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1
Cao, Liming. "Protein Separation with Ion-exchange Membrane Chromatography." Link to electronic thesis, 2005. http://www.wpi.edu/Pubs/ETD/Available/etd-050405-174109/.
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Hsu, Kuang-Hsin. "Scale-up of affinity chromatography for protein purifications." Ohio : Ohio University, 2000. http://www.ohiolink.edu/etd/view.cgi?ohiou1172002240.
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Ramat, Fabien M. "Protein purification using expanded bed chromatography." Digital WPI, 2004. https://digitalcommons.wpi.edu/etd-theses/92.
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Expanded bed chromatography using ion-exchange media is a powerful first step in purification processes. Expanded bed chromatography can be used to extract components from complex and viscous solution. This can be achieved because of the void created between adsorbent particles where as in packed bed chromatography, the adsorbent is too compact and dense for a complex feed stock to flow through. Expanded bed chromatography was used to purify bovine serum albumin (BSA) from chicken egg white (CEW). The high viscosity of CEW presents a unique challenge for efficient large-scale protein purification. This project aimed to optimize and evaluate a separation method that is believed to be particularly suitable for high viscosity solutions: expanded-bed ion exchange chromatography. The BSA was admixed into the CEW and the solution was pumped through the column for purification. The media used in the column was Streamline DEAE which is an anion-exchanger. The yield obtained was 85% and the purity was 57%. A mathematical model to understand and predict the behavior of expanded bed chromatography was developed to provide an estimation of the breakthrough curves obtained for BSA. A small sized porous dense adsorbent was also synthesized to enhance the purification process. This zirconia-based adsorbent allows use of higher flow velocities that is a key factor when working with viscous fluids such as chicken egg white.
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Batas, Borislav. "Protein refolding using size exclusion chromatography." Thesis, University of Bath, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337817.
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Ramat, Fabien M. "Protein purification using expanded bed chromatography." Link to electronic thesis, 2003. http://www.wpi.edu/Pubs/ETD/Available/etd-0114104-114704.
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Thesis (M.S.)--Worcester Polytechnic Institute.Keywords: zirconia; protein purification; anion exchange; chicken egg white; expanded bed chromatography. Includes bibliographical references (p. 83-86).
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Tuan, Chik Syed Mohd Saufi. "Mixed Matrix Membrane Chromatography for Bovine Whey Protein Fractionation." Thesis, University of Canterbury. Chemical and Process Engineering, 2010. http://hdl.handle.net/10092/3647.
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Whey protein fractionation is an important industrial process that requires effective large-scale processes. Although packed bed chromatography has been used extensively, it suffers from low processing rates due to high back-pressures generated at high flow rates. Batch chromatography has been applied but generally has a low efficiency. More recently, adsorptive membranes have shown great promise for large-scale protein purification, particularly from large-volume dilute feedstocks. A new method for producing versatile adsorptive membranes by combining membrane and chromatographic resin matrices has been developed but not previously applied to whey protein fractionation. In this work, a series of mixed matrix membranes (MMMs) were developed for membrane chromatography using ethylene vinyl alcohol (EVAL) based membranes and various types of adsorbent resin. The feasibility of MMM was tested in bovine whey protein fractionation processes.
Flat sheet anion exchange MMMs were cast using EVAL and crushed Lewatit® MP500 (Lanxess, Leverkusen, Germany) anion resin, expected to bind the acidic whey proteins β-lactoglobulin (β-Lac), α-lactalbumin (α-Lac) and bovine serum albumin (BSA). The MMM showed a static binding capacity of 120 mg β-Lac g⁻¹ membrane (36 mg β-Lac mL⁻¹ membrane) and 90 mg α-Lac g⁻¹ membrane (27 mg α-Lac mL⁻¹ membrane). It had a selective binding towards β-Lac in whey with a binding preference order of β-Lac > BSA > α-Lac. In batch whey fractionation, average binding capacities of 75.6 mg β-Lac g⁻¹ membrane, 3.5 mg α-Lac g⁻¹ membrane and 0.5 mg BSA g⁻¹ membrane were achieved with a β-Lac elution recovery of around 80%.
Crushed SP Sepharose™ Fast Flow (GE Healthcare Technologies, Uppsala, Sweden) resin was used as an adsorbent particle in preparing cation exchange MMMs for lactoferrin (LF) recovery from whey. The static binding capacity of the cationic MMM was 384 mg LF g⁻¹membrane or 155 mg LF mL⁻¹ membrane, exceeding the capacity of several commercial adsorptive membranes. Adsorption of lysozyme onto the embedded ion exchange resin was visualized by confocal laser scanning microscopy. In LF isolation from whey, cross-flow operation was used to minimize membrane fouling and to enhance the protein binding capacity. LF recovery as high as of 91% with a high purity (as judged by the presence of a single band in gel electrophoresis) was achieved from 150 mL feed whey.
The MMM preparation concept was extended, for the first time, to produce a hydrophobic interaction membrane using crushed Phenyl Sepharose™ (GE Healthcare Technologies, Uppsala, Sweden) resin and tested for the feasibility in whey protein fractionation. Phenyl Sepharose MMM showed binding capacities of 20.54 mg mL⁻¹ of β-Lac, 45.58 mg mL⁻¹ of α-Lac, 38.65 mg mL⁻¹ of BSA and 42.05 mg mL⁻¹ of LF for a pure protein solution (binding capacity values given on a membrane volume basis). In flow through whey fractionation, the adsorption performance of the Phenyl Sepharose MMM was similar to the HiTrap™ Phenyl hydrophobic interaction chromatography column. However, in terms of processing speed and low pressure drop across the column, the benefits of using MMM over a packed bed column were clear.
A novel mixed mode interaction membrane was synthesized in a single membrane by incorporating a certain ratio of SP Sepharose cation resin and Lewatit MP500 anion resin into an EVAL base polymer solution. The mixed mode cation and anion membrane chromatography developed was able to bind basic and acidic proteins simultaneously from a solution. Furthermore, the ratio of the different types of adsorptive resin incorporated into the membrane matrix could be customised for protein recovery from a specific feedstream. The customized mixed mode MMM consisting of 42.5 wt% of MP500 anionic resin and 7.5 wt% SP Sepharose cationic resin showed a binding capacity of 7.16 mg α-Lac g⁻¹ membrane, 11.40 mg LF g⁻¹ membrane, 59.21 mg β-Lac g⁻¹ membrane and 6.79 mg IgG g⁻¹ membrane from batch fractionation of 1 mL LF-spiked whey. A tangential flow process using this membrane was predicted to be able to produce 125 g total whey protein per L membrane per h.
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Polykarpou, E. "Optimisation of chromatography for downstream protein processing." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1334599/.
