-T) was reported by simple thermal cross-linking chitosan and polyacrylic acid which exhibits excellent performances for removing insoluble oil, soluble toxic dyes and heavy metal ions from water, simultaneously. The adsorption capacities are 990.1mgg -1 for methylene blue (MB) and 135.9mgg -1 for Cu 2+ , which are higher than most of present advanced absorbents. The adsorption towards organic dyes possesses high selectivity which makes CH-PAA-T be able to efficiently separate dye mixtures. The stable superoleophobicity under water endows CH-PAA-T good performance to separate toluene-in-water emulsion stabilized by Tween 80. Moreover, CH-PAA-T can be recycled for 10 times with negligible reduction of efficiency. Such versatile bio-based material is a potential candidate for water purification. Copyright © 2017. Published by Elsevier Ltd.

Synthesis and use of 2-[ 18F]fluoromalondialdehyde, an accessible synthon for bioconjugation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hooker, Jacob M.

We proposed methods for the synthesis and purification of 2-[ 18F]fluoromalondialdehyde, which will be a readily accessible synthon for bioconjugation. Our achievements in these areas will specifically address a stated goal of the DOE providing a transformational technology for macromolecule radiolabeling. Accomplishment of our aims will serve both DOE mission-related research as well as nuclear medicine research supported by the NIH and industry. At the heart of our proposal is the aim to “improve synthetic methodology for rapidly and efficiently incorporating radionuclides into a wide range of organic compounds.”

Real-time modulated nanoparticle separation with an ultra-large dynamic range.

PubMed

Zeming, Kerwin Kwek; Thakor, Nitish V; Zhang, Yong; Chen, Chia-Hung

2016-01-07

Nanoparticles exhibit size-dependent properties which make size-selective purification of proteins, DNA or synthetic nanoparticles essential for bio-analytics, clinical medicine, nano-plasmonics and nano-material sciences. Current purification methods of centrifugation, column chromatography and continuous-flow techniques suffer from particle aggregation, multi-stage process, complex setups and necessary nanofabrication. These increase process costs and time, reduce efficiency and limit dynamic range. Here, we achieve an unprecedented real-time nanoparticle separation (51-1500 nm) using a large-pore (2 μm) deterministic lateral displacement (DLD) device. No external force fields or nanofabrication are required. Instead, we investigated innate long-range electrostatic influences on nanoparticles within a fluid medium at different NaCl ionic concentrations. In this study we account for the electrostatic forces beyond Debye length and showed that they cannot be assumed as negligible especially for precise nanoparticle separation methods such as DLD. Our findings have enabled us to develop a model to simultaneously quantify and modulate the electrostatic force interactions between nanoparticle and micropore. By simply controlling buffer solutions, we achieve dynamic nanoparticle size separation on a single device with a rapid response time (<20 s) and an enlarged dynamic range (>1200%), outperforming standard benchtop centrifuge systems. This novel method and model combines device simplicity, isolation precision and dynamic flexibility, opening opportunities for high-throughput applications in nano-separation for industrial and biological applications.

Purification of polymorphic components of complex genomes

DOEpatents

Stodolsky, M.

1991-07-16

A method is disclosed for processing related subject and reference macromolecule populations composed of complementary strands into their respective subject and reference populations of representative fragments and effectuating purification of unique polymorphic subject fragments. 1 figure.

One-step purification of nisin A by immunoaffinity chromatography.

PubMed Central

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-01-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method. PMID:9406424

Standardization of Procedures for the Preparation of (177)Lu- and (90)Y-labeled DOTA-Rituximab Based on the Freeze-dried Kit Formulation.

PubMed

Wojdowska, Wioletta; Karczmarczyk, Urszula; Maurin, Michal; Garnuszek, Piotr; Mikołajczak, Renata

2015-01-01

Rituximab when radiolabelled with (177)Lu or (90)Y has been investigated for the treatment of patients with Non-Hodgkin's Lymphoma. In this study, we optimized the preparation of antibody conjugates with chelating agent in the freeze-dried kit. It shortens procedures needed for the successful radiolabeling with lutetium-177 and yttrium-90 and assures reproducible labelling yields. Various molar ratios of Rituximab:DOTA (from 1:5 to 1:100) were used at the conjugation step and different purification method to remove unbound DOTA were investigated (size-exclusion chromatography, dialysis, ultrafiltration). The final monoclonal antibody concentration was quantified by Bradford method, and the number of DOTA molecules was determined by radiolabeling assay using (64)Cu. The specific activity of (177)Lu-DOTA-Rituximab and (90)Y-DOTA-Rituximab were optimized using various amounts of radiometal. Quality control (SE-HPLC, ITLC) and stability study were performed. An average of 4.2 ± 0.8 p-SCN-Bz-DOTA molecules could be randomly conjugated to a single molecule of Rituximab. The ultrafiltration system was the most efficient for purification and resulted in the highest recovery efficiency (77.2%). At optimized conditions the (177)Lu-DOTARituximab and (90)Y-DOTA-Rituximab were obtained with radiochemical purity >99% and specific activity ca. 600 MBq/mg. The radioimmunoconjugates were stable in human serum and 0.9% NaCl. After 72 h of incubation the radiochemical purity of (177)Lu-DOTA-Rituximab decreased to 94% but it was still more than 88% for (90)Y-DOTA-Rituximab. The radioimmunoconjugate showed stability after six months storage at 2 - 8(0)C, as a lyophilized formulation. Our study shows that Rituximab-DOTA can be efficiently radiolabeled with (177)Lu and (90)Y via p-SCN-Bn-DOTA using a freezedried kit.

Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification

PubMed Central

Liao, Pin-Chao; Boldogh, Istvan R.; Siegmund, Stephanie E.

2018-01-01

Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods. PMID:29698455

Qualitative and quantitative analysis of an alkaloid fraction from Piper longum L. using ultra-high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry.

PubMed

Li, Kuiyong; Fan, Yunpeng; Wang, Hui; Fu, Qing; Jin, Yu; Liang, Xinmiao

2015-05-10

In a previous research, an alkaloid fraction and 18 alkaloid compounds were prepared from Piper longum L. by series of purification process. In this paper, a qualitative and quantitative analysis method using ultra-high performance liquid chromatography-diode array detector-mass spectrometry (UHPLC-DAD-MS) was developed to evaluate the alkaloid fraction. Qualitative analysis of the alkaloid fraction was firstly completed by UHPLC-DAD method and 18 amide alkaloid compounds were identified. A further qualitative analysis of the alkaloid fraction was accomplished by UHPLC-MS/MS method. Another 25 amide alkaloids were identified according to their characteristic ions and neutral losses. At last, a quantitative method for the alkaloid fraction was established using four marker compounds including piperine, pipernonatine, guineensine and N-isobutyl-2E,4E-octadecadienamide. After the validation of this method, the contents of above four marker compounds in the alkaloid fraction were 57.5mg/g, 65.6mg/g, 17.7mg/g and 23.9mg/g, respectively. Moreover, the relative response factors of other three compounds to piperine were calculated. A comparative study between external standard quantification and relative response factor quantification proved no remarkable difference. UHPLC-DAD-MS method was demonstrated to be a powerful tool for the characterization of the alkaloid fraction from P. longum L. and the result proved that the quality of alkaloid fraction was efficiently improved after appropriate purification. Copyright © 2015. Published by Elsevier B.V.

Direct digestion of proteins in living cells into peptides for proteomic analysis.

PubMed

Chen, Qi; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

2015-01-01

To analyze the proteome of an extremely low number of cells or even a single cell, we established a new method of digesting whole cells into mass-spectrometry-identifiable peptides in a single step within 2 h. Our sampling method greatly simplified the processes of cell lysis, protein extraction, protein purification, and overnight digestion, without compromising efficiency. We used our method to digest hundred-scale cells. As far as we know, there is no report of proteome analysis starting directly with as few as 100 cells. We identified an average of 109 proteins from 100 cells, and with three replicates, the number of proteins rose to 204. Good reproducibility was achieved, showing stability and reliability of the method. Gene Ontology analysis revealed that proteins in different cellular compartments were well represented.

Combined electron-beam and coagulation purification of molasses distillery slops. Features of the method, technical and economic evaluation of large-scale facility

NASA Astrophysics Data System (ADS)

Pikaev, A. K.; Ponomarev, A. V.; Bludenko, A. V.; Minin, V. N.; Elizar'eva, L. M.

2001-04-01

The paper summarizes the results obtained from the study on combined electron-beam and coagulation method for purification of molasses distillery slops from distillery produced ethyl alcohol by fermentation of grain, potato, beet and some other plant materials. The method consists in preliminary mixing of industrial wastewater with municipal wastewater, electron-beam treatment of the mixture and subsequent coagulation. Technical and economic evaluation of large-scale facility (output of 7000 m 3 day -1) with two powerful cascade electron accelerators (total maximum beam power of 400 kW) for treatment of the wastewater by the above method was carried out. It was calculated that the cost of purification of the wastes is equal to 0.25 US$ m -3 that is noticeably less than in the case of the existing method.

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

PubMed

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

2014-12-01

Efficient loading of immunoglobulin G in mixed-mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed-mode ligand, 4-(1H-imidazol-1-yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15-64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparative isolation and purification of capsaicin and dihydrocapsaicin from Capsici Fructus using supercritical fluid extraction combined with high speed countercurrent chromatography.

PubMed

Yan, Rongwei; Zhao, Leilei; Tao, Junfei; Zou, Yong; Xu, Xinjun

2018-05-01

Supercritical fluid extraction with CO 2 (SFE-CO 2 ) was utilized for extraction of capsaicin (CA) and dihydrocapsaicin (DHCA) from Capsici Fructus, and then a two-step enrichment method for separating capsaicinoids from SFE-CO 2 extracts was developed. The process involved extraction with aqueous methanol and crystallization by alkali extraction and acid precipitation. Finally, a consecutive high-speed countercurrent chromatography (HSCCC) separation method was successfully applied in the purification of CA and DHCA from capsaicinoid crystal. The extraction pressure, extraction temperature and volume of co-solvent were optimized at 33 MPa, 41 °C and 75 mL, respectively, using response surface methodology; the extraction rates of CA and DHCA were about 93.18% and 93.49%, respectively. 407.43 mg capsaicinoid crystal was isolated from the SFE-CO 2 extracts obtained from 100 g capsicum powder by the two-step enrichment method. About 506 mg and 184 mg CA and DHCA with purities up to 98.31% and 96.68%, respectively, were obtained from 1 g capsaicinoid crystal in one HSCCC of three consecutive sample loadings without exchanging any solvent system. This method comprising SFE-CO 2 , a two-step enrichment and HSCCC was efficient, powerful and practical for the large-scale preparation of CA and DHCA from Capsici Fructus with high purity and high yield. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Development and validation of a method for purification of mallein for the diagnosis of glanders in equines

PubMed Central

2012-01-01

Background The allergic test of mallein is one of the most frequently used tests, together with the Complement Fixation Test (CFT), for the diagnosis of glanders in endemic areas. Mallein, a purified protein derivative (PPD), is produced similarly to PPD tuberculin and the end product is a primarily proteic antigen, which is only poorly purified. The immuno-allergic activity of mallein is believed to be due to a high molecular weight group of proteins present in the antigen. To improve the quality of the antigen, in terms of sensitivity and specificity, a new method of mallein production was developed, in which purification was accomplished by ultrafiltration in a Tangential Flow Filtration system (TFF). Results The TFF methodology efficiently separated the high and low molecular weight protein groups of mallein. The five TFF-purified malleins, produced from Burkholderia mallei strains isolated from clinical cases of glanders in Brazil, proved to be more potent than standard mallein in the induction of an allergic reaction in sensitized animals. Regarding specificity, two of the purified malleins were equivalent to the standard and three were less specific. Conclusion Some of the TFF-purified malleins showed considerable potential to be used as an auxiliary test in the diagnosis of glanders. PMID:22937975

[Isolation and purification of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5 using chromatography].

PubMed

Ma, Lina; Wu, Dan; Bian, Liujiao

2012-08-01

The Kringle 5 domain of plasminogen is one of the most potent angiogenesis inhibitors known to date, which can inhibit cell proliferation and migration efficiently. In the study, on the foundation of successful clone and expression of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5, a two-step chromatographic method, including the use of SP Sepharose Fast Flow cation exchanger and Sephacryl S-100 HR size exclusion chromatography in sequence, was established to separate and purify angiogenesis inhibitor Kringle 5. On the SP Sepharose Fast Flow column, the buffer A consisted of 50.0 mmol/L acetic acid-sodium acetate (pH 5.2), and the buffer B consisted of buffer A with the addition of 0.5 mol/L sodium chloride (pH 5.2); on Sephacryl S-100 HR column, the elution buffer was 5.0 mmol/L phosphate solution (pH 7.0). Through the two-step chromatographic purification process, the purity of the obtained Kringle 5 was more than 98%. In addition, it was found that the obtained Kringle 5 could inhibit the blood vessel growth of chick embryo chorioallantoic membrane effectively. Finally it is concluded that this method can effectively separate active recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5.

Highly scalable, closed-loop synthesis of drug-loaded, layer-by-layer nanoparticles.

PubMed

Correa, Santiago; Choi, Ki Young; Dreaden, Erik C; Renggli, Kasper; Shi, Aria; Gu, Li; Shopsowitz, Kevin E; Quadir, Mohiuddin A; Ben-Akiva, Elana; Hammond, Paula T

2016-02-16

Layer-by-layer (LbL) self-assembly is a versatile technique from which multicomponent and stimuli-responsive nanoscale drug carriers can be constructed. Despite the benefits of LbL assembly, the conventional synthetic approach for fabricating LbL nanoparticles requires numerous purification steps that limit scale, yield, efficiency, and potential for clinical translation. In this report, we describe a generalizable method for increasing throughput with LbL assembly by using highly scalable, closed-loop diafiltration to manage intermediate purification steps. This method facilitates highly controlled fabrication of diverse nanoscale LbL formulations smaller than 150 nm composed from solid-polymer, mesoporous silica, and liposomal vesicles. The technique allows for the deposition of a broad range of polyelectrolytes that included native polysaccharides, linear polypeptides, and synthetic polymers. We also explore the cytotoxicity, shelf life and long-term storage of LbL nanoparticles produced using this approach. We find that LbL coated systems can be reliably and rapidly produced: specifically, LbL-modified liposomes could be lyophilized, stored at room temperature, and reconstituted without compromising drug encapsulation or particle stability, thereby facilitating large scale applications. Overall, this report describes an accessible approach that significantly improves the throughput of nanoscale LbL drug-carriers that show low toxicity and are amenable to clinically relevant storage conditions.

CD25 Preselective Anti-HIV Vectors for Improved HIV Gene Therapy

PubMed Central

Kalomoiris, Stefanos; Lawson, Je'Tai; Chen, Rachel X.; Bauer, Gerhard; Nolta, Jan A.

2012-01-01

Abstract As HIV continues to be a global public health problem with no effective vaccine available, new and innovative therapies, including HIV gene therapies, need to be developed. Due to low transduction efficiencies that lead to low in vivo gene marking, therapeutically relevant efficacy of HIV gene therapy has been difficult to achieve in a clinical setting. Methods to improve the transplantation of enriched populations of anti-HIV vector-transduced cells may greatly increase the in vivo efficacy of HIV gene therapies. Here we describe the development of preselective anti-HIV lentiviral vectors that allow for the purification of vector-transduced cells to achieve an enriched population of HIV-resistant cells. A selectable protein, human CD25, not normally found on CD34+ hematopoietic progenitor cells (HPCs), was incorporated into a triple combination anti-HIV lentiviral vector. Upon purification of cells transduced with the preselective anti-HIV vector, safety was demonstrated in CD34+ HPCs and in HPC-derived macrophages in vitro. Upon challenge with HIV-1, improved efficacy was observed in purified preselective anti-HIV vector-transduced macrophages compared to unpurified cells. These proof-of-concept results highlight the potential use of this method to improve HIV stem cell gene therapy for future clinical applications. PMID:23216020

Current strategies for protein production and purification enabling membrane protein structural biology.

