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Purification Step (purification + step)
Selected AbstractsA New Indirect Electroanalytical Method to Monitor the Contamination of Natural Waters with 4-Nitrophenol Using Multiwall Carbon NanotubesELECTROANALYSIS, Issue 9 2009Cruz Moraes, Fernando Abstract The electrochemical detection of the hazardous pollutant 4-nitrophenol (4-NP) at low potentials, in order to avoid matrix interferences, is an important research challenge. This study describes the development, electrochemical characterization and utilization of a multiwall carbon nanotube (MWCNT) film electrode for the quantitative determination of 4-NP in natural water. Electrochemical impedance spectroscopy measurements showed that the modified surface exhibits a decrease of ca. 13 times in the charge transfer resistance when compared with a bare glassy carbon (GC) surface. Voltammetric experiments showed the possibility to oxidize a hydroxylamine layer (produced by the electrochemical reduction of 4-NP on the GC/MWNCT surface) in a potential region which is approximately 700,mV less positive than that needed to oxidize 4-NP, thus minimizing the interference of matrix components. The limit of detection for 4-NP obtained using square-wave voltammetry (0.12,,mol L,1) was lower than the value advised by EPA. A natural water sample from a dam located in Săo Carlos (Brazil) was spiked with 4-NP and analyzed by the standard addition method using the GC/MWCNT electrode, without any further purification step. The recovery procedure yielded a value of 96.5% for such sample, thus confirming the suitability of the developed method to determine 4-NP in natural water samples. The electrochemical determination was compared with that obtained by HPLC with UV-vis detection. [source] Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detectionELECTROPHORESIS, Issue 13 2005Michael Cornelius Abstract We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation. [source] New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb,GLIA, Issue 2 2001Holly H. Nash Abstract Ensheathing cells exclusively enfold olfactory axons. The ability of olfactory axons to reinnervate the adult mammalian olfactory bulb throughout the lifetime of an organism is believed to result from the presence of this unique glial cell in the olfactory system. This theory has been substantiated by research demonstrating the ability of transplanted ensheathing cells to promote axonal regrowth in areas of the central nervous system that are normally nonpermissive. A simple method for purifying ensheathing cells resulting in a large yield of cells is therefore invaluable for transplantation studies. We have developed such a method based on the differing rates of attachment of the various harvested cell types. The greatest percentage of cells (70.4%) that attached during the first step of the separation was determined to be fibroblasts. The remainder of the cells were classified as astrocytes (20.8%) and ensheathing cells (6.8%). The percentage of attached astrocytes (67.6%) was greatly increased during the second purification step while the percentage of fibroblasts decreased greatly (27.9%) and the percentage of ensheathing cells (5.3%) slightly decreased. In the final cultures, 93.2 % of the attached cells were ensheathing cells, while astrocytes (5.9%) and fibroblasts (1.4%) were only minor components. This simple, inexpensive method of purifying ensheathing cells will facilitate their use in central nervous system regeneration research. GLIA 34:81,87, 2001. © 2001 Wiley-Liss, Inc. [source] Purification of Aspergillus carbonarius polygalacturonase using polymeric membranesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2008E. Nakkeeran Abstract BACKGROUND: Microfiltration (MF: 70,450 nm) and ultrafiltration (UF: 10,500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes. RESULTS: MF resulted in removal of 2,25% carbohydrates with an enzyme recovery of 69,82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg,1 of dry matter. CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright © 2008 Society of Chemical Industry [source] Synthesis of a technetium-99m labelled L -tyrosine derivative with the fac - 99mTc(I)(CO)3 -core using a simple kit-procedureJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 8 2004Ralf Schirrmacher Abstract The synthesis of a novel technetium-99m labelled derivative of L -tyrosine as a potential tumour imaging agent for nuclear medicine diagnosis is reported. The synthesis involved the labelling precursor fac -[99mTc(OH2)(CO)3]+ which was synthesized using the commercially available Isolink® -labelling kit and the tyrosine derivative O-(N,N-bis(carboxymethyl)aminoethyl)- L -tyrosine trifluoroacetate. The labelled compound O -(99mTc(I)-tricarbonyl- N,N-bis(carboxymethyl)aminoethyl)- L -tyrosine was obtained in a radiochemical yield of 70,80% within 60 min with a radiochemical purity greater than 98% without any HPLC purification step. Purification was achieved merely by solid phase extraction. Chemical as well as chiral purity was determined using gradient- and chiral HPLC. Copyright © 2004 John Wiley & Sons, Ltd. [source] Extraction of pure lycopene from industrial tomato by-products in water using a new high-pressure processJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2008Daniele Naviglio Abstract BACKGROUND: Lycopene, a precursor of ,-carotene with a well-known antioxidant activity, contained in many natural products such as tomato (Lycopersicon esculentum Mill.), watermelon, red pepper and papaya, is usually recovered from natural vegetal sources using organic solvents and a purification step. In this paper an innovative process for the extraction of pure lycopene from tomato waste in water that uses the Naviglio® extractor and water as extracting phase is presented. RESULTS: Lycopene was obtained in the all- trans form at a very high grade of purity, not less than 98% (w/w), with an average recovery of 14% (w/w). The availability of high-purity trans -lycopene allowed measurement of the molar absorption coefficient. An alternative procedure for high-performance liquid chromatographic analysis using a phenyl-hexyl silicone phase as inverse phase and a linear gradient in water and acetonitrile is also described. CONCLUSIONS: The use of water as extracting phase considerably reduces the cost of the entire process when compared with the commonly used solvent-based procedure or with the newer supercritical extraction process of lycopene from tomato waste. Lycopene, not soluble in water, was recovered in a quasi-crystalline solid form and purified by solid-phase extraction using a small amount of organic solvent. Copyright © 2008 Society of Chemical Industry This article was published online on September 15, 2008. Errors in Figures 2 - 4 were subsequently identified. The publishers wish to apologise for these errors. This notice is included in the online and print versions to indicate that both have been corrected [September 19, 2008] [source] Activity-guided isolation of a novel protein from Croton tiglium with antifungal and antibacterial activitiesPHYTOTHERAPY RESEARCH, Issue 12 2008Muhammad Shahid Abstract This study describes the activity-guided isolation and purification of a novel antimicrobial protein from the seed of Croton tiglium Linn. Purification was carried out by (NH4)2SO4 precipitation, gel filtration and DEAE-cellulose ion-exchange chromatography. Antifungal and antibacterial activities were determined after each purification step. SDS-polyacrylamide gel electrophoresis revealed that the purified protein was a monomer with molecular mass of 50 kDa. This is a first report on purification of a protein from Croton tiglium, which possesses a strong and broad spectrum antimicrobial activity. Copyright © 2008 John Wiley & Sons, Ltd. [source] Technical note: Removal of metal ion inhibition encountered during DNA extraction and amplification of copper-preserved archaeological bone using size exclusion chromatographyAMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY, Issue 2 2009Carney D. Matheson Abstract A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results. Am J Phys Anthropol, 2009. © 2009 Wiley-Liss, Inc. [source] Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maizeRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2003Aldo Laganŕ A reliable, sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to determine four trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X and 3-acetyldeoxynivalenol) in maize. Sample preparation was performed by extracting the analytes with a mixture of acetonitrile and water, followed by a solid-phase extraction with Carbograph-4 cartridges as the purification step. For the LC/MS/MS analysis two interfacing systems, Turbo IonSpray (TISP) and atmospheric pressure chemical ionization (APCI), were compared in both negative and positive ion modes. LC and MS parameters were optimized to achieve better results and sensitivity. The effect of mobile phase modifiers such as ammonium acetate and formic acid on the ionization yield was also evaluated. The best results were obtained using the electrospray ionization (ESI) interface in negative ion mode and the multiple reaction monitoring mode (MRM) for the quantitation. The detection limits ranged between 10,ng/g for fusarenon X and 1.5,ng/g for deoxynivalenol. A linear working range was achieved with a standard deviation between 3 and 10% and recovery rates from the maize samples above 81%. The procedure was applied to the analysis of a set of maize samples collected from farms located in different areas of northern and central Italy. The investigated samples turned out to be contaminated primarily with deoxynivalenol and, to a minor extent, with its derivatives. Copyright © 2003 John Wiley & Sons, Ltd. [source] Efficacy of Bone Marrow Mononuclear Cells to Promote Bone Regeneration Compared With Isolated CD34+ Cells From the Same Volume of AspirateARTIFICIAL ORGANS, Issue 7 2010Shinji Yasuhara Abstract Autologous bone marrow mononuclear cell (BMMNC) transplantation is currently an emerging clinical treatment in the orthopedic as well as cardiovascular fields. It is believed that the therapeutic effect of the BMMNCs is due to neovascularization enhanced by the CD34+ cells