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Downstream bioprocessing and especially chromatographic steps, commonly used for the purification of multicomponent systems, are significant cost drivers in the production of therapeutic proteins. Lately, there has been an increased interest in the development of systematic methods where operating conditions are defined and chromatographic trains are selected. Several models have been developed previously, where chromatographic trains were selected under the assumption of 100% recovery of the desired product. Removing this assumption gives the opportunity not only to select chromatographic trains but also determine the timeline in which the product is selected. Initially, a mixed integer non-linear (MINLP) programming mathematical model was developed to tackle that problem and was tested using three illustrative examples. Later on, this model was linearised by applying piecewise linear approximation techniques and computational efficiency was improved. Next, an alternative MILP model was developed by discretising the recovery levels of the product and computational efficiency improved even by 100-fold. Finally, the equilibrium dispersive model was used in a simple 4-protein mixture and the MINLP model was validated. This research represents a significant step towards efficient downstream process operation and synthesis
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Conti, Sofia Alessandra. "Monoclonal antibodies purification via Protein G and protein A affinity chromatography." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2021.
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realization of a pilot scale affinity protein G chromatography for the purification of small volumes of supernatant from different harvests from the same antibody clone. The objective was to make a study on the productivity of each harvest, investigating for a dilution effect in case of an increasing number of harvests, and to have a rapid retention kit for the quality control of the purified antibody. Moreover, an identical column packed with protein A has been tested with the same products, in order to see if there was space for a yield improvement, and a further scale-up.
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To, Chi Shung Brian. "Protein retention and transport in hydrophobic interaction chromatography." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 2.12 Mb., 319 p, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3205434.
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Johnson, David H. "High-throughput self-interaction chromatography applications in formulation prediction for proteins /." Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2009r/johnson.pdf.
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Thesis (M.S.)--University of Alabama at Birmingham, 2008.Title from PDF title page (viewed Sept. 21, 2009). Additional advisors: Martha W. Bidez, W. Michael Carson, Richard A. Gray, W. William Wilson. Includes bibliographical references.
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Cochran, Keith Jacob. "Combined fermentation and recovery using expanded bed chromatography." Worcester, Mass. : Worcester Polytechnic Institute, 2006. http://www.wpi.edu/Pubs/ETD/Available/etd-081806-184321/.
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Edwardson, P. A. D. "High Performance Liquid Chromatography of polynucleotides and proteins." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376534.
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Watkins, Adam. "Protein interactions with model chromatography surfaces using FTIR ATR." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272744.
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De, Sousa Silva Jouciane <1984>. "Comparison of affinity chromatography supports for separation of protein." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/6090/.
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Chromatography is the most widely used technique for high-resolution separation and analysis of proteins. This technique is very useful for the purification of delicate compounds, e.g. pharmaceuticals, because it is usually performed at milder conditions than separation processes typically used by chemical industry. This thesis focuses on affinity chromatography. Chromatographic processes are traditionally performed using columns packed with porous resin. However, these supports have several limitations, including the dependence on intra-particle diffusion, a slow mass transfer mechanism, for the transport of solute molecules to the binding sites within the pores and high pressure drop through the packed bed. These limitations can be overcome by using chromatographic supports like membranes or monoliths. Dye-ligands are considered important alternatives to natural ligands. Several reactive dyes, particularly Cibacron Blue F3GA, are used as affinity ligand for protein purification. Cibacron Blue F3GA is a triazine dye that interacts specifically and reversibly with albumin. The aim of this study is to prepare dye-affinity membranes and monoliths for efficient removal of albumin and to compare the three different affinity supports: membranes and monoliths and a commercial column HiTrapTM Blue HP, produced by GE Healthcare. A comparison among the three supports was performed in terms of binding capacity at saturation (DBC100%) and dynamic binding capacity at 10% breakthrough (DBC10%) using solutions of pure BSA. The results obtained show that the CB-RC membranes and CB-Epoxy monoliths can be compared to commercial support, column HiTrapTM Blue HP, for the separation of albumin. These results encourage a further characterization of the new supports examined.
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Hey, Carolyn McKenzie. "Antibody Purification from Tobacco by Protein A Affinity Chromatography." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/42645.
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Antibodies represent the largest group of biopharmaceuticals. Due to the nature of
their clinical applications, they often need to be produced in large quantities. Plants have
distinct advantages of producing large quantities of recombinant proteins, and tobacco is
arguably the most promising plant for plant-made-pharmaceuticals (PMP) due to its high
biomass yields and robust transformation technology. However, to produce proteins using
transgenic tobacco for human applications, purification of the proteins is challenging. On
the other hand, Protein A, a bacterial cell wall protein isolated from Staphylococcus
aureus that binds to the Fc regions of immunoglobulins, is useful to the isolation and
purification of antibodies. An affinity chromatography purification step utilizing Protein
A resin introduced early in the purification process can reduce successive unit operations,
thereby reducing the overall process cost. However, directly applying tobacco extract to
Protein A chromatography columns may be problematic due to the non-specific binding
of native tobacco proteins (NTP). In this project, three different Protein A resins, ProSepvA
High Capacity, ProSep-vA Ultra, and ProSep Ultra Plus, marketed by Millipore, were
studied to provide valuable information for future downstream processes for antibody
purification from transgenic tobacco. The efficiency of the post load wash buffer to
reduce non-specific binding of NTP to the ProSep A resins were evaluated by altering the
ionic strength and pH. Lower salt concentrations of sodium chloride (NaCl) in the post
load wash preformed best at reducing the non-specific binding of NTP to the ProSep A
resins, while higher salt concentrations were more effective at reducing the amount of
NTP contaminants present during elution of the columns. Using a post load wash buffer
with an intermediate pH between the binding buffer and the elution buffer was more
efficient at eluting our model antibody, human IgG. However, lowering the ionic strength
and the pH of the post load wash buffer resulted in a greater presence of IgG prematurely
eluting from the ProSep A resins. The non-specific binding of NTP to the resins reduced
the dynamic binding capacity (DBC) of the resins after repeated cycles of tobacco extract
samples were loaded onto the column. Nevertheless, cleaning the columns with
denaturing solutions, such as urea or guanidine hydrochloride, every 8-10 cycles was
effective in regenerating the DBC of the resins and prolonging the life cycle of the resins.
This is important to evaluating the economic feasibility of directly using Protein A
chromatography to recover antibodies from tobacco extract. Of the three Protein A resins
studied, ProSep Ultra Plus performed best for antibody purification from tobacco using a
PBS wash buffer with a lower ionic strength of 140mM NaCl and an intermediate pH of
5.Master of Science
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Garg, Nandita. "Polymer shielded dye-affinity chromatography and temperature induced phase separation two strategies to simplify protein affinity separation processes /." Lund : Dept. of Biotechnology, Lund University, 1995. http://books.google.com/books?id=Qw5rAAAAMAAJ.