PubMed

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

2016-12-01

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

Identification of minor inner-membrane components of the Shigella type III secretion system 'needle complex'.

PubMed

Zenk, Sebastian F; Stabat, David; Hodgkinson, Julie L; Veenendaal, Andreas K J; Johnson, Steven; Blocker, Ariel J

2007-08-01

Type III secretion systems (T3SSs or secretons) are central virulence factors of many Gram-negative bacteria, used to inject protein effectors of virulence into eukaryotic host cells. Their overall morphology, consisting of a cytoplasmic region, an inner- and outer-membrane section and an extracellular needle, is conserved in various species. A portion of the secreton, containing the transmembrane regions and needle, has been isolated biochemically and termed the 'needle complex' (NC). However, there are still unsolved questions concerning the nature and relative arrangement of the proteins assembling the NC. Until these are resolved, the mode of function of the NC cannot be clarified. This paper describes an affinity purification method that enables highly efficient purification of Shigella NCs under near-physiological conditions. Using this method, three new minor components of the NC were identified by mass spectrometry: IpaD, a known component of the needle tip complex, and two predicted components of its central inner-membrane export apparatus, Spa40 and Spa24. A further minor component of the NC, MxiM, is only detected by immunoblotting. MxiM is a 'pilotin'-type protein for the outer-membrane 'secretin' ring formed of MxiD. As expected, it localized to the outer rim of the upper ring of NCs, validating the other findings.

Development and validation of a method for purification of mallein for the diagnosis of glanders in equines.

PubMed

de Carvalho Filho, Maurício Baltazar; Ramos, Rodrigo Mauro; Fonseca, Antônio Augusto; de Lima Orzil, Lívia; Sales, Mariana Lázaro; de Assis Santana, Vania Lucia; de Souza, Marcilia Maria Alves; Dos Reis Machado, Evandro; Filho, Paulo Rodrigues Lopes; Leite, Rômulo Cerqueira; Dos Reis, Jenner Karlisson Pimenta

2012-09-02

The allergic test of mallein is one of the most frequently used tests, together with the Complement Fixation Test (CFT), for the diagnosis of glanders in endemic areas. Mallein, a purified protein derivative (PPD), is produced similarly to PPD tuberculin and the end product is a primarily proteic antigen, which is only poorly purified. The immuno-allergic activity of mallein is believed to be due to a high molecular weight group of proteins present in the antigen. To improve the quality of the antigen, in terms of sensitivity and specificity, a new method of mallein production was developed, in which purification was accomplished by ultrafiltration in a Tangential Flow Filtration system (TFF). The TFF methodology efficiently separated the high and low molecular weight protein groups of mallein. The five TFF-purified malleins, produced from Burkholderia mallei strains isolated from clinical cases of glanders in Brazil, proved to be more potent than standard mallein in the induction of an allergic reaction in sensitized animals. Regarding specificity, two of the purified malleins were equivalent to the standard and three were less specific. Some of the TFF-purified malleins showed considerable potential to be used as an auxiliary test in the diagnosis of glanders.

Current strategies for protein production and purification enabling membrane protein structural biology

PubMed Central

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E.; Liu, Xiang-Qin; Rainey, Jan K.

2017-01-01

Membrane proteins are still heavily underrepresented in the protein data bank (PDB) due to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles due to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and/or amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10–15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). PMID:27010607

Plasmonic heating from indium nanoparticles on a floating microporous membrane for enhanced solar seawater desalination.

PubMed

Zhang, Lulu; Xing, Jun; Wen, Xinglin; Chai, Jianwei; Wang, Shijie; Xiong, Qihua

2017-09-14

Passive solar evaporation represents a promising and environmentally benign method of water purification/desalination. Plasmonic nanoparticles have been demonstrated as an effective approach for enhancing solar steam generation through a plasmonic heating effect, nonetheless the efficiency is constrained by unnecessary bulk heating of the entire liquid volume, while the noble metals commonly used are not cost-effective in terms of availability and their sophisticated preparation. Herein, a paper-like plasmonic device consisting of a microporous membrane and indium nanoparticles (In NPs/MPM) is fabricated through a simple thermal evaporation method. Due to the light-weight and porous nature of the device, the broadband light absorption properties, and theoretically the excellent plasmonic heating effect from In NP which could be even higher than gold, silver and aluminium nanoparticles, our device can effectively enhance solar water evaporation by floating on the water surface and its utility has been demonstrated in the solar desalination of a real seawater sample. The durability of the device in solar seawater desalination has also been investigated over multiple cycles with stable performances. This portable device could provide a solution for individuals to do water/seawater purification in under-developed areas with limited/no access to electricity or a centralized drinking water supply.

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.

PubMed

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.

The LUX prototype detector: Heat exchanger development

DOE PAGES

Akerib, D. S.; Bai, X.; Bedikian, S.; ...

2013-01-24

The LUX (large underground xenon) detector is a two-phase xenon time projection chamber (TPC) designed to search for WIMP–nucleon dark matter interactions. As with all noble element detectors, continuous purification of the detector medium is essential to produce a large (> 1 ms) electron lifetime; this is necessary for efficient measurement of the electron signal which in turn is essential for achieving robust discrimination of signal from background events. Here, we describe the development of a novel purification system deployed in a prototype detector. The results from the operation of this prototype indicated heat exchange with an efficiency above 94%more » up to a flow rate of 42 slpm, allowing for an electron drift length greater than 1 m to be achieved in approximately 2 days and sustained for the duration of the testing period.« less

Sanitary and bacteriological aspects of sewage treatment.

PubMed

Filipkowska, Zofia

2003-01-01

A study into the removal of contamination load and indicator bacteria was carried out in 1992-1996 in the mechanical, biological and chemical waste-water treatment plant WTP in Lezany, in the County of Reszel, in the Province of Warmia and Mazury in Poland. The results of chemical analyses found a high efficiency of removal of carbon compounds, COD (90%) and BOD (98%), in the process of purification of household sewage. In addition, a high effectiveness of total nitrogen, on average 71%, and unsatisfactory removal of ammonia nitrogen and phosphorus compounds were found. The results of microbiological analyses confirmed the high efficiency of removal of indicator bacteria in the process of sewage treatment from 94 to 97%. In the sewage after the final phase of purification in stabilization ponds, the following pathogenic bacteria were identified with the use of the EPL 21tests: Escherichia coli, Enterobacter agglomerans, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter georgoriae, Citrobacter freundii, Klebsiella pnemoniae, Klebsiella oxytoca, Klebsiella ozaenae, Ervinia herbicola, Edwardsiella tarda, Serratia odoriefra, Serratia marcescens, Providencia alcalifaciens, Hafnia alvei, Yersina pestis, Yersina pseudotuberculosis, Yersinia fredericksenii, Salmonella spp., Shigella dysenteriae, Aeromons hydrophila, Pseudomonas aerulginosa. The obtained results show that although the sewage purification system is efficient and reduces the contamination load to the level required by the regulations (Ministry of Environmental Protection, Natural Resources and Forestry from 20 September 1991) and removes a great percentage of indicator bacteria, the purified sewage may be a source of pathogenic bacteria in inland waters.

Comparative study of photocatalytic oxidation on the degradation of formaldehyde and fuzzy mathematics evaluation of filters

NASA Astrophysics Data System (ADS)

Yu, Huili; Zhang, Jieting

2012-04-01

In this study, formaldehyde, one of the major volatile organic compounds, is chosen as the target pollutant. The polytetrafluoroethylene (PTFE) filter, a low cost and commonly used material in industry, is employed as the substrate for nano TiO2 photocatalyst coating at room temperature, which has been scarcely used compared to ceramics or glass beads. Furthermore, a specific experimental set-up that is similar to actual air purification system is developed for the testing. The degradation mechanisms of photolysis reaction, adsorption and photocatalytic oxidation reaction on volatile organic compounds are present respectively. The influences of three aspects mentioned above are compared by a serial of experimental data. The high efficiency of volatile organic compounds on the degradation of formaldehyde is assured. Furthermore, the purification characteristics of three kinds of activated carbon filters and PTFE filter with nano TiO2 are evaluated with the method of fuzzy mathematics. In the end, the result shows that the filter with nano TiO2 has the optimal comprehensive performances.

Comparative study of photocatalytic oxidation on the degradation of formaldehyde and fuzzy mathematics evaluation of filters

NASA Astrophysics Data System (ADS)

Yu, Huili; Zhang, Jieting

2011-11-01

In this study, formaldehyde, one of the major volatile organic compounds, is chosen as the target pollutant. The polytetrafluoroethylene (PTFE) filter, a low cost and commonly used material in industry, is employed as the substrate for nano TiO2 photocatalyst coating at room temperature, which has been scarcely used compared to ceramics or glass beads. Furthermore, a specific experimental set-up that is similar to actual air purification system is developed for the testing. The degradation mechanisms of photolysis reaction, adsorption and photocatalytic oxidation reaction on volatile organic compounds are present respectively. The influences of three aspects mentioned above are compared by a serial of experimental data. The high efficiency of volatile organic compounds on the degradation of formaldehyde is assured. Furthermore, the purification characteristics of three kinds of activated carbon filters and PTFE filter with nano TiO2 are evaluated with the method of fuzzy mathematics. In the end, the result shows that the filter with nano TiO2 has the optimal comprehensive performances.

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

PubMed

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

2015-08-01

Prenylated phenolics such as amorfrutins are recently identified potent anti-inflammatory and antidiabetic natural products. In this work, high-speed counter-current chromatography was investigated for the isolation and purification of prenylated phenolics from the fruits of Amorpha fruticosa by using a two-phase solvent system composed of n-hexane/ethanol/water (5:4:1, v/v). As a result, 14.2 mg of 5,7-dihydroxy-8-geranylflavanone, 10.7 mg of amorfrutin A and 17.4 mg of amorfrutin B were obtained from 200 mg of n-hexane-soluble crude extract in one step within 250 min. The purities of 5,7-dihydroxy-8-geranylflavanone, amorfrutins A and B were 95.2, 96.7 and 97.1%, respectively, as determined by ultra high performance liquid chromatography. The structural identification was performed by mass spectrometry and (1) H and (13) C NMR spectroscopy. The results indicated that the established method is an efficient and convenient way to purified prenylated phenolics from A. fruticosa extract. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparative two-step purification of recombinant H1.0 linker histone and its domains.

PubMed

Ivic, Nives; Bilokapic, Silvija; Halic, Mario

2017-01-01

H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various subtypes and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1.0 overexpressed in Escherichia coli, as well as its domains: N-terminal/globular domain and C-terminal intrinsically disordered domain. This purification protocol relies on a simple affinity chromatography step followed by cation exchange due to the highly basic properties of histone proteins. Therefore, this protocol can also be applied to other linker histones. Highly pure proteins in amounts sufficient for most biochemical experiments can be obtained. The functional quality of purified H1.0 histone and its domains has been confirmed by pull-down, gel-mobility shift assays and the nuclear import assay.

Microwave-assisted extraction and purification of chlorogenic acid from by-products of Eucommia Ulmoides Oliver and its potential anti-tumor activity.

PubMed

Shao, P; Zhang, J F; Chen, X X; Sun, P L

2015-08-01

An efficient method for the rapid extraction, separation and purification of chlorogenic acid (CGA) from by-products of Eucommia Ulmoides Oliver (E. ulmoides) by microwave-assisted extraction (MAE) coupled with high-speed counter-current chromatography (HSCCC) was developed. The optimal MAE parameters were evaluated by response surface methodology (RSM), and they were extraction time of 12 min, microwave power of 420 W, ethanol concentration of 75 %, solvent/sample ratio of 30:1 (mL/g), yield of CGA reached 3.59 %. The crude extract was separated and purified directly by HSCCC using ethyl acetate-butyl alcohol-water (3:1:4, v/v) as the two-phase solvent system. The 14.5 mg of CGA with the purity of 98.7 % was obtained in one-step separation from 400 mg of crude extract. The chemical structure of CGA was verified with IR, ESI-MS analysis. Meanwhile, the purified CGA extract was evaluated by MTT assay and results indicate that CGA extract exhibited potential anti-tumor activity for AGS gastric cancer cell.

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

PubMed

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

2016-12-23

A centrifugal partition chromatography (CPC) method was developed for the preparative-scale isolation and purification of xylindein from the wood-staining fungi, Chlorociboria aeruginosa. Xylindein, a blue-green pigment naturally secreted from the hyphae and fruiting bodies of the fungus, has great value in the decorative wood industry and textile coloration. Xylindein has great potential for use as a fluorescent labeling agent as well as in organic semiconductor applications. However, a primary limitation of xylindein is its poor solubility in most common HPLC solvents. Consequently, it is arduous to purify using preparative liquid chromatography or solid-phase extraction (SPE). Support-free, liquid-liquid chromatographic methods, including CPC, where solutes are separated based on their different distribution coefficients (K D ) between two immiscible solvent systems, are promising alternatives for the purification of the compound on a preparative scale. In this work, a new biphasic solvent system suitable for CPC separation of xylindein was developed. Various groups of solvents were assessed for their suitability as xylindein extractants. A new solvent system suitable for CPC separation of xylindein, composed of heptane/THF/MEK/acetonitrile/acetic acid/water, was developed. This solvent system yielded a K D value for xylindein of 1.54±0.04, as determined by HPLC (n=3). The compositions of the upper phase and lower phase of the solvent system were determined by Heteronuclear Single Quantum Correlation (HSQC) NMR and proton NMR. A CPC system, equipped with a fraction collector, was used for the isolation of xylindein from crude extracts. The xylindein fractions isolated by the CPC were then analyzed using HPLC and presented as a fractogram. Based on the CPC fractogram, the purified xylindein fractions were achieved after 30min CPC separation time, yielding 71% extraction efficiency. The developed CPC method allowed for isolation of this naturally sourced xylindein in amounts suitable for further study. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficient purification of paclitaxel from yews using high-performance displacement chromatography technique.

PubMed

Watchueng, Jean; Kamnaing, Pierre; Gao, Jin-Ming; Kiyota, Taira; Yeboah, Faustinus; Konishi, Yasuo

2011-05-20

Paclitaxel was purified using high-performance displacement chromatography (HPDC) technique, but not by the mechanism of HPDC. On small scale, paclitaxel was extracted with methanol from dry needles of Taxus canadensis and was enriched by extracting with chloroform after removing water-soluble hydrophilic components and hexane-soluble hydrophobic components. Then, 93-99% purity of paclitaxel was obtained using the HPDC technique. On large scale, taxanes were enriched by solvent partitioning between acetic acid/MeOH/H(2)O and hexane and extracted with CH(2)Cl(2). Taxanes except paclitaxel were further removed by extracting with methanol-water-trifluoroacetic acid (1.0:98.9:0.1, v/v/v). Applying HPDC technique to water-insoluble substances is problematic as this method requires a highly aqueous solvent system. In order to overcome this incompatibility, a system was set up where paclitaxel, although in low concentration, was extracted by methanol-water-trifluoroacetic acid (10.0:89.9:0.1, v/v/v). Recycling the extracting solvent to ensure minimal volume, the extracted paclitaxel was adsorbed on a C(18) trap column. A C(18) column of 4.6mm internal diameter was then connected to the trap column. The HPDC technique was thus carried out using an isocratic acetonitrile-water-trifluoroacetic acid (30.0:69.9:0.1, v/v/v) mobile phase consisting of a displacer cetylpyridinium trifluoroacetate (3mg/mL). Paclitaxel was co-eluted with the displacer and spontaneously crystallized. The crystal (114mg) showed 99.4% purity and only 10% of paclitaxel in the starting crude extract was lost during the enrichment/purification processes. This large scale purification method was successfully applied to purify paclitaxel from Chinese yew in small scale, suggesting general applicability of the method. This is the first report of purifying a water-insoluble natural product using HPDC technique. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Rapid purification of diastereoisomers from Piper kadsura using supercritical fluid chromatography with chiral stationary phases.