contained therein, which include endothelial progenitor cells. However, isolation of the CD34+ cell fraction for clinical application has many disadvantages such as cost and invasiveness related to cell mobilization with cytokine. To investigate whether a purification step is in fact necessary for bone regeneration, we separated BMMNCs, CD34+, and CD34 - cells from the same initial volume of rabbit bone marrow aspirates. We then transplanted them back into a femoral bone defect of the same rabbit together with atelocollagen gel and basic fibroblast growth factor (bFGF) and evaluated neovascularization and bone regeneration up to 8 weeks after transplantation. The greatest potential for neovascularization and bone regeneration medicated by cells from the same volume of bone marrow aspirate was found in the BMMNC group. Although purified CD34+ cells might be an ideal cell source, BMMNCs could be a practical and feasible cell source for bone regeneration in present clinical settings with limited cost, availability of materials, and technical issues for transplantation. [source] Protein crystallization in hydrogel beadsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2005Ronnie Willaert The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4,kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0,kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein,agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead. [source] Purification of bioethanol effluent in an UASB reactor system with simultaneous biogas formationBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2003M. Torry-Smith In this study, the prospect of using an Upflow Anaerobic Sludge Blanket (UASB) reactor for detoxification of process water derived from bioethanol production has been investigated. The bioethanol effluent (BEE) originated from wet oxidized wheat straw fermented by Saccharomyces cerevisiae and Thermoanaerobacter mathranii A3M4 to produce ethanol from glucose and xylose, respectively. In batch experiments the methane potential of BEE was determined to 529 mL-CH4/g-VS. In batch degradation experiments it was shown that the presence of BEE had a positive influence on the removal of the inhibitors 2-furoic acid, 4-hydroxyacetophenone, and acetovanillone as compared to conversion of the inhibitors as sole substrate in synthetic media. Furthermore, experiments were carried out treating BEE in a laboratory-scale UASB reactor. The results showed a Chemical Oxygen Demand (COD) removal of 80% (w/w) at an organic loading rate of 29 g-COD/(L · d). GC analysis of the lignocellulosic related potentially inhibitory compounds 2-furoic acid, vanillic acid, homovanillic acid, acetovanillone, syringic acid, acetosyringone, syringol, 4-hydroxybenzoic acid, and 4-hydroxybenzaldehyde showed that all of these compounds were removed from the BEE in the reactor. Implementation of a UASB purification step was found to be a promising approach to detoxify process water from bioethanol production allowing for recirculation of the process water and reduced production costs. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 7,12, 2003. [source] Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2005Ramelito C. Agapay The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8,Ĺ using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8,Ĺ, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters. [source] Engineering the prion protein using chemical synthesisCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2001H.L. Ball Abstract: In recent years, the technology of solid-phase peptide synthesis (SPPS) has improved to the extent that chemical synthesis of small proteins may be a viable complementary strategy to recombinant expression. We have prepared several modified and wild-type prion protein (PrP) polypeptides, of up to 112 residues, that demonstrate the flexibility of a chemical approach to protein synthesis. The principal event in prion disease is the conformational change of the normal, ,-helical cellular protein (PrPC) into a ,-sheet-rich pathogenic isoform (PrPSc). The ability to form PrPSc in transgenic mice is retained by a 106 residue ,mini-prion' (PrP106), with the deletions 23,88 and 141,176. Synthetic PrP106 (sPrP106) and a His-tagged analog (sPrP106HT) have been prepared successfully using a highly optimized Fmoc chemical methodology involving DCC/HOBt activation and an efficient capping procedure with N -(2-chlorobenzyloxycarbonyloxy) succinimide. A single reversed-phase purification step gave homogeneous protein, in excellent yield. With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP106 was indistinguishable from its recombinant analog (rPrP106). Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine (Bo) PrP(90,200), were successfully prepared using a combination of the highly activated coupling reagent HATU and t -Boc chemistry. To mimic the glycosylphosphatidyl inositol (GPI) anchor and target sPrP to cholesterol-rich domains on the cell surface, where the conversion of PrPC is believed to occur, a lipophilic group or biotin, was added to an orthogonally side-chain-protected Lys residue at the C-terminus of sPrP sequences. These groups enabled sPrP to be immobilized on either the cell surface or a streptavidin-coated ELISA plate, respectively, in an orientation analogous to that of membrane-bound, GPI-anchored PrPC. The chemical manipulation of such biologically relevant forms of PrP by the introduction of point mutations or groups that mimic post-translational modifications should enhance our understanding of the processes that cause prion diseases and may lead to the chemical synthesis of an infectious agent. [source] Biofuels , Economic AspectsCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 5 2008G. W. Festel Abstract Assuming an oil price of US$60 per barrel, both biodiesel and bioethanol produced from wheat are not profitable in Europe. The producers' high margins are only due to the current mineral oil tax concessions. At present, biomass-to-liquid (BTL) fuel also cannot be produced competitively. At the assumed oil price, only bioethanol and biobutanol produced on a large scale from lignocellulose-containing raw materials have the potential to be produced competitively. Analyses of the technologies used in this field show that in Europe there are interesting new technological developments for the hydrolysis, fermentation and purification step. [source] Assessment of protein-incorporated arginine methylation in biological specimens by CZE UV-detectionELECTROPHORESIS, Issue 23 2007Angelo Zinellu Dr. Abstract Protein arginine methyltransferases methylate post-translationally arginine residues in proteins to synthesize monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), or symmetric dimethylarginine. Protein arginine methylation is involved in the regulation of signal transduction, RNA export, and cell proliferation. Moreover, upon proteolysis, arginines are released into the cytosol in which they exert important biological effects. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Quantification of these analytes is commonly performed by HPLC after sample cleanup and derivatization. We propose a CE method in which these steps have been avoided and the procedure for sample preparation has been simplified. After acidic hydrolysis of proteins, samples were dried, resuspended in water, and directly injected in CE. A baseline separation of analytes was reached in a 60 cm×75,,m id uncoated silica capillary, by using a Tris-phosphate run buffer at pH,2.15. This method allows an accurate assessment of protein arginine methylation degree in different biological samples such as whole blood, plasma, red blood cells, cultured cells, and tissue. Moreover, its good sensitivity permits to evaluate the methylation of a single protein type after the opportune purification steps. A method applicability concerns both clinical laboratories, where the evaluation of blood protein from numerous samples could be rapidly performed, and research laboratories where the factors affecting the arginine protein methylation degree could be easily studied. [source] Novel 4-Aminoquinolines through Microwave-Assisted SNAr Reactions: a Practical Route to Antimalarial AgentsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 36 2007Sergio Melato Abstract 4-Aminoquinolines have recently been indicated to be an important class of chemotherapeutic agents for artemisinin-based antimalarial combination therapy. A rapid, cheap, possibly clean and scalable route to 4-aminoquinolines endowed with multiple diversity is therefore badly needed. Classically, they have been prepared by means of SNAr reactions, requiring hazardous or costly reagents and conditions and complex purification procedures. In this paper, microwave flash-heating chemistry is shown to allow the efficient conversion of the available 4,7-dichloroquinoline into a library of aminoquinolines in high yields and purities, with no need for further purification steps and requiring very short reaction times. Some of the compounds in this library were active against chloroquine-sensitive and chloroquine-resistant parasite strains.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Purification of Aspergillus carbonarius polygalacturonase using polymeric membranesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2008E. Nakkeeran Abstract BACKGROUND: Microfiltration (MF: 70,450 nm) and ultrafiltration (UF: 10,500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes. RESULTS: MF resulted in removal of 2,25% carbohydrates with an enzyme recovery of 69,82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg,1 of dry matter. CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright © 2008 Society of Chemical Industry [source] Protein purification using chromatography: selection of type, modelling and optimization of operating conditionsJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2009J. A. Asenjo Abstract To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant , -1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (, -lactoalbumin, ovalbumin and , -lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. In order to optimize operating conditions, a cost function was built that included the effect of the different production stages, namely fermentation, purification and concentration. This cost function was also successfully used for the determination of the fraction of product to be collected (peak cutting) in chromatography. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters. Copyright © 2008 John Wiley & Sons, Ltd. [source] Solid-phase biotinylation of antibodies,JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2004Elizabeth Strachan Abstract Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide,PEO2 biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS,PEO4 biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2,M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin,alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100,,g). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels. Copyright © 2004 John Wiley & Sons, Ltd. [source] Synthesis of the C -terminal domain of the tissue inhibitor of metalloproteinases-1(TIMP-1)JOURNAL OF PEPTIDE SCIENCE, Issue 7 2003József Bódi Abstract According to recent investigations, the C -terminal domain of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) is responsible for some biological effects that are independent of the enzyme-inhibiting effect of the N -terminal domain of the molecule. The C -terminal domain has been prepared for structure,biological activity investigations. After the chemical synthesis and the folding of the linear peptide, LC-MS and MALDI-MS analysis revealed that two isomers with different disulphide bond arrangements were formed. Since more than 30 folding experiments resulted in products with a very similar HPLC-profile, it was concluded that in the absence of the TIMP-1 N -terminal domain no entirely correct folding of the C -terminal domain occurred. Furthermore, it was observed that, in spite of several purification steps, mercury(II) ions were bound to the 6SH-linear peptide; it was demonstrated,using disulphide bonded TIMP-1(Cys145 -Cys166) as a model,that mercury(II) ions can cause peptide degradation at pH 7.8 as well as in 0.1% trifluoroacetic acid. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Isolation of high-quality RNA from white spruce tissue using a three-stage purification method and subsequent cloning of a transcript from the PR-10 gene familyPHYTOCHEMICAL ANALYSIS, Issue 4 2003Nathalie Mattheus Abstract Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280,nm and 260/230,nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5,µg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picg1, that was very similar to members of the PR10 gene family. Copyright © 2003 John Wiley & Sons, Ltd. [source] Flow cytometry-assisted purification and proteomic analysis of the corticotropes dense-core secretory granulesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2008Daniel J. Gauthier Abstract The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, 1-DE or 2-DE, gel staining, in-gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS. [source] A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopyBIOTECHNOLOGY JOURNAL, Issue 9-10 2008Patrick Garidel Dr. Abstract Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed. [source] Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen-related proteinsBIOTECHNOLOGY PROGRESS, Issue 5 2009Cheng Zhang Abstract Corn continues to be considered an attractive transgenic host for producing recombinant therapeutic and industrial proteins because of its potential for producing recombinant proteins at large volume and low cost as coproducts of corn seed-based biorefining. Efforts to reduce production costs have been primarily devoted to increasing accumulation level, optimizing protein extraction conditions, and simplifying the purification. In the present work, we evaluated two grain fractionation methods, dry milling and wet milling, to enrich two recombinant collagen-related proteins; thereby, reducing the amount and type of corn-derived impurities in subsequent protein extraction and purification steps. The two proteins were a full-length human recombinant collagen type I alpha 1(rCI,1) chain with telopeptides and peptide foldon to effect triple helix formation and a 44-kDa rCI,1 fragment. For each, ,60% of the rCI,1s in the seed was recovered in the dry-milled germ-rich fractions making up ca. 25% of the total kernel mass. For wet milling, ,60% of each was recovered in three fractions accounting for 20,25% of the total kernel mass. The rCI,1s in the dry-milled germ-rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCI,1s were enriched 4,10 times in selected wet-milled fractions. The recovered starch from wet milling was almost free of rCI,1. Therefore, it was possible to generate rCI,1-enriched fractions by both dry and wet milling along with rCI,1-free starch using wet milling. Because of its simplicity, the dry milling procedure could be accomplished on-farm thus minimizing the risk of inadvertent release of viable transgenic seeds. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Optimal Synthesis of Protein Purification ProcessesBIOTECHNOLOGY PROGRESS, Issue 4 2001Elsa Vásquez-Alvarez There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches. [source] | ||||||||||||||||