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Gülich, Susanne. "Engineering Proteinaceous Ligands for Improved Performance in Affinity Chromatography Applications." Doctoral thesis, KTH, Biotechnology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3327.
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Wang, Liwen. "Characterization and identification of protein posttranslational modifications using protein enrichment and mass spectrometry." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1234422949.
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Jaffal, Jad M. "Isolation of potential protein targets of MS-818 using affinity chromatography." Honors in the Major Thesis, University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1428.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Medicine
Molecular and Microbiology
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Myers, Terence Anthony. "Assessment of chitosans as support matrices for dye-ligand affinity chromatography." Thesis, Queen's University Belfast, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295399.
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McCreath, Graham Edward. "Development and applications of perfluorocarbon affinity emulsions." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307052.
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Sines, Brian James. "Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents." Diss., Virginia Tech, 2000. http://hdl.handle.net/10919/29841.
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Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This study is concerned with the isolation of these important therapeutics from complex media. Immunoaffinity chromatography has been an important method in the isolation of these products, typically being implemented as the final cleanup step yielding an extremely pure, homogenous final product. However, the use of immunoaffinity chromatography in large-scale purification processes have been precluded due to high capital costs and the inherent lability of immunosorbents. Peptide-based affinity sorbents are being developed in order to surmount the inherent limitations posed by monoclonal antibodies that are used as ligands in immunosorbents.
The objective of this research is to quantitatively assess the impact of affinity ligand orientation, local density and transport phenomena on peptide-based affnity sorbent performance. The peptides under study herein can form high-affinity complexes with their protein targets, thus these ligands are one of the newest technologies arising from combinatorial chemistry with applications to the difficult problem of purifying high-molecular weight proteins from complex mixtures. Two types of structural motifs which are common to small peptide affinity ligands derived from combinatorial chemistry are studied here: a linear peptide which is comprised of the affinity recognition sequence in its entirety and a chain structure which displays multiple branches of the recognition sequence emanating from a central lysinic core structure. Two recognition sequences are studied here which bind plasma proteins. One peptide recognition sequence forms a high affinity complex with fibrinogen. Another peptide recognition sequence binds the Fc region of immunoglobulins. Immunglobulins are plasma proteins which range in molecular weight from 155 to 900-kDa and are valuable for therapeutic uses for imparting passive immunity.
This study seeks to identify factors analogous to those manifested in immunosorbent performance that may also be important in the optimal design of peptide-based affinity sorbents. In general, previous research with the design of immunosorbents have found that immunosorbent performance, i.e., target-binding efficiency or activity, is substantially dependent upon several factors which include effects associated with ligand orientation, and local density as related to steric incumbrance of target binding sites, and transport phenomena as related to under utilization of intra matrix volume. In summary, this study asks the questions: (1) What factors regarding ligand orientation, local ligand density, and intraparticle transport phenomena, are important in the optimal design of peptide affinity sorbents?; and (2) Are these effects analogous to those manifested in immunosorbent performance?
This study seeks to investigate the use of techniques used to mitigate the effects associated with these negative factors upon immunosorbent performance in order to elucidate the nature of these same effects upon peptide-based affinity sorbents. For example, oriented ligand immobilization can be facilitated through selective coupling chemistries and the premasking of ligand binding domains prior to immobilization. In addition, the manipulation of local ligand density using novel spatially controlled matrix activation and ligand immobilization methods can be assessed and implemented for the optimization of the performance and design of peptide-based affinity sorbents. This study has found that enhanced transport phenomena into the matrix interior volume can be achieved by using low solids content cellulose matrices having a low extent of crosslinking. This study demonstrates the effective use of these large-particle diameter, low-solids content cellulose hydrogel matrices in immunoaffinity, peptide-based affinity and ion exchange chromatography in the separation of high-molecular weight therapeutic proteins.Ph. D.
This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes the molecular accessibility of the cellulose support to high molecular weight proteins relative to the mechanical stability of the support at high operating linear velocities by the manipulation of bead particle diameter and solids content. A novel epoxidegradient activation method (epoxy-GAM) is developed for creating a gradient of support activation for support crosslinking and affinity ligand installation in the preparation of DEAE hydrogel matrices. These cellulose hydrogel supports were evaluated with regards to structure, dynamic and static binding capacity, pressure-flow stability, chemical stability and intraparticle transport phenomena. The utility of the low-solids content, large-particle diameter DEAE hydrogel matrices was demonstrated in a column-mode protein purification using albumin and fibrinogen mixtures.
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Pathange, Lakshmi Prasad. "Characterization of protein microstructure by various chromatographic techniques." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/26851.
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Due to the rising health care costs and with the advent of biogenerics, there is a growing demand to develop new and reliable techniques to characterize proteins and biopharmaceuticals. In addition, characterization aids in understanding the intricate relationship between a protein's structure and its function. To address this challenge, two protein structural parameters, 1) amino acid surface area and 2) amino acid microstructure, were chosen to be investigated. Two chromatographic techniques, 1) ion exchange chromatography (IEC) and 2) immobilized metal affinity chromatography (IMAC), were used to characterize the above-mentioned protein structure parameters.
The model protein chosen for our work is T4 lysozyme. The protein consists of 164 amino acids with molecular weight ~ 18 kD. SYBYL 7.1 software was used to generate in silico point mutants. Two categories of protein variants (point mutants) were generated using site-directed protein mutagenesis. The goal for generating point mutants was to obtain mutants that vary in the two structural parameters. The first category point mutants vary in the surface accessibility of a surface accessible histidine residue. The second category point mutants predominantly vary in protein net charge and the amino acid microstructure. In total, seventeen point mutants were generated: 1) category I consists of seven variants that vary predominantly in their histidine surface accessibility, and were obtained by replacing a charged amino acid residue at different locations on the surface of the protein molecule, and 2) category II consists of ten variants that vary in both net charge and charge distribution were obtained by replacing charged and neutral amino acid residues at different locations (different microenvironments) on the protein surface.
PCR technique was used to generate the point mutants. Gene and protein sequencing were employed to confirm the veracity of point mutation. CD and Lysozyme activity assays were performed to determine whether or not the 3D structure of all the protein variants was intact. Zonal analysis was used to obtain the binding strength values of all seventeen variants in IMAC with copper as the immobilized metal ions, and gradient elution method was used to obtain the relative retention times (rRT) values of all the variants in IEC.