PubMed

Xin, Huaxia; Dai, Zhuoshun; Cai, Jianfeng; Ke, Yanxiong; Shi, Hui; Fu, Qing; Jin, Yu; Liang, Xinmiao

2017-08-04

Supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an advanced solution for the separation of achiral compounds in Piper kadsura. Analogues and stereoisomers are abundant in natural products, but there are obstacles in separation using conventional method. In this paper, four lignan diastereoisomers, (-)-Galbelgin, (-)-Ganschisandrin, Galgravin and (-)-Veraguensin, from Piper kadsura were separated and purified by chiral SFC. Purification strategy was designed, considering of the compound enrichment, sample purity and purification throughput. Two-step achiral purification method on chiral preparative columns with stacked automated injections was developed. Unconventional mobile phase modifier dichloromethane (DCM) was applied to improve the sample solubility. Four diastereoisomers was prepared at the respective weight of 103.1mg, 10.0mg, 152.3mg and 178.6mg from 710mg extract with the purity of greater than 98%. Copyright © 2017 Elsevier B.V. All rights reserved.

Carbon-11 choline: synthesis, purification, and brain uptake inhibition by 2-dimethylaminoethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosen, M.A.; Jones, R.M.; Yano, Y.

We report an improved method for the synthesis and purification of (11C)methylcholine from the precursors (11C)methyliodide and 2-dimethylaminoethanol (deanol). Preparation time, including purification, is 35 min postbombardment. Forty millicuries of purified injectable (11C)choline were produced with a measured specific activity of greater than 300 Ci/mmol and a radiochemical purity greater than 98%. The decay corrected radiochemical yield for the synthesis and purification was approximately 50%. Residual precursor deanol, which inhibits brain uptake of choline, is removed by a rapid preparative high performance liquid chromatography (HPLC) method using a reverse phase cyano column with a biologically compatible 100% water eluent. Evaporationmore » alone did not completely remove the deanol precursor. Brain uptake of the (11C)choline product was six times greater after HPLC removal of deanol because doses of less than 1 microgram/kg significantly inhibit (14C)choline brain uptake.« less

Choices of capture chromatography technology in antibody manufacturing processes.

PubMed

DiLeo, Michael; Ley, Arthur; Nixon, Andrew E; Chen, Jie

2017-11-15

The capture process employed in monoclonal antibody downstream purification is not only the most critically impacted process by increased antibody titer resulting from optimized mammalian cell culture expression systems, but also the most important purification step in determining overall process throughput, product quality, and economics. Advances in separation technology for capturing antibodies from complex feedstocks have been one focus of downstream purification process innovation for past 10 years. In this study, we evaluated new generation chromatography resins used in the antibody capture process including Protein A, cation exchange, and mixed mode chromatography to address the benefits and unique challenges posed by each chromatography approach. Our results demonstrate the benefit of improved binding capacity of new generation Protein A resins, address the concern of high concentration surge caused aggregation when using new generation cation exchange resins with over 100mg/mL binding capacity, and highlight the potential of multimodal cation exchange resins for capture process design. The new landscape of capture chromatography technologies provides options to achieve overall downstream purification outcome with high product quality and process efficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

Experimental purification of two-atom entanglement.

PubMed

Reichle, R; Leibfried, D; Knill, E; Britton, J; Blakestad, R B; Jost, J D; Langer, C; Ozeri, R; Seidelin, S; Wineland, D J

2006-10-19

Entanglement is a necessary resource for quantum applications--entanglement established between quantum systems at different locations enables private communication and quantum teleportation, and facilitates quantum information processing. Distributed entanglement is established by preparing an entangled pair of quantum particles in one location, and transporting one member of the pair to another location. However, decoherence during transport reduces the quality (fidelity) of the entanglement. A protocol to achieve entanglement 'purification' has been proposed to improve the fidelity after transport. This protocol uses separate quantum operations at each location and classical communication to distil high-fidelity entangled pairs from lower-fidelity pairs. Proof-of-principle experiments distilling entangled photon pairs have been carried out. However, these experiments obtained distilled pairs with a low probability of success and required destruction of the entangled pairs, rendering them unavailable for further processing. Here we report efficient and non-destructive entanglement purification with atomic quantum bits. Two noisy entangled pairs were created and distilled into one higher-fidelity pair available for further use. Success probabilities were above 35 per cent. The many applications of entanglement purification make it one of the most important techniques in quantum information processing.

Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

PubMed Central

Gupta, Sanjeev K.; Shukla, Pratyoosh

2017-01-01

The protein productions strategies are crucial towards the development of application based research and elucidating the novel purification strategies for industrial production. Currently, there are few innovative avenues are studies for cloning, upstream, and purification through efficient bioprocess development. Such strategies are beneficial for industries as well as proven to be vital for effectual therapeutic protein development. Though, these techniques are well documented, but, there is scope of addition to current knowledge with novel and new approaches and it will pave new avenues in production of recombinant microbial and non-microbial proteins including secondary metabolites. In this review, we have focussed on the recent development in clone selection, various modern fermentation and purification technologies and future directions in these emerging areas. Moreover, we have also highlighted notable perspectives and challenges involved in the bioengineering of such proteins, including quality by design, gene editing and pioneering ideas. The biopharmaceutical industries continue to shift towards more flexible, automated platforms and economical product development, which in turn can help in developing the cost effective processes and affordable drug development for a large community. PMID:28725194

Renaissance of protein crystallization and precipitation in biopharmaceuticals purification.

PubMed

Dos Santos, Raquel; Carvalho, Ana Luísa; Roque, A Cecília A

The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Copyright © 2016 Elsevier Inc. All rights reserved.

Optimization of Ammonium Sulfate Concentration for Purification of Colorectal Cancer Vaccine Candidate Recombinant Protein GA733-FcK Isolated from Plants.

PubMed

Park, Se-Ra; Lim, Chae-Yeon; Kim, Deuk-Su; Ko, Kisung

2015-01-01

A protein purification procedure is required to obtain high-value recombinant injectable vaccine proteins produced in plants as a bioreactor. However, existing purification procedures for plant-derived recombinant proteins are often not optimized and are inefficient, with low recovery rates. In our previous study, we used 25-30% ammonium sulfate to precipitate total soluble proteins (TSPs) in purification process for recombinant proteins from plant leaf biomass which has not been optimized. Thus, the objective in this study is to optimize the conditions for plant-derived protein purification procedures. Various ammonium sulfate concentrations (15-80%) were compared to determine their effects on TSPs yield. With 50% ammonium sulfate, the yield of precipitated TSP was the highest, and that of the plant-derived colorectal cancer-specific surface glycoprotein GA733 fused to the Fc fragment of human IgG tagged with endoplasmic reticulum retention signal KDEL (GA733(P)-FcK) protein significantly increased 1.8-fold. SDS-PAGE analysis showed that the purity of GA733(P)-FcK protein band appeared to be similar to that of an equal dose of mammalian-derived GA733-Fc (GA733(M)-Fc). The binding activity of purified GA733(P)-FcK to anti-GA733 mAb was as efficient as the native GA733(M)-Fc. Thus, the purification process was effectively optimized for obtaining a high yield of plant-derived antigenic protein with good quality. In conclusion, the purification recovery rate of large quantities of recombinant protein from plant expression systems can be enhanced via optimization of ammonium sulfate concentration during downstream processes, thereby offering a promising solution for production of recombinant GA733-Fc protein in plants.

Use of anionic denaturing detergents to purify insoluble proteins after overexpression

PubMed Central

2012-01-01

Background Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS) is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. Results Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS) and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. Conclusion Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology. PMID:23231964

Procedures for making gaseous industrial waste safe

NASA Astrophysics Data System (ADS)

Matros, Yu Sh; Noskov, Aleksandr S.

1990-10-01

The application of various methods (adsorption, absorption, thermal afterburning, catalytic purification, and others) for the removal of sulphur and nitrogen oxides, toxic organic compounds, hydrogen sulphide, and carbon monoxide from industrial waste gases is described. Much attention is devoted to the catalytic procedure for making the gases safe using an energy collecting non-stationary method (reversible process). The advantages and limitations of various gas purification methods are considered. The bibliography includes 279 references.

Efficient ASK-assisted system for expression and purification of plant F-box proteins.

PubMed

Li, Haiou; Yao, Ruifeng; Ma, Sui; Hu, Shuai; Li, Suhua; Wang, Yupei; Yan, Chun; Xie, Daoxin; Yan, Jianbin

2017-11-01

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.

PubMed

Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto

2011-09-12

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

Production of phytase under solid-state fermentation using Rhizopus oryzae: novel strain improvement approach and studies on purification and characterization.

PubMed

Rani, Richa; Ghosh, Sanjoy

2011-11-01

Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (∼34 kDa) showed optimal temperature of 45 °C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×10(6) M(-1) s(-1)). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method. Copyright © 2011. Published by Elsevier Ltd.

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.

PubMed

Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi

2012-12-01

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

Broad application and optimization of a single wash-step for integrated endotoxin depletion during protein purification.

PubMed

Koziel, David; Michaelis, Uwe; Kruse, Tobias

2018-08-01

Endotoxins contaminate proteins that are produced in E. coli. High levels of endotoxins can influence cellular assays and cause severe adverse effects when administered to humans. Thus, endotoxin removal is important in protein purification for academic research and in GMP manufacturing of biopharmaceuticals. Several methods exist to remove endotoxin, but often require additional downstream-processing steps, decrease protein yield and are costly. These disadvantages can be avoided by using an integrated endotoxin depletion (iED) wash-step that utilizes Triton X-114 (TX114). In this paper, we show that the iED wash-step is broadly applicable in most commonly used chromatographies: it reduces endotoxin by a factor of 10 3 to 10 6 during NiNTA-, MBP-, SAC-, GST-, Protein A and CEX-chromatography but not during AEX or HIC-chromatography. We characterized the iED wash-step using Design of Experiments (DoE) and identified optimal experimental conditions for application scenarios that are relevant to academic research or industrial GMP manufacturing. A single iED wash-step with 0.75% (v/v) TX114 added to the feed and wash buffer can reduce endotoxin levels to below 2 EU/ml or deplete most endotoxin while keeping the manufacturing costs as low as possible. The comprehensive characterization enables academia and industry to widely adopt the iED wash-step for a routine, efficient and cost-effective depletion of endotoxin during protein purification at any scale. Copyright © 2018. Published by Elsevier B.V.

Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes.

PubMed

Gagaoua, Mohammed; Hoggas, Naouel; Hafid, Kahina

2015-02-01

The present work describes for the first time an elegant non-chromatographic method, the three phase partitioning for the purification and recovery of zingibain, a milk-clotting enzyme, from Zingiber officinale rhizomes. Factors affecting partitioning efficiency such as (NH4)2SO4 saturation, crude extract to t-butanol ratio and pH on zingibain partitioning were investigated. Optimal purification parameters were 50% (NH4)2SO4 saturation with 1.0:1.0 ratio of crude extract:t-butanol at pH 7.0, which gave 14.91 purification fold with 215% recovery of zingibain. The enzyme was found to be exclusively partitioned in the aqueous phase. The enzyme showed a prominent single band on SDS-PAGE. It is a monomeric protein of 33.8 kDa and its isoelectric point is 4.38. The enzyme exhibited maximal proteolytic activity at a temperature of 60 °C and pH 7.0. It was found to be stable at 40-65 °C during 2 h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca(2+), K(+) and Na(+). It was completely inhibited by heavy metal ions such as Cu(2+) and Hg(2+) and partially by Cd(+). Zingibain milk-clotting activity (MCA) was found to be highly stable when stored under freezing (-20 °C) for 30 days compared at 4 °C. Copyright © 2014 Elsevier B.V. All rights reserved.

Review of the workshop on low-cost polysilicon for terrestrial photovoltaic solar cell applications

NASA Technical Reports Server (NTRS)

Lutwack, R.

1986-01-01

Topics reviewed include: polysilicon material requirements; effects of impurities; requirements for high-efficiency solar cells; economics; development of silane processes; fluidized-bed processor development; silicon purification; and marketing.

Copper(I)/TEMPO Catalyzed Aerobic Oxidation of Primary Alcohols to Aldehydes with Ambient Air

PubMed Central

Hoover, Jessica M.; Steves, Janelle E.; Stahl, Shannon S.

2012-01-01

This protocol describes a practical laboratory-scale method for aerobic oxidation of primary alcohols to aldehydes, using a chemoselective CuI/TEMPO catalyst system. The catalyst is prepared in situ from commercially available reagents, and the reactions are performed in a common organic solvent (acetonitrile) with ambient air as the oxidant. Three different reaction conditions and three procedures for the isolation and purification of the aldehyde product are presented. The oxidations of eight different alcohols, described here, include representative examples of each reaction condition and purification method. Reaction times vary from 20 min to 24 h, depending on the alcohol, while the purification methods each take about 2 h. The total time necessary for the complete protocol ranges from 3 – 26 h. PMID:22635108

Effect of work of adhesion on deep bed filtration process

NASA Astrophysics Data System (ADS)

Przekop, Rafał; Jackiewicz, Anna; WoŻniak, Michał; Gradoń, Leon

2016-06-01

Collection of aerosol particles in the particular steps of the technology of their production, and purification of the air at the workplace and atmospheric environment, requires the efficient method of separation of particulate matter from the carrier gas. There are many papers published in last few years in which the deposition of particles on fibrous collectors is considered, Most of them assume that collisions between particle and collector surface is 100% effective. In this work we study the influence of particles and fiber properties on the deposition efficiency. For the purpose of this work the lattice-Boltzmann model describes fluid dynamics, while the solid particle motion is modeled by the Brownian dynamics. The interactions between particles and surface are modelled using energy balanced oscillatory model. The work of adhesion was estimated using Atomic Force Microscopy.

Versatile Poly(Diallyl Dimethyl Ammonium Chloride)-Layered Nanocomposites for Removal of Cesium in Water Purification.

PubMed

Jang, Sung-Chan; Kang, Sung-Min; Kim, Gi Yong; Rethinasabapathy, Muruganantham; Haldorai, Yuvaraj; Lee, Ilsong; Han, Young-Kyu; Renshaw, Joanna C; Roh, Changhyun; Huh, Yun Suk

2018-06-12

In this work, we elucidate polymer-layered hollow Prussian blue-coated magnetic nanocomposites as an adsorbent to remove radioactive cesium from environmentally contaminated water. To do this, Fe₃O₄ nanoparticles prepared using a coprecipitation method were thickly covered with a layer of cationic polymer to attach hollow Prussian blue through a self-assembly process. The as-synthesized adsorbent was confirmed through various analytical techniques. The adsorbent showed a high surface area (166.16 m²/g) with an excellent cesium adsorbent capacity and removal efficiency of 32.8 mg/g and 99.69%, respectively. Moreover, the superparamagnetism allows effective recovery of the adsorbent using an external magnetic field after the adsorption process. Therefore, the magnetic adsorbent with a high adsorption efficiency and convenient recovery is expected to be effectively used for rapid remediation of radioactive contamination.