The seven lysozyme variants generated in category I each contains one surface histidine residue. In IMAC, there is a correlation between the surface accessibility of the lone surface histidine and the protein's binding strength with R²⁺= 0.76. In IEC, the correlation between the protein's microstructure, which predominantly consists the surface accessibility of the histidine residue, and the protein's retention times was R²⁺= 0.95. However, there were few outlier variants (e.g. variant K83H) which did not follow the correlations. The variations presented by few outlier variants can be attributed to the presence of intramolecular bonds, which restrict the mobility of the amino acid side chains and subsequently hinder the specific interaction between the amino acid residue and chromatographic media.
For category II variants, short and medium range charge perturbations around the sole histidine residue in T4 lysozyme were engineered within 15 Ã distance of histidine. There was a strong correlation (R²⁺ = 0.96) between the theoretical (DeltaDeltaGElec) values, calculated using simple Coulomb's law, and the experimental (DeltaDeltaGB) values, which were obtained by measuring the protein binding strength values using IMAC. Similar correlation (R²⁺= 0.93) was obtained between the change in net charge (-2 to +2 units) and the relative retention times in IEC. Similarly, there were few variants (e.g. S136K, R76D) that did not follow the trends. The deviations of the few outlier variants can be attributed to the presence of unique microstructure effects around the histidine residue. These microstructure effects were quantified in IMAC as (DeltaDeltaGMicro), and in IEC they were quantified by the change in rRT values.
In summary, all seventeen variants had different binding strengths and rRT values indicating the variation in the protein structure around the histidine residue. Our work reveals that it is possible to capture the microstructural effects of a protein through the combination of protein molecular modeling and simple chromatographic experiments.Ph. D.
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Emanuelsson, Ida, Anna-Karin Jansson, and Katarina Risö. "Characterising of chromatography gels for purification of erythropoietin." Thesis, Högskolan i Borås, Institutionen Ingenjörshögskolan, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-19097.
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Erythropoietin is a human natural hormone which task is to regulate the amount of red blood cells in the body. At Centro de Inmunología Molecular, situated in Havana, erythropoietin is produced by recombinant DNA-technique. The protein is purified through several chromatography steps. Among other things, Centro de Inmunología Molecular uses affinity chromatography and ion exchange chromatography. To both of these chromatographic methods, gel is used as stationary phase. The aim of this study was to investigate and determine parameters for characterising of two gels, this because Centro de Inmunología Molecular have to exchange the gels. The reason for the gel exchange is that the currently used gels will not be manufactured any more. The gel used in the affinity chromatography is Chelating Sepharose Fast Flow and the gel used in the ion exchange chromatography is Q Sepharose Fast Flow. For both of this gels kinetic parameters and isotherm parameters were determined by experiments. The isotherm parameters qmax and Kd were calculated from an adsorption isotherm. To be able to calculate qmax and Kd for both Q Sepharose Fast Flow gel and Chelating Sepharose Fast Flow gel different experiments were made. A kinetic adsorption and an isotherm adsorption were made on each gel. The kinetic adsorption was made in due to find out how long the two different processes were supposed to run and to understand which part of the mass transfer that is controlling the rate. There is no use to let the process to be in progress any longer than until the adsorption ceases. For the Q Sepharose Fast Flow gel this was after 200 seconds. The adsorption with the Chelating Sepharose Fast Flow gel never ceased completely, but after 1000 seconds the adsorption was so slow that it would be no use to continue the process. If the processes continue after the calculated times only money, hours and recourses will be wasted. The data that were achieved was plotted in two different isotherm adsorption models both the Freundlich- and the Langmuir model, this to determine which model that had the best fit. One could see that the Q Sepharose Fast Flow gel was following the model of Langmuir better and because of this the Langmuir equation was used to calculate qmax and Kd. The qmax for the Q Sepharose Fast Flow gel agreed a lot with the value that Centro de Inmunología Molecular had assumed. When it came to the Chelating Sepharose Fast Flow gel, the same kind of plotting was made. But one could see that this time the data was following the model of Freundlich much better. Therefore a calculation of the desired qmax was impossible. Only the value of Kd was calculated. Because the company Centro de Inmunología Molecular still needed the value of qmax an assumption that the gel was following the model of Langmuir was made. qmax was calculated but without any satisfied results. The programs Excel, Statgraphic and Matlab have been used in all calculations.Uppsatsnivå: C
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Atkinson, Ian E. "Mass Spectrometric Analysis of Environmental Contaminants, Protein Structure and Expression." Cleveland State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1231174291.
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Serracchiani, Marcel Mafei 1986. "Estratégia de purificação por IMAC de fragmento Fab de IgG humana adicionado a extrato proteico de soja." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/266613.
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Orientador: Sonia Maria Alves BuenoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: A produção de proteínas recombinantes em plantas transgênicas tem se mostrado uma forma segura, eficiente e barata de obtenção em grande escala de várias proteínas de interesse, tais como vacinas, enzimas industriais, bioativos, biofarmacos e anticorpos e seus fragmentos. Contudo, para que a produção de proteínas recombinantes em plantas se torne viável é necessário o desenvolvimento de um processo de recuperação e separação da molécula alvo que seja eficiente e reprodutivo, pois a produção de biomoléculas em plantas transgênicas, assim como os métodos tradicionais de produção, apresentam componentes indesejáveis que devem ser removidos. Neste trabalho, avaliou-se a purificação de fragmentos Fab de IgG humana adicionados artificialmente (spiking) a extrato protéico de soja não transgênica utilizando-se a técnica de cromatografia de afinidade por íons metálicos imobilizados (IMAC). Estudou-se o efeito dos quelatos IDA-Ni(II) e TREN-Ni(II), dos sistemas tamponantes Tris-HCl, fosfato de sódio e Mes, na presença e na ausência de sal (NaCl) na purificação de fragmentos Fab. Primeiramente foram realizados experimentos cromatográficos com as proteínas nativas do extrato proteico do grão de soja. Dos resultados obtidos, selecionou-se, para o adsorvente agarose-TREN-Ni(II), o sistema tamponante Tris-HCl/Tris-HCl na ausência de NaCl, e para o adsorvente agarose-IDA-Ni(II), selecionou-se o sistema tamponante fosfato de sódio/acetato de sódio contendo NaCl. Estes sistemas tamponantes e adsorventes foram utilizados para purificação dos fragmentos Fab adicionados (spiking) ao extrato proteico de grãos de soja. Em agarose-TREN-Ni(II), os fragmentos Fab foram purificados por cromatografia negativa, obtendo-se 66% dos fragmentos Fab na etapa de flowthrough e lavagem, com um grau de pureza de 91% e fator de purificação de 1,4. Para o adsorvente agarose-IDA-Ni(II), os fragmentos Fab foram purificados por cromatografia tradicional (eluição a pH 5,8), obtendo-se alto grau de pureza do fragmento Fab purificados, com um fator de purificação de 2,5. Este estudo contribuiu para obter conhecimento de base para posterior aplicação da técnica de IMAC na purificação de proteínas recombinantes produzidas em sementes de plantas transgênicas