Effect of work of adhesion on deep bed filtration process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Przekop, Rafał; Jackiewicz, Anna; Gradoń, Leon

2016-06-08

Collection of aerosol particles in the particular steps of the technology of their production, and purification of the air at the workplace and atmospheric environment, requires the efficient method of separation of particulate matter from the carrier gas. There are many papers published in last few years in which the deposition of particles on fibrous collectors is considered, Most of them assume that collisions between particle and collector surface is 100% effective. In this work we study the influence of particles and fiber properties on the deposition efficiency. For the purpose of this work the lattice-Boltzmann model describes fluid dynamics,more » while the solid particle motion is modeled by the Brownian dynamics. The interactions between particles and surface are modelled using energy balanced oscillatory model. The work of adhesion was estimated using Atomic Force Microscopy.« less

Engineering of a highly efficient Xe₂*-excilamp (xenon excimer lamp, λmax=172 nm, η=40%) and qualitative comparison to a low-pressure mercury lamp (LP-Hg, λ=185/254 nm) for water purification.

PubMed

Al-Gharabli, Samer; Engeßer, Patrick; Gera, Diana; Klein, Sandra; Oppenländer, Thomas

2016-02-01

Excilamps are mercury-free gas-discharge sources of non-coherent VUV or UV radiation with high radiant power and a long lifetime. The most efficient excilamp that is currently available on the market is a VUV xenon excilamp system (Xe2(*)-excimer lamp, λ(max) = 172 nm) with a stated radiant efficiency η of 40% at an electrical input power P(el) of 20 W, 50 W or 100 W. In this paper, the use of this highly efficient Xe2(*)-excilamp (P(el) = 20 W) for water treatment is demonstrated using a recirculating laboratory photoreactor system with negative radiation geometry. The efficiency in the 172 nm initiated bleaching of aqueous solutions of Rhodamine B is compared to that initiated by a common low-pressure mercury (LP-Hg) lamp (185 nm, TNN 15/32). The dependence of the pseudo zero order rate constant k´ of decolorization of RhB on the flow rate and on the initial concentration of RhB was investigated. Both lamps exhibited dependences of k´ on the initial concentration of RhB, which represents a typical saturation kinetical behavior. The saturation kinetics was very prominent in the case of the Xe2(*)-excilamp. Also, the Xe2(*)-excilamp treatment exhibited a significant influence on the flow rate of the RhB aqueous solution, which was not the case during the LP-Hg lamp initiated bleaching of RhB. The results of this paper demonstrate that Xe2(*)-excilamps can be used for VUV-initiated water purification. However, to reach the maximum efficacy of the Xe2(*)-excilamp for photo-initiated water purification further engineering optimization of the photoreactor concept is necessary. Copyright © 2015 Elsevier Ltd. All rights reserved.

Emulsion Synthesis of Size-Tunable CH3NH3PbBr3 Quantum Dots: An Alternative Route toward Efficient Light-Emitting Diodes.

PubMed

Huang, Hailong; Zhao, Fangchao; Liu, Lige; Zhang, Feng; Wu, Xian-gang; Shi, Lijie; Zou, Bingsuo; Pei, Qibing; Zhong, Haizheng

2015-12-30

We report a facile nonaqueous emulsion synthesis of colloidal halide perovskite quantum dots by controlled addition of a demulsifier into an emulsion of precursors. The size of resulting CH3NH3PbBr3 quantum dots can be tuned from 2 to 8 nm by varying the amount of demulsifier. Moreover, this emulsion synthesis also allows the purification of these quantum dots by precipitation from the colloidal solution and obtains solid-state powder which can be redissolved for thin film coating and device fabrication. The photoluminescence quantum yields of the quantum dots is generally in the range of 80-92%, and can be well-preserved after purification (∼80%). Green light-emitting diodes fabricated comprising a spin-cast layer of the colloidal CH3NH3PbBr3 quantum dots exhibited maximum current efficiency of 4.5 cd/A, power efficiency of 3.5 lm/W, and external quantum efficiency of 1.1%. This provides an alternative route toward high efficient solution-processed perovskite-based light-emitting diodes. In addition, the emulsion synthesis is versatile and can be extended for the fabrication of inorganic halide perovskite colloidal CsPbBr3 nanocrystals.

Photocatalytic materials and technologies for air purification.

PubMed

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.

Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature.

PubMed

Xu, Juan; Luo, Hui; López, Claudia; Xiao, Jing; Chang, Yanhong

2015-10-01

The main goal of the present work is to investigate a novel process of purification and immobilization of a thermophilic catalase at high temperatures. The catalase, originated from Bacillus sp., was overexpressed in a recombinant Escherichia coli BL21(DE3)/pET28-CATHis and efficiently purified by heat treatment, achieving a threefold purification. The purified catalase was then immobilized onto an epoxy support at different temperatures (25, 40, and 55 °C). The immobilizate obtained at higher temperatures reached its maximum activity in a shorter time than that obtained at lower temperatures. Furthermore, immobilization at higher temperatures required a lower ionic strength than immobilization at lower temperatures. The characteristics of immobilized enzymes prepared at different temperatures were investigated. The high-temperature immobilizate (55 °C) showed the highest thermal stability, followed by the 40 °C immobilizate. And the high-temperature immobilizate (55 °C) had slightly higher operational stability than the 25 °C immobilizate. All of the immobilized catalase preparations showed higher stability than the free enzyme at alkaline pH 10.0, while the alkali resistance of the 25 °C immobilizate was slightly better than that of the 40 and 55 °C immobilizates.

Optimization in expression and purification of modified apoptin as selective anti-cancer therapy

NASA Astrophysics Data System (ADS)

Sahlan, Muhamad; Wiseso, Anggoro; Hermansyah, Heri; Yohda, Masafumi

2017-02-01

Cancer is a disease caused by abnormal growth of tissue cells of the body that turn into cancer cells. Apoptin from Chicken Anemia Virus is known to have the ability to trigger apoptosis in cancer cells in vitro and in vivo, but not in normal cells. The production of Apoptin was done on Escherichia coli via plasmid pET9a and modified to improve the efficiency and ease of purification using IMAC nickel, by adding a few tags and cleavage site. The expected result is modified Apoptin and evidence of proteins expressed through SDS-PAGE analysis.

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

NASA Astrophysics Data System (ADS)

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 dioxins and furans from flue gases by non-flammable adsorbents in a fixed bed.

PubMed

Fell, H J; Tuczek, M

1998-01-01

The presented adsorption--process KOMBISORBON is applied for high efficient off-gas purification, preferably of polychlorinated dioxins and furans from off-gas of incineration plants, which are generated, when these are operated under unfavourable conditions [2]. This off-gas purification process complies with german laws, which limit the concentration of these substances to less than 0.1 ng toxicity equivalents (TE) per cubic metre of gas [1]. The adsorbent, the adsorption process and its plant concept (fixed bed) is described in detail including economics and obtained operation results. Alternative removal technologies are briefly outlined.

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

PubMed

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

2015-04-01

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance. Copyright © 2015 Elsevier B.V. All rights reserved.

New Photocatalysis for Effective Degradation of Organic Pollutants in Water

NASA Astrophysics Data System (ADS)

Zarei Chaleshtori, M.; Saupe, G. B.; Masoud, S.

2009-12-01

The presence of harmful compounds in water supplies and in the discharge of wastewater from chemical industries, power plants, and agricultural sources is a topic of global concern. The processes and technologies available at the present time for the treatment of polluted water are varied that include traditional water treatment processes such as biological, thermal and chemical treatment. All these water treatment processes, have limitations of their own and none is cost effective. Advanced oxidation processes have been proposed as an alternative for the treatment of this kind of wastewater. Heterogeneous photocatalysis has recently emerged as an efficient method for purifying water. TiO2 has generally been demonstrated to be the most active semiconductor material for decontamination water. One significant factor is the cost of separation TiO2, which is generally a powder having a very small particle size from the water after treatment by either sedimentation or ultrafiltration. The new photocatalyst, HTiNbO5, has been tested to determine whether its photocatalytic efficiency is good enough for use in photocatalytic water purification since it has high surface area and relatively large particle size. The larger particle sizes of the porous materials facilitate catalyst removal from a solution, after purification has taken place. It can be separated from water easily than TiO2, a significant technical improvement that might eliminate the tedious final filtration necessary with a slurry. These materials are characterized and tested as water decontamination photocatalysts. The new catalyst exhibited excellent catalytic activity, but with a strong pH dependence on the photo efficiency. These results suggest that elimination of the ion exchange character of the catalyst may greatly improve its performance at various pHs. This new research proposes to study the effects of a topotactic dehydration reaction on these new porous material catalysts.

Removal of emerging micropollutants from water using cyclodextrin.

PubMed

Nagy, Zsuzsanna Magdolna; Molnár, Mónika; Fekete-Kertész, Ildikó; Molnár-Perl, Ibolya; Fenyvesi, Éva; Gruiz, Katalin

2014-07-01

Small scale laboratory experiment series were performed to study the suitability of a cyclodextrin-based sorbent (ß-cyclodextrin bead polymer, BCDP) for modelling the removal of micropollutants from drinking water and purified waste water using simulated inflow test solutions containing target analytes (ibuprofen, naproxen, ketoprofen, bisphenol-A, diclofenac, β-estradiol, ethinylestradiol, estriol, cholesterol at 2-6 μg/L level). This work was focused on the preliminary evaluation of BCDP as a sorbent in two different model systems (filtration and fluidization) applied for risk reduction of emerging micropollutants. For comparison different filter systems combined with various sorbents (commercial filter and activated carbon) were applied and evaluated in the filtration experiment series. The spiked test solution (inflow) and the treated outflows were characterized by an integrated methodology including chemical analytical methods gas chromatography-tandem mass spectrometry (GC-MS/MS) and various environmental toxicity tests to determine the efficiency and selectivity of the applied sorbents. Under experimental conditions the cyclodextrin-based filters used for purification of drinking water in most cases were able to absorb more than 90% of the bisphenol-A and of the estrogenic compounds. Both the analytical chemistry and toxicity results showed efficient elimination of these pollutants. Especially the toxicity of the filtrate decreased considerably. Laboratory experiment modelling post-purification of waste water was also performed applying fluidization technology by ß-cyclodextrin bead polymer. The BCDP removed efficiently from the spiked test solution most of the micropollutants, especially the bisphenol-A (94%) and the hormones (87-99%) The results confirmed that the BCDP-containing sorbents provide a good solution to water quality problems and they are able to decrease the load and risk posed by micropollutants to the water systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Extraction and purification methods in downstream processing of plant-based recombinant proteins.

PubMed

Łojewska, Ewelina; Kowalczyk, Tomasz; Olejniczak, Szymon; Sakowicz, Tomasz

2016-04-01

During the last two decades, the production of recombinant proteins in plant systems has been receiving increased attention. Currently, proteins are considered as the most important biopharmaceuticals. However, high costs and problems with scaling up the purification and isolation processes make the production of plant-based recombinant proteins a challenging task. This paper presents a summary of the information regarding the downstream processing in plant systems and provides a comprehensible overview of its key steps, such as extraction and purification. To highlight the recent progress, mainly new developments in the downstream technology have been chosen. Furthermore, besides most popular techniques, alternative methods have been described. Copyright © 2015 Elsevier Inc. All rights reserved.

[Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil].

PubMed

Pirog, T P; Shevchuk, T A; Voloshinka, I N; Gregirchak, N N

2005-01-01

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5-99.8%.

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

PubMed Central

Aboulhouda, Soufiane; Di Santo, Rachael; Therizols, Gabriel; Weinberg, David

2017-01-01

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues. PMID:29170751

Bioconversion of Sugarcane Biomass into Ethanol: An Overview about Composition, Pretreatment Methods, Detoxification of Hydrolysates, Enzymatic Saccharification, and Ethanol Fermentation

PubMed Central

Canilha, Larissa; Chandel, Anuj Kumar; Suzane dos Santos Milessi, Thais; Antunes, Felipe Antônio Fernandes; Luiz da Costa Freitas, Wagner; das Graças Almeida Felipe, Maria; da Silva, Silvio Silvério

2012-01-01

Depleted supplies of fossil fuel, regular price hikes of gasoline, and environmental damage have necessitated the search for economic and eco-benign alternative of gasoline. Ethanol is produced from food/feed-based substrates (grains, sugars, and molasses), and its application as an energy source does not seem fit for long term due to the increasing fuel, food, feed, and other needs. These concerns have enforced to explore the alternative means of cost competitive and sustainable supply of biofuel. Sugarcane residues, sugarcane bagasse (SB), and straw (SS) could be the ideal feedstock for the second-generation (2G) ethanol production. These raw materials are rich in carbohydrates and renewable and do not compete with food/feed demands. However, the efficient bioconversion of SB/SS (efficient pretreatment technology, depolymerization of cellulose, and fermentation of released sugars) remains challenging to commercialize the cellulosic ethanol. Among the technological challenges, robust pretreatment and development of efficient bioconversion process (implicating suitable ethanol producing strains converting pentose and hexose sugars) have a key role to play. This paper aims to review the compositional profile of SB and SS, pretreatment methods of cane biomass, detoxification methods for the purification of hydrolysates, enzymatic hydrolysis, and the fermentation of released sugars for ethanol production. PMID:23251086

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Fluidic conduits for highly efficient purification of target species in EWOD-driven droplet microfluidics.

PubMed

Shah, Gaurav J; Kim, Chang-Jin Cj

2009-08-21

Due to the lack of continuous flows that would wash unwanted specifies and impurities off from a target location, droplet microfluidics commonly employs a long serial dilution process to purify target species. In this work, we achieve high-purity separation for the case of electrowetting-on-dielectric (EWOD) based droplet microfluidics by introducing a "fluidic conduit" between a sample droplet and a buffer droplet. The long and slender fluidic path minimizes the diffusion and fluidic mixing between the two droplets (thus eliminating non-specific transport) but provides a conduit between them for actively transported particles (thus allowing the specific transport). The conduit is purely fluidic, stabilized chemically (e.g. using surfactants) and controlled by EWOD. The effectiveness of the technique is demonstrated by eliminating approximately 97% non-magnetic beads in just one purification step, while maintaining high collection efficiency (>99%) of magnetic beads.

Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

NASA Astrophysics Data System (ADS)

Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

1992-08-01

The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

Air purification from a mixture VOCs in the pilot-scale trickle-bed bioreactor (TBB)

NASA Astrophysics Data System (ADS)

Sarzyński, Rafał; Gąszczak, Agnieszka; Janecki, Daniel; Bartelmus, Grażyna

2017-10-01

The efficiency of the air bio-purification from the mixture of two volatile organic compounds (styrene and p-xylene) was studied. The process was carried out in a pilot-scale trickle-bed bioreactor installation designed to purify ˜200 m3h-1 of the polluted air. The bioreactor operated at concurrent flow of gas and liquid (mineral salt solution) through packing (polypropylene Ralu rings) covered with a thin layer of microorganisms (bacterial consortium of Pseudomonas sp. E-022150 and Pseudomonas putida mt-2). The experiments, carried out for various values of a reactor load with pollutant, confirmed the great efficiency of the investigated process. At the tested bed load with pollution (inlet specific pollutant load was changed within the range of 41 - 84 gm-3 h -1), styrene conversion degree changed within the range of 80-87% and p-xylene conversion degree within the range of 42-48%.

Methods for purifying carbon materials

DOEpatents

Dailly, Anne [Pasadena, CA; Ahn, Channing [Pasadena, CA; Yazami, Rachid [Los Angeles, CA; Fultz, Brent T [Pasadena, CA

2009-05-26

Methods of purifying samples are provided that are capable of removing carbonaceous and noncarbonaceous impurities from a sample containing a carbon material having a selected structure. Purification methods are provided for removing residual metal catalyst particles enclosed in multilayer carbonaceous impurities in samples generate by catalytic synthesis methods. Purification methods are provided wherein carbonaceous impurities in a sample are at least partially exfoliated, thereby facilitating subsequent removal of carbonaceous and noncarbonaceous impurities from the sample. Methods of purifying carbon nanotube-containing samples are provided wherein an intercalant is added to the sample and subsequently reacted with an exfoliation initiator to achieve exfoliation of carbonaceous impurities.

Determination of acrylamide in coffee and coffee products by GC-MS using an improved SPE clean-up.

PubMed

Soares, C; Cunha, S; Fernandes, J

2006-12-01

An improved gas chromatography-mass spectrometry (GC-MS) method to determine acrylamide (AA) in coffee and coffee products was developed. The method was based on two main purification steps: the first with ethanol and Carrez solutions in order to precipitate polysaccharides and proteins, respectively; and the second with a layered solid-phase extraction (SPE) column which proved to be efficient in the elimination of the main chromatographic interferences. The method is applicable to a wide range of coffee products. Twenty-six samples of different coffee products were analysed. The levels of AA were in the range 11.4-36.2 microg l-1 for 'espresso coffee' and 200.8-229.4 microg l-1 for coffee blends with cereals. The results indicate that the presence of cereals significantly increased the levels of AA.

Evaluation of toxicity of ‘Vatsanabha’ (Aconitum ferox, Ranunculaceae) Before and After Shodhana

PubMed Central

Deore, S.L.; Moon, K.V.; Khadabadi, S.S.; Deokate, U.A.; Baviskar, B.A.

2013-01-01

Ayurvedic preparations contain toxic elements like heavy metals and other chemicals exceeding their permissible limits. Ayurvedic method of detoxification of such products involves Shodhana. Hence, in present paper it has been decided to replace Ayurvedic Shodhana process by chemical purification method and to study the benefits and/or drawbacks of the traditional Ayurvedic Shodhana process. Crude aconite root, Ayurvedic Shodhana treated aconite root and chemical Shodhana treated aconite root samples were evaluated for toxicity and changes by animal studies and thin layer chromatography (TLC) respectively. The results of the toxicity study suggest that the modified method of Shodhana is less efficient as compared to the traditional Ayurvedic Shodhana process. TLC studies have shown that pseudoaconitine and aconitine were converted into far less toxic substances like veratroyl pseudoaconine and benzoylaconine respectively only in traditional Ayurvedic Shodhana. PMID:24023444

Database and new models based on a group contribution method to predict the refractive index of ionic liquids.

PubMed

Wang, Xinxin; Lu, Xingmei; Zhou, Qing; Zhao, Yongsheng; Li, Xiaoqian; Zhang, Suojiang

2017-08-02

Refractive index is one of the important physical properties, which is widely used in separation and purification. In this study, the refractive index data of ILs were collected to establish a comprehensive database, which included about 2138 pieces of data from 1996 to 2014. The Group Contribution-Artificial Neural Network (GC-ANN) model and Group Contribution (GC) method were employed to predict the refractive index of ILs at different temperatures from 283.15 K to 368.15 K. Average absolute relative deviations (AARD) of the GC-ANN model and the GC method were 0.179% and 0.628%, respectively. The results showed that a GC-ANN model provided an effective way to estimate the refractive index of ILs, whereas the GC method was simple and extensive. In summary, both of the models were accurate and efficient approaches for estimating refractive indices of ILs.

Soft-Bake Purification of SWCNTs Produced by Pulsed Laser Vaporization

NASA Technical Reports Server (NTRS)

Yowell, Leonard; Nikolaev, Pavel; Gorelik, Olga; Allada, Rama Kumar; Sosa, Edward; Arepalli, Sivaram

2013-01-01

The "soft-bake" method is a simple and reliable initial purification step first proposed by researchers at Rice University for single-walled carbon nanotubes (SWCNT) produced by high-pressure carbon mon oxide disproportionation (HiPco). Soft-baking consists of annealing as-produced (raw) SWCNT, at low temperatures in humid air, in order to degrade the heavy graphitic shells that surround metal particle impurities. Once these shells are cracked open by the expansion and slow oxidation of the metal particles, the metal impurities can be digested through treatment with hydrochloric acid. The soft-baking of SWCNT produced by pulsed-laser vaporization (PLV) is not straightforward, because the larger average SWCNT diameters (.1.4 nm) and heavier graphitic shells surrounding metal particles call for increased temperatures during soft-bake. A part of the technology development focused on optimizing the temperature so that effective cracking of the graphitic shells is balanced with maintaining a reasonable yield, which was a critical aspect of this study. Once the ideal temperature was determined, a number of samples of raw SWCNT were purified using the soft-bake method. An important benefit to this process is the reduced time and effort required for soft-bake versus the standard purification route for SWCNT. The total time spent purifying samples by soft-bake is one week per batch, which equates to a factor of three reduction in the time required for purification as compared to the standard acid purification method. Reduction of the number of steps also appears to be an important factor in improving reproducibility of yield and purity of SWCNT, as small deviations are likely to get amplified over the course of a complicated multi-step purification process.

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

PubMed

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.

New Contribution to the Method of Van Arkel for the Purification of Metals on Incandescent Filaments; NUEVAS APORTACIONES AL METODO DE VAN ARKEL PARA LA PURIFICACION DE METALES SOBRE FILAMENTOS INCANDESCENTES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez, M.L.; Martorell, J.T.

1962-01-01

The purification of zirconium in a cyclical static process using ZrI/sub 4/ as the volatile compound and W filaments was studied after a review of previous works on the subject. The equations corresponding to the isothermal process are given, in some detail. The optimum conditions of temperature and velocity for the maximum purification of the metal were determined. (J.S.R.)

Comparative aspects of the purification and properties of cholinesterases

PubMed Central

Augustinsson, Klas-Bertil

1971-01-01

Recent years have seen great progress in the purification and characterization of cholinesterases. Investigation has indicated the existence of two principal groups: a fairly homogeneous group of acetylcholinesterases and a group of enzymes that utilize butyrylcholine, propionycholine, or benzoylcholine as substrates and that differ widely in their properties. This paper reviews the different types of cholinesterase and their sources, the importance of a proper choice of substrate in cholinesterase studies, methods for the purification of cholinesterases, and some of the properties of these enzymes. PMID:4938026

Purification non-aqueous solution of quantum dots CdSe- CdS-ZnS from excess organic substance-stabilizer by use PE- HD membrane

NASA Astrophysics Data System (ADS)

Kosolapova, K.; Al-Alwani, A.; Gorbachev, I.; Glukhovskoy, E.

2015-11-01

Recently, a new simple method for the purification of CdSe-CdS-ZnS quantum dots by using membrane filtration, the filtration process, successfully separated the oleic acid from quantum dots through membranes purification after synthesis; purification of quantum dots is a very significant part of post synthetical treatment that determines the properties of the material. We explore the possibilities of the Langmuir-Blodgett technique to make such layers, using quantum dots as a model system. The Langmuir monolayer of quantum dots were then investigated the surface pressure-area isotherm. From isotherm, we found the surface pressure monolayer changed with time.

Preparation of drinking water used in water supply systems of the towns Zrenjanin and Temerin by electrochemical methods.

PubMed

Orescanin, Visnja; Kollar, Robert; Nad, Karlo; Mikulic, Nenad

2013-01-01

The aim of this work was the development and application of the pilot plant with the capacity of 1000 L/day for the purification of groundwater used for human consumption characterized with high concentration of arsenic and increased values of organic pollutants, ammonia, nitrites, color and turbidity. For that purpose, groundwater from the production wells supplying the towns Zrenjanin and Temerin (Vojvodina, Serbia) was used. Due to its complex composition, the purification system required the combination of the electroreduction/electrocoagulation, using iron and aluminum electrode plates with/without ozonation, followed by the electromagnetic treatment and the finally by the simultaneous ozonation/UV treatment. The electroreduction was used for the removal of nitrates, nitrites, and Cr(VI), while the removal of arsenic, heavy metals, suspended solids, color and turbidity required the application of the electrocoagulation with simultaneous ozonation. Organic contaminants and ammonia were removed completely in the last treatment step by applying the simultaneous ozonation/UV treatment. All measured parameters in the purified water were significantly lower compared to the regulated values. Under the optimum treatment conditions, the removal efficiencies for color, turbidity, suspended solids, total arsenic, total chromium, Ni(II), total copper, sulfates, fluorides, chemical oxygen demand, ammonia, nitrates, and nitrites were 100%. The removal efficiencies of the total manganese and iron were 85.19% and 97.44%, respectively, whilst the final concentrations were 4 and 7 μg/L, respectively.

Purification and switching protocols for dissipatively stabilized entangled qubit states

NASA Astrophysics Data System (ADS)

Hein, Sven M.; Aron, Camille; Türeci, Hakan E.

2016-06-01

Pure dephasing processes limit the fidelities achievable in driven-dissipative schemes for stabilization of entangled states of qubits. We propose a scheme which, combined with already existing entangling methods, purifies the desired entangled state by driving out of equilibrium auxiliary dissipative cavity modes coupled to the qubits. We lay out the specifics of our scheme and compute its efficiency in the particular context of two superconducting qubits in a cavity-QED architecture, where the strongly coupled auxiliary modes provided by collective cavity excitations can drive and sustain the qubits in maximally entangled Bell states with fidelities reaching 90% for experimentally accessible parameters.

An overview of the kinetic parameters of class B beta-lactamases.

PubMed Central

Felici, A; Amicosante, G; Oratore, A; Strom, R; Ledent, P; Joris, B; Fanuel, L; Frère, J M

1993-01-01

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific 'carbapenemase'. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column. Images Figure 1 PMID:8471035

An alternate approach to the production of radioisotopes for nuclear medicine applications

NASA Astrophysics Data System (ADS)

D'Auria, John M.; Keller, Roderich; Ladouceur, Keith; Lapi, Suzanne E.; Ruth, Thomas J.; Schmor, Paul

2013-03-01

There is a growing need for the production of radioisotopes for both diagnostic and therapeutic medical applications. Radioisotopes that are produced using the (n,γ) or (γ,n) reactions, however, typically result in samples with low specific activity (radioactivity/gram) due to the high abundance of target material of the same element. One method to effectively remove the isotopic impurity is electro-magnetic mass separation. An Ion Source Test Facility has been constructed at TRIUMF to develop high-intensity, high-efficiency, reliable ion sources for purification of radioactive isotopes, particularly those used in nuclear medicine. In progress studies are presented.

Proteomic Analysis of Cytoskeleton Proteins in Fish.

PubMed

Gotesman, Michael; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

2016-01-01

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.

An alternate approach to the production of radioisotopes for nuclear medicine applications.

PubMed

D'Auria, John M; Keller, Roderich; Ladouceur, Keith; Lapi, Suzanne E; Ruth, Thomas J; Schmor, Paul

2013-03-01

There is a growing need for the production of radioisotopes for both diagnostic and therapeutic medical applications. Radioisotopes that are produced using the (n,γ) or (γ,n) reactions, however, typically result in samples with low specific activity (radioactivity∕gram) due to the high abundance of target material of the same element. One method to effectively remove the isotopic impurity is electro-magnetic mass separation. An Ion Source Test Facility has been constructed at TRIUMF to develop high-intensity, high-efficiency, reliable ion sources for purification of radioactive isotopes, particularly those used in nuclear medicine. In progress studies are presented.

Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths.

PubMed

Krajacić, Mladen; Ivancic-Jelecki, Jelena; Forcic, Dubravko; Vrdoljak, Anto; Skorić, Dijana

2007-03-09

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens' presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.

A general method for the purification of restriction enzymes.

PubMed Central

Greene, P J; Heyneker, H L; Bolivar, F; Rodriguez, R L; Betlach, M C; Covarrubias, A A; Backman, K; Russel, D J; Tait, R; Boyer, H W

1978-01-01

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA. PMID:673857

Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification.

PubMed

Carnes, Aaron E; Hodgson, Clague P; Luke, Jeremy M; Vincent, Justin M; Williams, James A

2009-10-15

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.

Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing.

PubMed

Heider, Susanne; Muzard, Julien; Zaruba, Marianne; Metzner, Christoph

2017-07-01

Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles.

Photocatalytic wastewater purification with simultaneous hydrogen production using MoS2 QD-decorated hierarchical assembly of ZnIn2S4 on reduced graphene oxide photocatalyst.

PubMed

Zhang, Shuqu; Wang, Longlu; Liu, Chengbin; Luo, Jinming; Crittenden, John; Liu, Xia; Cai, Tao; Yuan, Jili; Pei, Yong; Liu, Yutang

2017-09-15

It is attractive to photocatalytically purify wastewater and simultaneously convert solar energy into clean hydrogen energy. However, it is still a challenge owing to the relatively low photocatalytic efficiency of photocatalysts. In this study, we synthesized a molybdenum disulfide (MoS 2 ) quantum dot-decorated 3D nanoarchitecture (MoS 2 QDs) of indium zinc sulfide (ZnIn 2 S 4 ) and reduced grapheme oxide (MoS 2 QDs@ZnIn 2 S 4 @RGO) photocatalyst using a simple solvothermal method. The RGO promotes the electron transfer, and the highly dispersed MoS 2 QDs provides numerous catalytic sites. The photocatalytic purification of rhodamine B (RhB), eosin Y (EY), fulvic acid (FA), methylene blue (MB) and p-nitrophenol (PNP) in simulated wastewaters were further tested. The degradation efficiencies and TOC removal were 91% and 75% for PNP, 92.2% and 72% for FA, 98.5% and 80% for MB, 98.6% and 84% for EY, and 98.8% and 88% for RhB, respectively (C organics  = 20 mg/L, C catalyst  = 1.25 g/L, t = 12 h, I light  = 3.36 × 10 -5  E L -1  s -1 ). Among these tests, the highest hydrogen production was achieved (45 μmol) during RhB degradation. Both experimental and calculational results prove that lower LUMO (lowest unoccupied molecular orbit) level of organic molecules was available for transferring electrons to catalysts, resulting in more efficient hydrogen production. Significantly, the removal efficiencies of natural organic substances in actual river water reached 76.3-98.4%, and COD reduced from 32 to 16 mg/L with 13.8 μmol H 2 production after 12 h. Copyright © 2017 Elsevier Ltd. All rights reserved.

Accelerated solvent extraction (ASE) for purification and extraction of silicone passive samplers used for the monitoring of organic pollutants.

PubMed

Brockmeyer, Berit; Kraus, Uta R; Theobald, Norbert

2015-12-01

Silicone passive samplers have gained an increasing attention as single-phased, practical and robust samplers for monitoring of organic contaminants in the aquatic environment in recent years. However, analytical challenges arise in routine application during the extraction of analytes as silicone oligomers are co-extracted and interfere severely during chemical analyses (e.g. gas chromatographic techniques). In this study, we present a fast, practical pre-cleaning method for silicone passive samplers applying accelerated solvent extraction (ASE) for the removal of silicone oligomers prior to the water deployment (hexane/dichloromethane, 100 °C, 70 min). ASE was also shown to be a very fast (10 min) and efficient extraction method for non-polar contaminants (non-exposed PRC recoveries 66-101 %) sampled by the silicone membrane. For both applications, temperature, extraction time and the solvent used for ASE have been optimized. Purification of the ASE extract was carried out by silica gel and high-pressure liquid size exclusion chromatography (HPLC-SEC). The silicone oligomer content was checked by total reflection X-ray fluorescence spectroscopy (TXRF) in order to confirm the absence of the silicone oligomers prior to analysis of passive sampler extracts. The established method was applied on real silicone samplers from the North- and Baltic Sea and showed no matrix effects during analysis of organic pollutants. Internal laboratory standard recoveries were in the same range for laboratory, transport and exposed samplers (85-126 %).

The use of fortified soil-clay as on-site system for domestic wastewater purification.