Abstract: The recombinant proteins production using transgenic plants have been considered a safe, efficient and cheap way of producing on large scale innumerous proteins of interest, such as vaccines, industrial enzymes, bioactives, biopharmaceuticals and antibody and it fragments. However, for the production of recombinant proteins in plants become viable is necessary to develop a process for recovery and separation of the target molecule that is efficient and reproductive, because the production of biomolecules in transgenic plants, as well as traditional production methods, have components reactions which must be removed. In this work, the purification of Fab fragments of human IgG added artificially (spiking) to extract non-transgenic soy protein, using the technique of affinity chromatography on immobilized metal ions (IMAC), was evaluated. The effect of chelating agents IDA-Ni(II) and TREN-Ni(II), and the buffers Tris, sodium phosphate and Mes, in presence and the absent of NaCl, was studied for the Fab purification. First, the chromatographic experiments with the native proteins of soybean extract were performed. From the results obtained, it was selected, for the adsorbent TREN-Ni(II), the buffers system Tris-HCl/Tris-HCl without NaCl, and for the adsorbent IDA-Ni(II), the buffer system sodium phosphate/ sodium acetate with NaCl was selected. These adsorbents and buffer systems were used for purification of Fab fragments added (spiking) to soybean extract proteins. In the agarose adsorbent TREN-Ni (II), Fab fragments were purified by negative chromatography, obtaining 66% of Fab fragments in flowthrough and wash steps with a purity of 91% and purification factor of 1.4. For the adsorbent agarose-IDA-Ni (II), Fab fragments were purified by traditional chromatography (elution at pH 5.8), obtaining a high purity of the purified FAB, with a purification factor of 2.5. This study contributed to obtain knowledge base for application of the technique on the IMAC purification of recombinant proteins produced in transgenic plant seeds
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química
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Fernando, Samantha. "Monoclonal antibody (mAb) purification by counter current chromatography (CCC)." Thesis, Brunel University, 2011. http://bura.brunel.ac.uk/handle/2438/6522.
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Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave-like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built 'g' shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard.
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Jönsson, Daniel. "Evaluation of Rate Constants from Protein-Ligand Interactions with Weak Affinity Chromatography." Thesis, Linnéuniversitetet, Institutionen för naturvetenskap, NV, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-19974.
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The paradigm of drug discovery have been to find the strongest possible binder to the target by high-throughput screening (HTS) but high affinity interactions are related to low kinetic off rates and thus result in severe side-effects and non-approved drugs. Lead molecules working in a transient manner (KD > µM) will allow for rapid off rates and possibly less side-effects. In this study the peak profile method applied to weak affinity chromatography (WAC) was evaluated as a simple way to provide the kinetics of the interaction and thereby allowing for high-throughput determinations. In the peak profile formula all band-broadening effects except the stationary mass transfer is subtracted which simplifies the calculations for the kinetics of the interaction tremendously. The technique was evaluated by screening of 3 different benzamidines at 3 linear flow-rates using zonal chromatography and human α-thrombin as immobilized target protein. The kinetics of the interaction could unfortunately not be determined. This was possibly due to the flow-rates not being high enough as indicated by a low critical ratio (η < 1). Higher flow-rates would increase the contribution to band-broadening due to kinetic effects but would also require more precise estimation of peak variance.
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Xu, Xuankuo. "Protein adsorption isotherms and their effects on transport in cation-exchange chromatography." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 244 p, 2008. http://proquest.umi.com/pqdweb?did=1459924621&sid=13&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Castiblanco, Adriana P. "Expression and Purification of Engineered Calcium Binding Proteins." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/chemistry_theses/20.
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Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.
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Ansong, Godfred. "Analysis of plant polyphenols by high performance liquid chromatography/mass spectrometry and protein binding." Oxford, Ohio : Miami University, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1083081905.
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Monaco, Enzo. "pH Transients in Hydroxyapatite chromatography columns." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2018.
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Ceramic Hydroxyapatite (CHT), with empirical formula (Ca5(PO4)3OH)2, is a material used as ion exchange resin in chromatographic applications. The material, having both positive and negative charged sites, can be used in many different contexts encountered in the protein purification processes. Nevertheless, the resin shows an intrinsic limitation for this kind of applications: even if the material solubility in water is very low at pH values higher than 6.5, it sharply increases in more acidic environments, reducing the life of the material and increasing the operative costs of the process, making problematic the CHT application at these conditions.
There is a further complication related to the CHT application: it is experimentally reported that the result of the resin interactions with salts is a pH transient occurring in the liquid phase of the chromatographic column.
These pH variations are temporary but remarkable, they may influence in an important manner the material solubility, affecting the number of utilization cycle of the fixed bed and, eventually, the stability of the proteins involved in the separation process.
In this work the principal aspects contributing at the selection of the operating pH and of the buffering specie are analyzed, discussing also the effect of the buffer itself on the material solubility.
Then, it is reported a mathematical model useful for the description of the dynamic behavior of the column and it is proposed an interaction mechanism between the salts of the mobile phase and the stationary phase. In particular, it is emphasized the discussion regarding the different affinity shown by the stationary phase for the different counter-ions which may accompanies the buffering specie.
Finally, a series of equations are developed at the scope to obtain a model useful to describe the observed pH transients.
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Hamnell-Pamment, Ylva. "Novel methods for the identification of cellular S-glutathionylated proteins and sites of glutathionedependent modification using affinity chromatography and proteomic analyses /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-248-9/.
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Gilbert, Max [Verfasser], and Waltraud [Akademischer Betreuer] Schulze. "Prediction of protein-protein complexes by combining size exclusion chromatography and mass spectrometric analysis / Max Gilbert ; Betreuer: Waltraud Schulze." Hohenheim : Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim, 2021. http://nbn-resolving.de/urn:nbn:de:bsz:100-opus-19403.
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Fahey, Edward Michael. "A study of the refolding of urokinase plasminogen activator by size exclusion chromatography and batch dilution." Thesis, University of Bath, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323582.
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Thomas, Sarah Jane. "New analytical and synthetic tools for the study of protein-gycosaminoglycan interactions." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/16215.