PubMed

Oladoja, N A; Ademoroti, C M A

2006-02-01

The quest for simple, low-cost and high-performance decentralized wastewater treatment system for domestic application in developing nations necessitated this study. Clay samples collected from different deposits in Nigeria were characterized by studying the mineralogical and geochemical composition using X-ray diffraction (XRD) and atomic absorption spectroscopy (AAS), respectively. Three major clay minerals of kaolinite, illite and smectite were identified. The geochemical studies showed the abundance of SiO2, Al2O3 and H2O+ in each of the clay samples. Performance efficiency studies were conducted to determine the best combination ratio of pebbles/soil-clay. Soil-clay fortified by pebbles in combination ratios of 1:3 (i.e. pebbles:soil-clay = 1:3 (w/w) showed the optimum water purification, while the combination 3:1 gave the least. The flow rate studies showed that the wastewater had a longer residence time in non-fortified soil-clay than in fortified soil-clay. Two modes of treatment methods were employed-single and double column treatment methods (SCT and DCT). The two methods gave effluents of good quality characteristics, but those from the DCT were of better quality. The quality of effluents also varies from one clay type to another. The quality of effluents from media containing smectite clay mineral was better than those from other columns. Repeated usage of the fortified clay column showed a decrease of pH, TS and DO, and an increase of COD when monitored over a period of 10 days.

Effects of Average Signed Area Between Two Item Characteristic Curves and Test Purification Procedures on the DIF Detection via the Mantel-Haenszel Method

ERIC Educational Resources Information Center

Wang, Wen-Chung; Su, Ya-Hui

2004-01-01

In this study we investigated the effects of the average signed area (ASA) between the item characteristic curves of the reference and focal groups and three test purification procedures on the uniform differential item functioning (DIF) detection via the Mantel-Haenszel (M-H) method through Monte Carlo simulations. The results showed that ASA,…

Coherent-state information concentration and purification in atomic memory

NASA Astrophysics Data System (ADS)

Herec, Jiří; Filip, Radim

2006-12-01

We propose a feasible method of coherent-state information concentration and purification utilizing quantum memory. The method allows us to optimally concentrate and purify information carried by many noisy copies of an unknown coherent state (randomly distributed in time) to a single copy. Thus nonclassical resources and operations can be saved, if we compare information processing with many noisy copies and a single copy with concentrated and purified information.

Argon purification studies and a novel liquid argon re-circulation system

NASA Astrophysics Data System (ADS)

Mavrokoridis, K.; Calland, R. G.; Coleman, J.; Lightfoot, P. K.; McCauley, N.; McCormick, K. J.; Touramanis, C.

2011-08-01

Future giant liquid argon (LAr) time projection chambers (TPCs) require a purity of better than 0.1 parts per billion (ppb) to allow the ionised electrons to drift without significant capture by any electronegative impurities. We present a comprehensive study of the effects of electronegative impurity on gaseous and liquid argon scintillation light, an analysis of the efficiency of various purification chemicals, as well as the Liverpool LAr setup, which utilises a novel re-circulation purification system. Of the impurities tested - Air, O2, H2O, N2 and CO2 in the range of between 0.01 ppm to 1000 ppm - H2O was found to have the most profound effect on gaseous argon scintillation light, and N2 was found to have the least. Additionally, a correlation between the slow component decay time and the total energy deposited with 0.01 ppm - 100 ppm O2 contamination levels in liquid argon has been established. The superiority of molecular sieves over anhydrous complexes at absorbing Ar gas, N2 gas and H2O vapour has been quantified using BET isotherm analysis. The efficiency of Cu and P2O5 at removing O2 and H2O impurities from 1 bar N6 argon gas at both room temperature and -130 °C was investigated and found to be high. A novel, highly scalable LAr re-circulation system has been developed. The complete system, consisting of a motorised bellows pump operating in liquid and a purification cartridge, were designed and built in-house. The system was operated successfully over many days and achieved a re-circulation rate of 27 litres/hour and high purity.

[Characteristics of microbial community and operation efficiency in biofilter process for drinking water purification].

PubMed

Xiang, Hong; Lü, Xi-Wu; Yang, Fei; Yin, Li-Hong; Zhu, Guang-Can

2011-04-01

In order to explore characteristics of microbial community and operation efficiency in biofilter (biologically-enhanced active filter and biological activated carbon filter) process for drinking water purification, Biolog and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) techniques were applied to analyze the metabolic function and structure of microbial community developing in biofilters. Water quality parameters, such as NH; -N, NO; -N, permanganate index, UV254 and BDOC etc, were determined in inflow and outflow of biofilters for investigation of operation efficiency of the biofilters. The results show that metabolic capacity of microbial community of the raw water is reduced after the biofilters, which reflect that metabolically active microbial communities in the raw water can be intercepted by biofilters. After 6 months operation of biofilters, the metabolic profiles of microbial communities are similar between two kinds of biologically-enhanced active filters, and utilization of carbon sources of microbial communities in the two filters are 73.4% and 75.5%, respectively. The metabolic profiles of microbial communities in two biological activated carbon filters showed significant difference. The carbon source utilization rate of microbial community in granule-activated carbon filter is 79.6%, which is obviously higher than 53.8% of the rate in the columnar activated carbon filter (p < 0.01). The analysis results of PCR-SSCP indicate that microbial communities in each biofilter are variety, but the structure of dominant microorganisms is similar among different biofilters. The results also show that the packing materials had little effect on the structure and metabolic function of microbial community in biologically-enhanced active filters, and the difference between two biofilters for the water purification efficiency was not significant (p > 0.05). However, in biological activated carbon filters, granule-activated carbon is conducive to microbial growth and reproduction, and the microbial communities in the biofilter present high metabolic activities, and the removal efficiency for NH4(+)-N, permanganate index and BDOC is better than the columnar activated carbon filter(p < 0.05). The results also suggest that operation efficiency of biofilter is related to the metabolic capacity of microbial community in biofilter.

Analysis of Gas Membrane Ultra-High Purification of Small Quantities of Mono-Isotopic Silane

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Almeida, Valmor F.; Hart, Kevin J.

A small quantity of high-value, crude, mono-isotopic silane is a prospective gas for a small-scale, high-recovery, ultra-high membrane purification process. This is an unusual application of gas membrane separation for which we provide a comprehensive analysis of a simple purification model. The goal is to develop direct analytic expressions for estimating the feasibility and efficiency of the method, and guide process design; this is only possible for binary mixtures of silane in the dilute limit which is a somewhat realistic case. Among the common impurities in crude silane, methane poses a special membrane separation challenge since it is chemically similarmore » to silane. Other potential problematic surprises are: ethylene, diborane and ethane (in this order). Nevertheless, we demonstrate, theoretically, that a carefully designed membrane system may be able to purify mono-isotopic, crude silane to electronics-grade level in a reasonable amount of time and expenses. We advocate a combination of membrane materials that preferentially reject heavy impurities based on mobility selectivity, and light impurities based on solubility selectivity. We provide estimates for the purification of significant contaminants of interest. To improve the separation selectivity, it is advantageous to use a permeate chamber under vacuum, however this also requires greater control of in-leakage of impurities in the system. In this study, we suggest cellulose acetate and polydimethylsiloxane as examples of membrane materials on the basis of limited permeability data found in the open literature. We provide estimates on the membrane area needed and priming volume of the cell enclosure for fabrication purposes when using the suggested membrane materials. These estimates are largely theoretical in view of the absence of reliable experimental data for the permeability of silane. Last but not least, future extension of this work to the non-dilute limit may apply to the recovery of silane from rejected streams of natural silicon semi-conductor processes.« less

Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.

PubMed

Amarouche, Nassima; Giraud, Matthieu; Forni, Luciano; Butte, Alessandro; Edwards, F; Borie, Nicolas; Renault, Jean-Hugues

2014-04-11

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid-liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by (1)H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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.

Determination of multiple pesticides in fruits and vegetables using a modified quick, easy, cheap, effective, rugged and safe method with magnetic nanoparticles and gas chromatography tandem mass spectrometry.

PubMed

Li, Yan-Fei; Qiao, Lu-Qin; Li, Fang-Wei; Ding, Yi; Yang, Zi-Jun; Wang, Ming-Lin

2014-09-26

Based on a modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation method with Fe3O4 magnetic nanoparticles (MNPs) as the adsorbing material and gas chromatography-tandem mass spectrometry (GC-MS/MS) determination in multiple reaction monitoring (MRM) mode, we established a new method for the determination of multiple pesticides in vegetables and fruits. It was determined that bare MNPs have excellent function as adsorbent when purified, and it is better to be separated from the extract. The amount of MNPs influenced the clean-up performance and recoveries. To achieve the optimum performance of modified QuEChERS towards the target analytes, several parameters including the amount of the adsorbents and purification time were investigated. Under the optimum conditions, recoveries were evaluated in four representative matrices (tomato, cucumber, orange and apple) with the spiked concentrations of 10 μg kg(-1), 50 μg kg(-1)and 200 μg kg(-1) in all cases. The results showed that the recovery of 101 pesticides ranged between 71.5 and 111.7%, and the relative standard deviation was less than 10.5%. The optimum clean-up system improved the purification efficiency and simultaneously obtained satisfactory recoveries of multiple pesticides, including planar-ring pesticides. In short, the modified QuEChERS method in addition to MNPs used for removing impurities improved the speed of sample pre-treatment and exhibited an enhanced performance and purifying effect. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification processes of xenogeneic bone substitutes and their impact on tissue reactions and regeneration.

PubMed

Perić Kačarević, Zeljka; Kavehei, Faraz; Houshmand, Alireza; Franke, Jörg; Smeets, Ralf; Rimashevskiy, Denis; Wenisch, Sabine; Schnettler, Reinhard; Jung, Ole; Barbeck, Mike

2018-04-01

Xenogeneic bone substitute materials are widely used in oral implantology. Prior to their clinical use, purification of the former bone tissue has to be conducted to ensure the removal of immunogenic components and pathogens. Different physicochemical methods are applied for purification of the donor tissue, and temperature treatment is one of these methods. Differences in these methods and especially the application of different temperatures for purification may lead to different material characteristics, which may influence the tissue reactions to these materials and the related (bone) healing process. However, little is known about the different material characteristics and their influences on the healing process. Thus, the aim of this mini-review is to summarize the preparation processes and the related material characteristics, safety aspects, tissue reactions, resorbability and preclinical and clinical data of two widely used xenogeneic bone substitutes that mainly differ in the temperature treatment: sintered (cerabone ® ) and non-sintered (Bio-Oss ® ) bovine-bone materials. Based on the summarized data from the literature, a connection between the material-induced tissue reactions and the consequences for the healing processes are presented with the aim of translation into their clinical application.

Purification of Rubisco Activase from Leaves or after Expression in Escherichia coli.

USDA-ARS?s Scientific Manuscript database

Rubisco activase is a molecular chaperone that modulates the activation state of Rubisco by catalyzing the ATP-dependent removal of tightly-bound inhibitory sugar-phosphates from Rubisco’s catalytic sites. This chapter reports methods developed for the purification of native and recombinant Rubisco...

A study of the parameters affecting the effectiveness of Moringa oleifera in drinking water purification

NASA Astrophysics Data System (ADS)

Pritchard, M.; Craven, T.; Mkandawire, T.; Edmondson, A. S.; O'Neill, J. G.

The powder obtained from the seeds of the Moringa oleifera tree has been shown to be an effective primary coagulant for water treatment. When the seeds are dried, dehusked, crushed and added to water, the powder acts as a coagulant binding colloidal particles and bacteria to form agglomerated particles (flocs), which settle allowing the clarified supernatant to be poured off. Very little research has been undertaken on the parameters affecting the effectiveness of M. oleifera, especially in Malawi, for purification of drinking water and there is a great need for further testing in this area. Conclusive data needs to be compiled to demonstrate the effects of various water parameters have on the efficiency of the seeds. A parametric study was undertaken at Leeds Metropolitan University, UK, with the aim to establish the most appropriate dosing method; the optimum dosage for removal of turbidity; the influence of pH and temperature; together with the shelf life of the M. oleifera seeds. The study revealed that the most suitable dosing method was to mix the powder into a concentrated paste, hence forming a stock suspension. The optimum M. oleifera dose, for turbidity values between 40 and 200 NTU, ranged between 30 and 55 mg/l. With turbidity set at 130 NTU and a M. oleifera dose within the optimum range at 50 mg/l, pH levels were varied between 4 and 9. It was discovered that the coagulant performance was not too sensitive to pH fluctuations when conditions were within the optimum range. The most efficient coagulation, determined by the greatest reduction in turbidity, occurred at pH 6.5. Alkaline conditions were overall more favourable than acidic conditions; pH 9 had an efficiency of 65% of optimum, whilst at pH 5 the efficiency dropped to around 55%. The efficiency further dropped at pH 4, where the powder only produced results of around 10% of optimum conditions. A temperature range of 4-60 °C was studied in this research. Colder waters (<15 °C) were found to hinder the effectiveness of the coagulation process. The higher the temperature the more effective was the coagulation. It was also found that the age of the seeds, up to 18 months, did not have any noticeable effect on dose level and percentage reduction in turbidity, although at 18 months the seeds had a narrower dosing range to produce near-optimum reduction. Seeds aged 24 months showed a significant decline in coagulant efficiency.

Optimization of SHINE Process: Design and Verification of Plant-Scale AG 1 Anion-Exchange Concentration Column and Titania Sorbent Pretreatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stepinski, Dominique C.; Abdul, Momen; Youker, Amanda J.

2016-06-01

Argonne National Laboratory has developed a Mo-recovery and -purification system for the SHINE medical technologies process, which uses a uranyl sulfate solution for the accelerator-driven production of Mo-99. The objective of this effort is to reduce the processing time for the acidification of the Mo-99 product prior to loading onto a concentration column and concentration of the Mo-99 product solution. Two methods were investigated: (1) the replacement of the titania concentration column by an anion-exchange column to decrease processing time and increase the radioiodine-decontamination efficiency and (2) pretreatment of the titania sorbent to improve its effectiveness for the Mo-recovery andmore » -concentration columns. Promising results are reported for both methods.« less

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

PubMed

Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

2015-08-15

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

Cleaner production in the ammonia-soda industry: an ecological and economic study.

PubMed

Kasikowski, T; Buczkowski, R; Lemanowska, E

2004-12-01

Five methods to reduce the negative influence of soda ash factories on the natural environment are presented: 1. obtaining calcium-magnesium phosphates by treating the suspension from raw brine purification with orthophosphoric acid (H(3)PO(4)), 2. production of precipitated chalk from soda processing waste, 3. production of gypsum and semi-brine, 4. desulphurisation of fume gases from the factory power plant, 5. utilization of distiller waste. The tests, accomplished on a laboratory scale, showed the high efficiency of these methods. Economic analysis has proved that only four out of the five presented processes can have a positive financial effect on soda ash factories, as well as being well justified economically. The value of two of the innovations presented is confirmed by their implementation in factories.

Studies of proteoglycan involvement in CPP-mediated delivery.

PubMed

Wittrup, Anders; Zhang, Si-He; Belting, Mattias

2011-01-01

Cell-penetrating peptides (CPPs) are widely used to deliver macromolecular cargoes to intracellular sites of action. Many CPPs have been demonstrated to rely on cell surface heparan sulfate proteoglycans (HSPGs) for efficient cellular entry and delivery. In this chapter, we describe methods for the study of PG involvement in CPP uptake. We provide descriptions of how to determine whether uptake of a CPP of interest is dependent on PGs. We also provide detailed protocols for the purification of PGs by anion-exchange chromatography as well as the characterization of the HSPG core protein composition of a cell line of interest. Finally, we present methods for modulating the expression level of specific HSPG core proteins as a means to determine the core protein specificity in the uptake of a particular CPP.

Determination of 3,6-dinitrobenzo[e]pyrene in surface soil and airborne particles by high-performance liquid chromatography with fluorescence detection.