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Glycosaminoglycans (GAGs) are linear polysaccharides found on most animal cell surfaces and in extracellular matrices. Their key biological roles include cell signalling, cell-to-cell recognition, bacterial and viral adhesion, and antibody production. They are composed of disaccharide repeating units, containing uronic acid and amino sugar residues, and may be highly heterogeneously sulfated. Protein-GAG complexes are thought to play an important role in a number of aspects of cancer development, but are an under-studied area due to a lack of enabling tools to facilitate their analysis as discussed in Chapter 1. To obtain homogenous GAG structures, a range of conditions for the separation of GAG oligosaccharides by Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) were developed using commercial chondroitin sulfate standards; these are discussed in Chapter 2. Mixed-modal separation mechanisms were explored across a different buffer compositions and elution programs to optimise the conditions to suit individual classes of native and modified glycosaminoglycans. These methods were then applied to the separation of chondroitin sulfate mixtures and heparin oligosaccharides. ZIC-HILIC may also be coupled to Mass Spectrometry to produce an online analytical method for GAG mixtures. A range of optimised conditions for the analysis of low molecular weight glycosaminoglycan oligosaccharides by Electrospray Mass Spectrometry were developed using commercial chondroitin sulfate disaccharide standards; as discussed in Chapter 3. These conditions were then employed in the tandem mass spectrometry (MS/MS) of chondroitin sulfate to identify diagnostic fragmentation patterns and this was applied in the characterisation of chondroitin sulfate disaccharides derived from enzymatic cleavage. The position of ring substituents can also be identified using this methodology, which is desirable in the analysis of chemically-labelled GAG structures. To allow for high resolution EPR and NMR studies of protein-GAG interactions, synthetic procedures for the incorporation of paramagnetic centres into GAG oligosaccharides and proteins were developed and these are discussed in Chapter 4. A propargyl-modified lysine was synthesised for recombinant expression into myoglobin. Copper-Catalysed Azide-Alkyne Cycloaddition (CuAAC) was employed in the spin labelling of myoglobin, however traditional experimental conditions were found to reduce the TEMPO-based spin labels used. Alternative conditions were developed using glutathione to stabilise the copper (II) catalyst without reducing the spin label. Spin labelling of the modified lysine was monitored by EPR. Modification of the non-reducing end was explored for the spin labelling of GAG oligosaccharides, to avoid the ring opening that typically occurs in reducing end labelling, and make use of the unsaturated uronic acid that is derived when obtaining GAGs through enzymatic depolymerisation. A hydrazide labelling of uronic acids was initially adopted, however due to concerns regarding selectivity and availability of hydrazide spin labels, an alternative Thiol-Ene Click (TEC) method was developed. TEC labelling of monosaccharides and unnatural amino acids was found to proceed efficiently under aqueous conditions and was monitored by NMR and HPLC. Preliminary studies in the TEC labelling of chondroitin sulfate disaccharides proved promising, however further optimisation is required for this method to be utilised in the study of protein-GAG interactions by NMR.
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Silva, Maria Zelandia Rocha. "Insulation immunoaffinity chromatography and antifungal activity osmotinas of latex fluid." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=14503.
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CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel SuperiorPlants are constantly exposed to a variety of stresses conditions. However, they react to biotic stresses by triggering a set of defense mechanisms including the synthesis of defensive substances as the pathogenesis-related (PR) proteins. The PR-protein named osmotin can be induced under osmotic stress and water shortage conditions. Osmotin-like proteins have been purified from latex and some of them are related to antifungal activity. The aim of this study was to investigate the osmotin in the following species laticifers: C. grandiflora, P. rubra, T. peruviana, H. drasticus and C. papaya to isolate and evaluate its antifungal activities. Immunoaffinity column chromatography with anti-CpOsm antibodies were performed in order to purify these osmotin-like. They were detected in latex of C. grandiflora and P. rubra by immunoassays the ELISA, Dot Blot and Western Blot using anti-CpOsm antibody (the osmotin of C. procera latex). Osmotin of C. procera, C. grandiflora, P. rubra and H. drasticus were identified by mass spectrometry. However, the osmotin from C. procera was co-purified with cysteine proteases. The co-purified cysteine protease from C. procera was identified as Procerain B. The alignment and the 3-D structure analysis of Procerain B and CpOsm revealed the presence of a similar sequence in both proteins. This sequence might be an epitope which allows the anti-antibody recognition. The osmotin from C. grandiflora, and P. rubra did not show antifungal activity against Fusarium solani and Colletotrichum gloesporioides. Since no correlation between the antifungal activity and the presence of these osmotins were found, the proteolytic activities of these latex protein fractions were evaluated in order to correlate with the antifungal activity C. procera and C. grandiflora showed a strong proteolytic activity. In latex, the cysteine proteases are more often related to antifungal activity than osmotin, which might explain, at least in part, the antifungal activity performed by C. grandifora and not for its osmotin. Further studies on the role of osmotin in physiology laticifers plants are needed.
As plantas estÃo constantemente sujeitas a diversos tipos de estresse, tanto biÃticos como abiÃticos, resultando em respostas de defesa. Decorrente disto, os vegetais sintetizam certas proteÃnas denominadas de proteÃnas relacionadas à patogÃnese (PR proteÃna). As Pr-proteÃnas chamadas de osmotinas podem ser induzidas sob condiÃÃes de estresse osmÃtico, frio e escassez de Ãgua. Osmotinas tem sido purificadas de fluidos laticÃferos e algumas delas estÃo relacionadas com a atividade antifÃngica. O objetivo do presente trabalho foi prospectar osmotinas, bem como isolÃ- las e avaliar suas atividades antifÃngicas, nos fluidos laticÃferos das seguintes espÃcies: C. grandiflora, P. rubra, T. peruviana, H. drasticus e C. papaya. Nos lÃtex de C. grandiflora e P. rubra foram detectadas osmotinas atravÃs de imunoensaios em placa de ELISA, Dot Blot e Westen Blot, utilizando os anticorpos anti-CpOsm (osmotina do lÃtex de C. procera). Cromatografia de imunoafinidade em coluna com anticorpos anti-CpOsm foram realizadas com o intuito de purificar estas osmotinas. AnÃlises por meio de espectrometria de massas, revelaram a presenÃa de osmotina em C. procera, C. grandiflora, P. rubra e H. drasticus. No entanto, a osmotina de C. procera foram co-purificadas com proteases cisteÃnicas. A protease cisteÃnica co- purificada no lÃtex de C. procera foi identificada como Proceraina B. O alinhamento e a anÃlise da estrutura tridimensional da Proceraina B e CpOsm revelaram a presenÃa de uma sequÃncia semelhante em ambas as proteÃnas, que pode ser um epÃtopo disponÃvel ao reconhecimento do anticorpo anti-CpOsm. As osmotinas isoladas de C. grandiflora e P. rubra nÃo apresentaram atividade antifÃngica contra F. solani e C. gloesporioides. Desde que nÃo houve correlaÃÃo entre a atividade antifÃngica e à presenÃa destas osmotinas, as atividades proteolÃticas das fraÃÃes proteicas foram avaliadas a fim de correlaciona-las à atividade antifÃngica. Nos fluidos laticÃferos, as proteases cisteÃnicas estÃo mais frequentemente relacionadas à atividade antifÃngica do que as osmotinas. Estudos mais aprofundados sobre a funÃÃo das osmotinas na fisiologia de plantas laticÃferas sÃo necessÃrios.