PubMed

Hasei, Tomohiro; Watanabe, Tetsushi; Hirayama, Teruhisa

2006-11-24

We developed a sensitive analytical method and an efficient clean-up method to quantify 3,6-dinitrobenzo[e]pyrene (3,6-DNBeP) in surface soil and airborne particles. After purification using a silica gel column and two reversed-phase columns, 3,6-DNBeP was reduced to 3,6-diaminobenzo[e]pyrene by a catalyst column and analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector. 3,6-DNBeP was detected in all of the soil samples and airborne particles examined. The concentration of 3,6-DNBeP in surface soil and airborne particles was determined in the ranges of 347-5007 pg/g of soil and 137-1238 fg/m3, respectively.

Simultaneous separation and purification of flavonoids and oleuropein from Olea europaea L. (olive) leaves using macroporous resin.

PubMed

Li, Chen; Zheng, Yuanyuan; Wang, Xiaofei; Feng, Shilan; Di, Duolong

2011-12-01

This study developed a feasible process to simultaneously separate and purify polyphenols, including flavonoids and oleuropein, from the leaves of Olea europaea L. Macroporous resins were used as the separation and purification materials. The performance and separation capabilities of eight resins (D101, DM130, HPD450, LSA-21, LSA-40, 07C, LSD001 and HPD600) were systematically evaluated. The contents of target polyphenols in different extracts were determined using ultraviolet (for flavonoids) and high-performance liquid chromatographic (for oleuropein) methods. The static adsorption and desorption results showed that resin LSA-21 had better adsorption properties among the eight resins. Influential factors such as extraction method, pH value of feeding solution, desorption solution, adsorption kinetics and adsorption isotherm, etc. to the extraction and purification of these polyphenols were successively investigated on resin LSA-21. The target flavonoids and oleuropein were selectively purified using resin LSA-21. Compared with the contents in raw leaves, the contents of total flavonoids and oleuropein in the final purified products were increased 13.2-fold (from 16 to 211 g kg(-1) ) and 7.5-fold (from 120 to 902 g kg(-1) ) with recovery yields of 87.9% and 85.6%, respectively. This extraction and purification method could be used in the large-scale enrichment or purification of flavonoids, oleuropein and other polyphenols from O. europaea L. leaves or other herbal materials in industrial, food processing and medical manufacture. Copyright © 2011 Society of Chemical Industry.

Water treatment plants assessment at Talkha power plant.

PubMed

El-Sebaie, Olfat D; Abd El-Kerim, Ghazy E; Ramadan, Mohamed H; Abd El-Atey, Magda M; Taha, Sahr Ahmed

2002-01-01

Talkha power plant is the only power plant located in El-Mansoura. It generates electricity using two different methods by steam turbine and gas turbine. Both plants drew water from River Nile (208 m3 /h). The Nile raw water passes through different treatment processes to be suitable for drinking and operational uses. At Talkha power plant, there are two purification plants used for drinking water supply (100 m3/h) and for water demineralization supply (108 m3/h). This study aimed at studying the efficiency of the water purification plants. For drinking water purification plant, the annual River Nile water characterized by slightly alkaline pH (7.4-8), high annual mean values of turbidity (10.06 NTU), Standard Plate Count (SPC) (313.3 CFU/1 ml), total coliform (2717/100 ml), fecal coliform (0-2400/100 ml), and total algae (3 x 10(4) org/I). The dominant group of algae all over the study period was green algae. The blue green algae was abundant in Summer and Autumn seasons. The pH range, and the annual mean values of turbidity, TDS, total hardness, sulfates, chlorides, nitrates, nitrites, fluoride, and residual chlorine for purified water were in compliance with Egyptian drinking water standards. All the SPC recorded values with an annual mean value of 10.13 CFU/1 ml indicated that chlorine dose and contact time were not enough to kill the bacteria. However, they were in compliance with Egyptian decree (should not exceed 50 CFU/1 ml). Although the removal efficiency of the plant for total coliform and blue green algae was high (98.5% and 99.2%, respectively), the limits of the obtained results with an annual mean values of 40/100 ml and 15.6 org/l were not in compliance with the Egyptian decree (should be free from total coliform, fecal coliform and blue green algae). For water demineralization treatment plant, the raw water was characterized by slightly alkaline pH. The annual mean values of conductivity, turbidity, and TDS were 354.6 microS/cm, 10.84 NTU, and 214.6 mg/I, respectively. There was an increase in the results of conductivity, turbidity, total hardness, and TDS in carbon filter effluent which was attributed to the desorption of adsorbed ions on the carbon media. The removal efficiencies of turbidity, total hardness, and TDS indicated the high efficiency of the cationic filter. The annual removal efficiencies of conductivity, turbidity, chloride, and TDS proved the efficiency of the anionic filter for removing the dissolved and suspended ions. All of the recorded values of the pH, conductivity, turbidity, chlorides, hardness, and TDS of the mixed bed effluent indicated that the water at this stage was of high quality for boiler feed. The study recommended adjustment of coagulant and residual chlorine doses as well as contact time, and continuous monitoring and maintenance of the different units.

Analysis of O-Glycopeptides by Acetone Enrichment and Capillary Electrophoresis-Mass Spectrometry.

PubMed

Mancera-Arteu, Montserrat; Giménez, Estela; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victòria

2017-11-03

Acetone precipitation was evaluated as a rapid, simple, low-cost, and efficient method for the selective purification of O-glycopeptides from enzymatic digests of glycoproteins. Ovalbumin (OVA), human and bovine α 1 -acid glycoprotein (hAGP and bAGP), human apolipoprotein C-III (APO-C3), and recombinant human erythropoietin (rhEPO) were used to obtain enzymatic digests with a broad and varied set of peptides, N-glycopeptides, and O-glycopeptides. After digestion and before capillary electrophoresis mass spectrometry (CE-MS) analysis, the amount of ice-cold acetone added to the digests was optimized to maximize recoveries of O-glycopeptides. Furthermore, the different behavior of peptides, N- and O-glycopeptides was explained by studying with multivariate data analysis methods the influence of several physicochemical parameters and properties related to their composition and structure. Principal component analysis (PCA) and, afterward, partial least-squares discriminant analysis (PLS-DA) were used to identify the most significant variables and their importance to differentiate between peptides, N-glycopeptides and O-glycopeptides, or within these classes. This information was useful to understand precipitation of these compounds after addition of acetone and for the selection of the optimal conditions for purification of specific O-glycopeptide biomarkers. Special attention was paid to O 126 -glycopeptide glycoforms of rhEPO because of their applicability in biopharmaceutical quality control and doping analysis.

High pH solubilization and chromatography-based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies.

PubMed

Li, Ming; Fan, Hua; Liu, Jiahua; Wang, Minhong; Wang, Lili; Wang, Chaozhan

2012-03-01

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

Physicochemical modifications and applications of carbon nano-onions for electrochemical energy storage

NASA Astrophysics Data System (ADS)

Borgohain, Rituraj

Carbon nano-onions (CNOs), concentrically multilayered fullerenes, are prepared by several different methods. We are studying the properties of two specific CNOs: A-CNOs and N-CNOs. A-CNOs are synthesized by underwater arc discharge, and N-CNOs are synthesized by high-temperature graphitization of commercial nanodiamond. In this study the synthesis of A-CNOs are optimized by designing an arc discharge aparatus to control the arc plasma. Moreover other synthesis parameters such as arc power, duty cycles, temperature, graphitic and metal impurities are controlled for optimum production of A-CNOs. Also, a very efficient purification method is developed to screen out A-CNOs from carboneseous and metal impurities. In general, A-CNOs are larger than N-CNOs (ca. 30 nm vs. 7 nm diameter). The high surface area, appropriate mesoporosity, high thermal stability and high electrical conductivity of CNOs make them a promising material for various applications. These hydrophobic materials are functionalized with organic groups on their outer layers to study their surface chemistry and to decorate with metal oxide nanoparticles. Both CNOs and CNO nanocomposites are investigated for application in electrochemical capacitors (ECs). The influences of pH, concentration and additives on the performance of the composites are studied. Electrochemical measurements demonstrate high specific capacitance and high cycling stability with high energy and power density of the composite materials in aqueous electrolyte. Key words: Carbon Nano-onions, Arc discharge, Purification, Functionalization, Supercapacitor.

Prospecting for Cellulolytic Activity in Insect Digestive Fluids

USDA-ARS?s Scientific Manuscript database

Efficient cellulolytic enzymes are needed to degrade recalcitrant plant biomass during ethanol purification and make lignocellulosic biofuels a cost-effective alternative to fossil fuels. Despite the large number of insect species that feed on lignocellulosic material, limited availability of quant...

Simple, Rapid, and Selective Isolation of 2S Albumins from Allergenic Seeds and Nuts.

PubMed

Hummel, Marlene; Wigger, Tina; Höper, Tessa; Westkamp, Imke; Brockmeyer, Jens

2015-07-08

The 2S albumins belong to the group of seed storage proteins present in different seeds and nuts. Due to their pronounced allergenic potential, which is often associated with severe allergic reactions, this protein family is of special interest in the field of allergen research. Here we present a simple, rapid, and selective method for the purification of 2S albumins directly from allergenic seeds and nuts. We systematically optimized the parameters "buffer system", "extraction temperature", "buffer molarity", and "pH " and were able to achieve 2S albumin purities of about 99% without further purification and demonstrate transferability of this method to nine different allergenic food matrices. Compared to conventional isolation routines, significant reduction of hands-on time and required laboratory equipment is achieved, but nonetheless higher protein yields are obtained. The presented method allows for the rapid purification of different 2S albumins including the corresponding isoforms from natural material.

Rare cancer cell analyzer for whole blood applications: automated nucleic acid purification in a microfluidic disposable card.

PubMed

Kokoris, M; Nabavi, M; Lancaster, C; Clemmens, J; Maloney, P; Capadanno, J; Gerdes, J; Battrell, C F

2005-09-01

One current challenge facing point-of-care cancer detection is that existing methods make it difficult, time consuming and too costly to (1) collect relevant cell types directly from a patient sample, such as blood and (2) rapidly assay those cell types to determine the presence or absence of a particular type of cancer. We present a proof of principle method for an integrated, sample-to-result, point-of-care detection device that employs microfluidics technology, accepted assays, and a silica membrane for total RNA purification on a disposable, credit card sized laboratory-on-card ('lab card") device in which results are obtained in minutes. Both yield and quality of on-card purified total RNA, as determined by both LightCycler and standard reverse transcriptase amplification of G6PDH and BCR-ABL transcripts, were found to be better than or equal to accepted standard purification methods.

Two-step purification of scutellarin from Erigeron breviscapus (vant.) Hand. Mazz. by high-speed counter-current chromatography.

PubMed

Gao, Min; Gu, Ming; Liu, Chun-Zhao

2006-07-11

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate-n-butanol-acetonitrile-0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.

Experimental studies on islets isolation, purification and function in rats

PubMed Central

Pang, Xinlu; Xue, Wujun; Feng, Xinshun; Tian, Xiaohui; Teng, Yan; Ding, Xiaoming; Pan, Xiaoming; Guo, Qi; He, Xiaoli

2015-01-01

To develop a simple and effective method of islet isolation and purification in rats. Collagenase P was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The purified islets were identified by dithizone staining. The viability of islets was assessed by fluorescence staining of acridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. 738±193 islets were recovered after purification. The average purity was 77±13%, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was 24.31±5.47 mIU/L when stimulated by low concentration glucose and 37.62±4.29 mIU/L by high concentration glucose. There was significant difference between the two phases (P<0.05). The blood sugar concentration recovered to normal level after two days in the animals with islet transplantation. In conclusion, islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification using discontinuous gravity gradient. PMID:26885021

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review.

PubMed

Biniarz, Piotr; Łukaszewicz, Marcin; Janek, Tomasz

2017-05-01

Lipopeptide biosurfactants are surface active biomolecules that are produced by a variety of microorganisms. Microbial lipopeptides have gained the interest of microbiologists, chemists and biochemists for their high biodiversity as well as efficient action, low toxicity and good biodegradability in comparison to synthetic counterparts. In this report, we review methods for the production, isolation and screening, purification and structural characterization of microbial lipopeptides. Several techniques are currently available for each step, and we describe the most commonly utilized and recently developed techniques in this review. Investigations on lipopeptide biosurfactants in natural products require efficient isolation techniques for the characterization and evaluation of chemical and biological properties. A combination of chromatographic and spectroscopic techniques offer opportunities for a better characterization of lipopeptide structures, which in turn can lead to the application of lipopeptides in food, pharmaceutical, cosmetics, agricultural and bioremediation industries.

Algae separation from urban landscape water using a high density microbubble layer enhanced by micro-flocculation.

PubMed

Chen, Shuwen; Xu, Jingcheng; Liu, Jia; Wei, Qiaoling; Li, Guangming; Huang, Xiangfeng

2014-01-01

Eutrophication of raw water results in outbreaks of algae, which hinders conventional water treatment. In this study, high density microbubble layers combined with micro-flocculation was adopted to remove algae from urban landscape water, and the effects of pressure, hydraulic loading, microbubble layer height and flocculation dosage on the removal efficiency for algae were studied. The greatest removal efficiency for algae, chemical oxygen demand, nitrogen and phosphorus was obtained at 0.42 MPa with hydraulic loading at 5 m/h and a flocculation dosage of 4 mg/L using a microbubble layer with a height of 130 cm. Moreover, the size, clearance distance and concentration of microbubbles were found to be affected by pressure and the height of the microbubble layer. Based on the study, this method was an alternative for algae separation from urban landscape water and water purification.

One-Pot Synthesis of Reduced Graphene Oxide/Anatase Titanium Dioxide Composites for Photocatalytic Degradation of Methylene Blue.

PubMed

Lee, Hyo In; Park, Soo-Jin

2018-09-01

In this work, highly ordered TiO2-reduced graphene oxide sheets (TGS) were successfully fabricated via a one-pot solvothermal method with different amounts of graphene oxide (0.01, 0.03, 0.05, and 0.07 g). This was achieved by reacting graphene oxide (GO) layers with titanium isopropoxide as the TiO2 precursor. The TGS exhibited superior efficiency compared to pristine TiO2 and the best results were recorded for the TGS-0.05 sample. The presence of the reduced graphene oxide (rGO) component was determined to be an important factor governing the separation of the photogenerated electron-hole pair via interfacial charge transfer. The significantly increased activity of the TGS under simulated solar light in the degradation of methylene blue (MB) indicates that these materials are promising photocatalysts for efficient water purification.

Degradation of organic pollutants and microorganisms from wastewater using different dielectric barrier discharge configurations--a critical review.

PubMed

Mouele, Emile S Massima; Tijani, Jimoh O; Fatoba, Ojo O; Petrik, Leslie F

2015-12-01

The growing global drinking water crisis requires the development of novel advanced, sustainable, and cost-effective water treatment technologies to supplement the existing conventional methods. One such technology is advanced oxidation based on dielectric barrier discharge (DBD). DBD such as single and double planar and single and double cylindrical dielectric barrier configurations have been utilized for efficient degradation of recalcitrant organic pollutants. The overall performance of the different DBD system varies and depends on several factors. Therefore, this review was compiled to give an overview of different DBD configurations vis-a-viz their applications and the in situ mechanism of generation of free reactive species for water and wastewater treatment. Our survey of the literature indicated that application of double cylindrical dielectric barrier configuration represents an ideal and viable route for achieving greater water and wastewater purification efficiency.

The development of new purification methods to assess the circadian rhythm of body temperature in Mongolian gerbils.