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Nicolas-Simonnot, Marie-Odile. "Contribution à l'étude de la chromatographie frontale des protéines par échange d'anions : Application à l'albumine du serum bovin bovin." Vandoeuvre-les-Nancy, INPL, 1991. http://docnum.univ-lorraine.fr/public/INPL_T_1991_NICOLAS_SIMONNOT_M_O.pdf.
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La chromatographie d'échange d'anions mettant en jeu une protéine fait intervenir simultanément plusieurs phénomènes : l'échange d'ions proprement dit, ainsi que d'autres réactions, essentiellement de dissociation, en phase liquide ou solide. La démarche adoptée ici consiste à découpler toutes les contributions et à les examiner séparément, avant de les regrouper dans un modèle global. On étudie le comportement des supports échangeurs d'ions faibles en fonction de l'environnement ionique, les interactions entre les électrolytes faibles (tampons de pH) et les échangeurs d'anions, les interactions entre la protéine (albumine du sérum de bovin) et la solution (modélisation des courbes de dosage), et les interactions entre la protéine et des échangeurs d'anions (mesures d'isothermes en réacteur ferme et expériences en colonne en milieu tamponné ou non). On propose un modèle de la protéine (au niveau de l'échange d'ions), dont le comportement global s'approche de celui d'un électrolyte faible. Avant d’aborder les expériences en colonne avec une protéine-modèle (albumine de sérum bovin), on analyse les interactions entre cette protéine et la solution (modélisation de courbes de dosage). De même, on étudie les interactions entre la protéine et des échangeurs d’anions par la mesure d’isothermes dans différentes conditions. Enfin, des expériences de chromatographie frontale sont présentées. On commence par des systèmes « simples » du point de vue de l’échange d’ions (échanges binaires ou ternaires, en milieu non tamponné), avant de travailler en milieu tampon (où toutes les contributions sont mêlées). L’interprétation de ces expériences nous conduit à proposer un mécanisme d’échange. On montre que le comportement global de la protéine s’approche de celui d’un électrolyte faible, ce qui constitue un point de départ de vue de l’élaboration d’un modèle
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Hidayah, Siti Nurul [Verfasser]. "Improvement of liquid chromatography for analysis and purification of proteoforms via rational protein purification parameter screening and sample displacement chromatography / Siti Nurul Hidayah." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2021. http://d-nb.info/1241743037/34.
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Loog, Mart. "Studies on the Differential Specificity of Protein Kinases and Its Applications." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5116-0/.
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Onesti, Riccardo. "Characterization of a ceramic monolithic support for affinity protein chromatogrophy." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2017.
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Affinity chromatography is a process for the separation of biomolecules from complex mixtures. Its versatility, high selectivity and efficiency make it the most currently used method in the final stages of purification of pharmaceutical and food industries, where a high degree of purity is required.
Conventional affinity chromatography uses packed beads as solid support and suffers of several limitations, such as high material costs, high operating costs, intraparticle diffusion as primary transport phenomenon and difficulties in column packing.
Therefore, it is necessary to develop and characterize cheaper and more efficient materials as stationary phase for chromatographic separations. From this perspective, monolithic media have shown a significant potential.
In this research project a new ceramic composite monolithic support, a cellular Al2TiO5 and Al2TiO5-Al2O3 composite monolith, has been experimentally characterized.
First, several monolithic columns have been prepared, by polishing the material to obtain cylindrical samples of the desired height and dimensions. Then the columns have been characterized by calculating the main fluid dynamic parameters that govern the motion of a fluid in a porous material: permeability, porosity and axial dispersion coefficient. To this aim permeability tests and pulse tests have been performed.
One column has been functionalized with epoxy groups, by linking 3-glycidoxypropyltrimethoxysilane (GPTMS). This step, called activation, creates active sites for the immobilization on the support of protein A, that on turn is used for the affinity adsorption of immunoglobulin-G.
The number of active epoxy groups has been estimated by measuring the ability of the column to irreversibly bind the bovine serum albumin, BSA, in chromatographic experiments.
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Zhou, Feng. "Protein characterization by capillary isoelectric focusing electrophoresis, reversed phase liquid chromatography and mass spectrometry." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 103 p, 2008. http://proquest.umi.com/pqdweb?did=1456289241&sid=4&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Jones, Karen Lorraine. "Analysis of ferredoxin and flavodoxin in Anabaena and Trichodesmium using fast protein liquid chromatography." PDXScholar, 1988. https://pdxscholar.library.pdx.edu/open_access_etds/3812.
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Iron is an essential nutrient for growth of photosynthetic microorganisms such as cyanobacteria and algae. Iron is required for proteins involved in the important processes of carbon and nitrogen assimilation. Low concentrations of iron in cultures or natural waters can lead to iron limitation which affects many aspects of algal metabolism. In natural waters, iron limitation can have effects on the patterns and rates of primary productivity.
The cellular content of certain proteins can be affected by media iron concentrations. Methods have been used that assay components of the cell as an indirect measure of iron nutritional status. For example, spectroscopy can be performed to determine the cellular concentration of iron-containing proteins involved in photosynthesis. Organisms grown in media that imitate natural conditions, or organisms collected from their natural habitat are usually dilute. Methods that assay iron nutritional status such as spectroscopy and column chromatography require large sample sizes which are difficult to obtain from natural samples. In addition, methods that utilize techniques such as immunology or radioactive labelling are complex and time-consuming. These considerations led to the necessity of developing a technique that would be simple, rapid and effective on dilute samples. The method developed here utilized fast protein liquid chromatography (FPLC), which fulfilled these requirements. A complete analysis could be done within two to three hours with minimal sample treatment. The FPLC was simple to operate and was effective on a sample containing less than 100 μg of protein.
Some photosynthetic organisms, when iron-depleted, can produce the flavin-containing protein flavodoxin (Flv). This protein substitutes for the iron-containing protein ferredoxin (Fd) in Fd-dependent reactions such as the light-induced reduction of NADP. The FPLC technique identified and quantified, in relative terms, Fd and Flv in the cell. Optical spectroscopy was used to verify FPLC retention time assignments. The results illustrated how the FPLC could be used to observe the changes in relative Fd and Flv content as a function of media iron concentration in cultures of the cyanobacterium Anabaena grown in the laboratory. It was found that Fd content decreased and Flv content increased with decreasing media iron concentration. In addition, samples of the cyanobacterium Trichodesmium collected from the ocean near Barbados were analyzed using FPLC to assay relative Fd and Flv content. By analogy with Anabaena, Fd and Flv retention times were identified. Using this technique conclusions could be drawn regarding the changing iron nutritional status of Trichodesmium in its natural habitat .