PubMed

Weinert, D; Nevill, A; Weinandy, R; Waterhouse, J

2003-03-01

Six Mongolian gerbils were studied for 8-10d while housed in separate cages in a 12:12h light-dark (L-D) cycle (lights on at 07:00h). Recordings of body temperature, heart rate, and spontaneous activity were made throughout. The temperature and heart rate rhythms were "purified" to take into account the effects of activity, and then the rhythm of temperature was further purified to take into account other masking influences ("non-activity masking effects" or NAME,). The methods employed in the purification processes involved linear regression analysis or analysis of covariance, the latter using functions of activity and NAME as covariates. From these methods, it was possible to obtain not only an estimate of the endogenous component of the temperature rhythm but also a measure of circadian changes in the sensitivity of temperature to masking effects. Even though all purification methods removed many of the effects of spontaneous activity from the temperature record, there remained temperature fluctuations at the L-D and D-L transitions that appeared to be independent of activity. The NAME was of only very marginal value in the purification process. Comparison of the purification methods indicated that the linear methods were inferior (both from a biological viewpoint and when the results were compared mathematically) to those that allowed the rate of rise of temperature due to increasing amounts of activity to become progressively less. The sensitivity of temperature and heart rate to the masking effects of activity showed a circadian rhythm, with sensitivities in the resting phase being greater than those in the active phase. These findings are compatible with the view that thermoregulatory reflexes are induced by spontaneous activity of sufficient amount, and that there is a circadian rhythm in the body temperature at which these reflexes are initiated and in their effectiveness.

The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

ERIC Educational Resources Information Center

Falconer, A. C.; Hayes, L. J.

1986-01-01

Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

Synthesis and purification of 1,3,5-triamino-2,4,6-trinitrobenzene (TATB)

DOEpatents

Mitchell, Alexander R [Livermore, CA; Coburn, Michael D [Santa Fe, NM; Lee, Gregory S [San Ramon, CA; Schmidt, Robert D [Livermore, CA; Pagoria, Philip F [Livermore, CA; Hsu, Peter C [Pleasanton, CA

2006-06-06

A method to convert surplus nitroarene explosives (picric acid, ammonium picrate,) into TATB is described. The process comprises three major steps: conversion of picric acid/ammonium picrate into picramide; conversion of picramide to TATB through vicarious nucleophilic substitution (VNS) of hydrogen chemistry; and purification of TATB.

Pilot-scale test for electron beam purification of flue gas from coal-combustion boiler

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Shoji; Namba, Hideki; Tokunaga, Okihiro

1995-06-01

Construction of a pilot plant of the treatment capacity of 12,000 m{sup 3}N/h flue gas was completed in November, 1992 in the Shin-Nagoya Thermal Power Station, Nagoya for electron beam purification of flue-gas from coal combustion boiler and the operation had been continued during one year. The results obtained In the tests shows that the target removal efficiency for SO{sub 2} (94 %) and for NO{sub x} (80 %) was achieved with appropriate operation conditions (electron beam dose, temperature, amount of ammonia etc.). The effective collection of powdery by-products was performed by an electrostatic precipitator.

Selective manipulation of superparamagnetic nanoparticles for product purification and microfluidic diagnostics.

PubMed

Gädke, Johannes; Thies, Jan-Wilhelm; Kleinfeldt, Lennart; Schulze, Torben; Biedendieck, Rebekka; Rustenbeck, Ingo; Garnweitner, Georg; Krull, Rainer; Dietzel, Andreas

2018-05-01

The needs of scalable product purification as well as the demand for sensitive diagnostics for highly dilute entities can be addressed with the utilization of tailored superparamagnetic nanoparticles. Recent developments have led to more efficient fluidic systems at different scales with suspended nanoparticles or nanoparticle aggregates. However, magnetic nanoparticle systems differ widely in properties and their applications are characterized by very specific challenges. This review summarizes advances in the synthesis of superparamagnetic particles and displays states and trends in research making use of these particles in biotechnological downstream processing and in biosensing. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

PubMed Central

Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

Purification of diverse hemoglobins by metal salt precipitation.

PubMed

Zimmerman, Devon; Dienes, Jack; Abdulmalik, Osheiza; Elmer, Jacob J

2016-09-01

Although donated blood is the preferred material for transfusion, its limited availability and stringent storage requirements have motivated the development of blood substitutes. The giant extracellular hemoglobin (aka erythrocruorin) of the earthworm Lumbricus terrestris (LtEc) has shown promise as a blood substitute, but an efficient purification method for LtEc must be developed to meet the potential large demand for blood substitutes. In this work, an optimized purification process that uses divalent and trivalent metal salts to selectively precipitate human, earthworm, and bloodworm hemoglobin (HbA, LtEc, and GdHb, respectively) from crude solutions was developed. Although several metal ions were able to selectively precipitate LtEc, Zn(2+) and Ni(2+) provided the lowest heme oxidation and highest overall yield of LtEc. In contrast, Zn(2+) was the only metal ion that completely precipitated HbA and GdHb. Polyacrylamide gel electrophoresis (PAGE) analysis shows that metal precipitation removes several impurities to provide highly pure hemoglobin samples. Heme oxidation levels were relatively low for Zn(2+)-purified HbA and LtEc (2.4±1.3% and 5.3±2.1%, respectively), but slightly higher for Ni(2+)-purified LtEc (8.4±1.2%). The oxygen affinity and cooperativity of the precipitated samples are also identical to samples purified with tangential flow filtration (TFF) alone, indicating the metal precipitation does not significantly affect the function of the hemoglobins. Overall, these results show that hemoglobins from several different species can be highly purified using a combination of metal (Zn(2+)) precipitation and tangential flow filtration. Copyright © 2015 Elsevier Inc. All rights reserved.

Drinking water purification by electrosynthesis of hydrogen peroxide in a power-producing PEM fuel cell.

PubMed

Li, Winton; Bonakdarpour, Arman; Gyenge, Előd; Wilkinson, David P

2013-11-01

The industrial anthraquinone auto-oxidation process produces most of the world's supply of hydrogen peroxide. For applications that require small amounts of H2 O2 or have economically difficult transportation means, an alternate, on-site H2 O2 production method is needed. Advanced drinking water purification technologies use neutral-pH H2 O2 in combination with UV treatment to reach the desired water purity targets. To produce neutral H2 O2 on-site and on-demand for drinking water purification, the electroreduction of oxygen at the cathode of a proton exchange membrane (PEM) fuel cell operated in either electrolysis (power consuming) or fuel cell (power generating) mode could be a possible solution. The work presented here focuses on the H2 /O2 fuel cell mode to produce H2 O2 . The fuel cell reactor is operated with a continuous flow of carrier water through the cathode to remove the product H2 O2 . The impact of the cobalt-carbon composite cathode catalyst loading, Teflon content in the cathode gas diffusion layer, and cathode carrier water flowrate on the production of H2 O2 are examined. H2 O2 production rates of up to 200 μmol h(-1)  cmgeometric (-2) are achieved using a continuous flow of carrier water operating at 30 % current efficiency. Operation times of more than 24 h have shown consistent H2 O2 and power production, with no degradation of the cobalt catalyst. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Separation of porcine parvovirus from bovine serum albumin using PEG-salt aqueous two-phase system.

PubMed

Vijayaragavan, K Saagar; Zahid, Amna; Young, Jonathan W; Heldt, Caryn L

2014-09-15

Vaccine production faces a challenge in adopting conventional downstream processing steps that can efficiently purify large viral particles. Some major issues that plague vaccine purification are purity, potency, and quality. The industry currently considers 30% as an acceptable virus recovery for a vaccine purification process, including all downstream processes, whereas antibody recovery from CHO cell culture is generally around 80-85%. A platform technology with an improved virus recovery would revolutionize vaccine production. In a quest to fulfill this goal, we have been exploring aqueous two-phase systems (ATPSs) as an optional mechanism to purify virus. ATPS has been unable to gain wide implementation mainly due to loss of virus infectivity, co-purification of proteins, and difficulty of polymer recycling. Non-enveloped viruses are chemically resistant enough to withstand the high polymer and salt concentrations that are required for effective ATPS separations. We used infectious porcine parvovirus (PPV), a non-enveloped, DNA virus as a model virus to test and develop an ATPS separation method. We successfully tackled two of the three main disadvantages of ATPS previously stated; we achieved a high infectious yield of 64% in a PEG-citrate ATPS process while separating out the main contaminate protein, bovine serum albumin (BSA). The most dominant forces in the separation were biomolecule charge, virus surface hydrophobicity, and the ATPS surface tension. Highly hydrophobic viruses are likely to benefit from the discovered ATPS for high-purity vaccine production and ease of implementation. Copyright © 2014 Elsevier B.V. All rights reserved.

Tackling saponin diversity in marine animals by mass spectrometry: data acquisition and integration.

PubMed

Decroo, Corentin; Colson, Emmanuel; Demeyer, Marie; Lemaur, Vincent; Caulier, Guillaume; Eeckhaut, Igor; Cornil, Jérôme; Flammang, Patrick; Gerbaux, Pascal

2017-05-01

Saponin analysis by mass spectrometry methods is nowadays progressively supplementing other analytical methods such as nuclear magnetic resonance (NMR). Indeed, saponin extracts from plant or marine animals are often constituted by a complex mixture of (slightly) different saponin molecules that requires extensive purification and separation steps to meet the requirement for NMR spectroscopy measurements. Based on its intrinsic features, mass spectrometry represents an inescapable tool to access the structures of saponins within extracts by using LC-MS, MALDI-MS, and tandem mass spectrometry experiments. The combination of different MS methods nowadays allows for a nice description of saponin structures, without extensive purification. However, the structural characterization process is based on low kinetic energy CID which cannot afford a total structure elucidation as far as stereochemistry is concerned. Moreover, the structural difference between saponins in a same extract is often so small that coelution upon LC-MS analysis is unavoidable, rendering the isomeric distinction and characterization by CID challenging or impossible. In the present paper, we introduce ion mobility in combination with liquid chromatography to better tackle the structural complexity of saponin congeners. When analyzing saponin extracts with MS-based methods, handling the data remains problematic for the comprehensive report of the results, but also for their efficient comparison. We here introduce an original schematic representation using sector diagrams that are constructed from mass spectrometry data. We strongly believe that the proposed data integration could be useful for data interpretation since it allows for a direct and fast comparison, both in terms of composition and relative proportion of the saponin contents in different extracts. Graphical Abstract A combination of state-of-the-art mass spectrometry methods, including ion mobility spectroscopy, is developed to afford a complete description of the saponin molecules in natural extracts.

Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

PubMed

Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

2007-10-01

A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.

Rapid separation and purification of uranium and plutonium from dilute-matrix samples

DOE PAGES

Armstrong, Christopher R.; Ticknor, Brian W.; Hall, Gregory; ...

2014-03-11

This work presents a streamlined separation and purification approach for trace uranium and plutonium from dilute (carrier-free) matrices. The method, effective for nanogram quantities of U and femtogram to picogram quantities of Pu, is ideally suited for environmental swipe samples that contain a small amount of collected bulk material. As such, it may be applicable for processing swipe samples such as those collected in IAEA inspection activities as well as swipes that are loaded with unknown analytes, such as those implemented in interlaboratory round-robin or proficiency tests. Additionally, the simplified actinide separation could find use in internal laboratory monitoring ofmore » clean room conditions prior to or following more extensive chemical processing. We describe key modifications to conventional techniques that result in a relatively rapid, cost-effective, and efficient U and Pu separation process. We demonstrate the efficacy of implementing anion exchange chromatography in a single column approach. We also show that hydrobromic acid is an effective substitute in lieu of hydroiodoic acid for eluting Pu. Lastly, we show that nitric acid is an effective digestion agent in lieu of perchloric acid and/or hydrofluoric acid. A step by step procedure of this process is detailed.« less

Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds.

PubMed

Köksal, Ekrem; Gülçin, Ilhami

2008-01-01

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H(2)O(2), pyrogallol/H(2)O(2), ABTS/H(2)O(2), catechol/H(2)O(2) and 4-methyl catechol/H(2)O(2) substrate patterns. The molecular weight (M(w)) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. K(m) and V(max) values were calculated from Lineweaver-Burk graph for each substrate patterns.

A polyacrylamide-based silica stationary phase for the separation of carbohydrates using alcohols as the weak eluent in hydrophilic interaction liquid chromatography.

PubMed

Cai, Jianfeng; Cheng, Lingping; Zhao, Jianchao; Fu, Qing; Jin, Yu; Ke, Yanxiong; Liang, Xinmiao

2017-11-17

A hydrophilic interaction liquid chromatography (HILIC) stationary phase was prepared by a two-step synthesis method, immobilizing polyacrylamide on silica sphere particles. The stationary phase (named PA, 5μm dia) was evaluated using a mixture of carbohydrates in HILIC mode and the column efficiency reached 121,000Nm -1 . The retention behavior of carbohydrates on PA stationary phase was investigated with three different organic solvents (acetonitrile, ethanol and methanol) employed as the weak eluent. The strongest hydrophilicity of PA stationary phase was observed in both acetonitrile and methanol as the weak eluent, when compared with another two amide stationary phases. Attributing to its high hydrophilicity, three oligosaccharides (xylooligosaccharide, fructooligosaccharide and chitooligosaccharides) presented good retention on PA stationary phase using alcohols/water as mobile phase. Finally, PA stationary phase was successfully applied for the purification of galactooligosaccharides and saponins of Paris polyphylla. It is feasible to use safer and cheaper alcohols to replace acetonitrile as the weak eluent for green analysis and purification of polar compounds on PA stationary phase. Copyright © 2017. Published by Elsevier B.V.

Separation and Purification of Ombuoside from Gynostemma Pentaphyllum by Microwave-Assisted Extraction Coupled with High-Speed Counter-current Chromatography.

PubMed

Jiang, Wenhui; Shan, Hu; Song, Jiying; Lü, Haitao

2017-01-01

A rapid and efficient method for the separation and purification of ombuoside from Gynostemma pentaphyllum by microwave-assisted extraction coupled with high-speed counter-current chromatography (HSCCC) was successfully developed. Using an orthogonal array design L 9 (3 4 ), the extraction conditions, including microwave power, irradiation time, solid-to-liquid ratio and extraction times, were optimized. Ombuoside was isolated and purified from the crude extraction by HSCCC with two-phase solvent system composed of n-hexane:ethyl acetate:ethanol:water (5:6:5:5, v/v) in a single run. A 210 mg quantity of the crude extract containing 2.16% ombuoside was loaded, yielding 3.9 mg of ombuoside at 96.7% purity. The chemical structure of ombuoside was determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as UV, FT-IR, ESI-MS, 1 H NMR and 13 C NMR spectra. The purified ombuoside had strong 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radical scavenging activities. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mass spectrometric directed system for the continuous-flow synthesis and purification of diphenhydramine.

PubMed

Loren, Bradley P; Wleklinski, Michael; Koswara, Andy; Yammine, Kathryn; Hu, Yanyang; Nagy, Zoltan K; Thompson, David H; Cooks, R Graham

2017-06-01

A highly integrated approach to the development of a process for the continuous synthesis and purification of diphenhydramine is reported. Mass spectrometry (MS) is utilized throughout the system for on-line reaction monitoring, off-line yield quantitation, and as a reaction screening module that exploits reaction acceleration in charged microdroplets for high throughput route screening. This effort has enabled the discovery and optimization of multiple routes to diphenhydramine in glass microreactors using MS as a process analytical tool (PAT). The ability to rapidly screen conditions in charged microdroplets was used to guide optimization of the process in a microfluidic reactor. A quantitative MS method was developed and used to measure the reaction kinetics. Integration of the continuous-flow reactor/on-line MS methodology with a miniaturized crystallization platform for continuous reaction monitoring and controlled crystallization of diphenhydramine was also achieved. Our findings suggest a robust approach for the continuous manufacture of pharmaceutical drug products, exemplified in the particular case of diphenhydramine, and optimized for efficiency and crystal size, and guided by real-time analytics to produce the agent in a form that is readily adapted to continuous synthesis.