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Ashayeri, Diane L. "Soy protein-xanthan gum interaction:stability and rheology." Thesis, Virginia Polytechnic Institute and State University, 1987. http://hdl.handle.net/10919/91074.
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This study investigated the effects of ionic strength, pH, gum concentration, and protein type on protein - xanthan gum interactions. Commercial soy sauce and tamari sauce as well as model systems of soy protein isolate and whey protein concentrate were the sources of protein used for evaluation with xanthan gum.
Preliminary research indicated that when either soy sauce or tamari sauce were mixed with xanthan gum, stable solutions with notable viscosity synergisms resulted. The soy protein and whey protein systems were subsequently prepared with a range of 0 to 5% added sodium chloride. Results indicated that an equilibrium existed between proteins and xanthan gum such that increased sodium chloride initially increased solution stability; but when in excess, the sodium chloride led to a loss of protein - xanthan gum solution solubility and in some cases to precipitation. Precipitation was also noted at the pH extremes of 2,3, and 9 and when xanthan gum was present in excess, or at 0.25%.
The effects of sodium chloride, protein type, and pH on the rheological parameters of model solutions were also examined. Higher sodium chloride levels yielded greater viscosity synergisms. Those solutions made.with intact protein were generally higher in apparent viscosity than similar solutions made with hydrolyzed protein. Solutions at pH 5 were generally higher in viscosity than were similar solutions at pH 7.
Several factors that appeared to affect the stability, solubility, and the rheological parameters of protein - xanthan gum solutions were sodium chloride concentration, gum concentration, pH, and protein type.M.S.
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Malkin, Douglas Scott, and Douglas Scott Malkin. "An Investigation of a Novel Monolithic Chromatography Column, Silica Colloidal Crystal Packed Columns." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/193936.
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Many researchers have investigated ways to improve the separation power of conventional chromatography, most notable is the development of ultra-high performance liquid chromatography (UHPLC). However, only slight improvements in separation efficiency have been achieved up to this point, and unfortunately, modern reversed phase liquid chromatography (RPLC) methods do not have high enough resolving power to analyze complex proteomic mixtures.Uniformly sized silica particles from 10 nm to 1 micron are known to self-assemble into a highly ordered face centered cubic crystal. Silica colloidal crystals have shown recent promise in biological applications such as permselective nanoporous membranes, DNA sieving, reversed phase separation of small molecules on planar substrates, protein sieving, microarrays, total internal reflection fluorescence microscopy of live cells, and 3-D scaffolds for supported lipid films. In this work, silica colloidal crystals packed in capillaries are explored for their potential improvement in the efficiency of reversed phase chromatography.The silica colloidal crystal columns were chemically stabilized by with trichlorosilanes. The trichlorosilanes form chemical bonds between the particles and the particles and the substrate creating an increase in mechanical stability, and at the same time, providing an excellent chromatographic monolayer. After stabilization the fritless columns were able to withstand the pressure limit of the commercial UHPLC. Next, the columns were characterized using a small dye molecule, 1,1' - Didodecyl - 3,3,3',3' - tetramethylindocarbocyanine (DiIC12). The dye was run under capillary electrochromatography (CEC), and sub-micron plate heights were achieved. Further, a van Deemter plot of the dye molecule indicates that the plate height is largely due to the molecule's diffusion. This result suggests that the plate heights for proteins would be even smaller, since proteins have diffusion coefficients an order of magnitude smaller. The analysis of proteins by CEC yielded nanometer plate heights. Finally, pressure driven flow separations coupled with nano-electrospray ionization (n-ESI) MS have also been explored. The Poiseuille flow profile has been shown not to perturb the low plate heights. Gradient elution of peptides was also achieved, and the results demonstrate the highest chromatographic peak capacities for short analysis times to date.
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Weinberg, Justin B. "Competitive IgG Adsorption on Protein A Chromatography Resins and Improving Resin Performance with PEGylated Ligands." Research Showcase @ CMU, 2017. http://repository.cmu.edu/dissertations/1075.
Full textAbstract:
Protein A (ProA) chromatography is a bioseparations technique employed throughout the biopharmaceutical industry for the selective capture and purification of IgG-class monoclonal antibodies (mAbs) and Fc-fusion proteins. The rapid growth of mAbs as commercial therapeutics has motivated the need for improved, efficient, and high-throughput purification processes during manufacturing. In direct response, the work presented in thesis aims to 1) increase the scientific community’s understanding of IgG adsorption behavior on ProA chromatography resins and 2) improve the performance of ProA chromatography with ligands that are chemically modified using polyethylene glycol (PEGylated). The results of this thesis suggest that IgG molecules of varying binding strength, or varying elution pH, are capable of competing for binding sites on ProA chromatography resins in simultaneous or sequential adsorption. The competitive phenomenon derives from variance in IgG binding strength, or IgG elution pH, due to differences in sub-class behavior as well as secondary IgG binding interactions with the ProA ligand. Competition is readily apparent in the adsorption of human polyclonal IgG, which has a wide variety of IgG sub-classes and binding epitopes. Additionally, the results presented in this thesis suggest that ProA chromatography resins with PEGylated ligands are a viable path to increase resin robustness and real-world chromatographic selectivity. It is demonstrated that ligand PEGylation can increase resistance to proteolytic digestion, mitigate impurity interactions with mAbs that are bound to ProA, and increase process selectivity against Chinese Hamster Ovary host cell proteins by up to 37%. However, resins with large volumes of conjugated PEG significantly decrease IgG static binding capacity and decrease the available pore space for diffusion, resulting in losses in dynamic binding capacity and productivity. Lighter modifications appear to avoid losses in dynamic binding capacity, however, they do not appear to be effective at mitigating impurity interactions with mAbs that are bound to ProA, which is key to increasing process selectivity. PEGylation of ProA also universally increases the elution pH of IgG molecules by weakening the binding interaction. This last result opens another path of viability for PEGylated ProA ligands for purification of mAbs of Fc-fusion proteins that are sensitive to low pH environments.
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Jayapalan, Swapna. "Purification and structural analysis of Newcastle disease virus V protein and flowering locus T (FT) protein." Master's thesis, Mississippi State : Mississippi State University, 2007. http://library.msstate.edu/etd/show.asp?etd=etd-09242007-090456.
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Holler, Christopher J. "Purification of an acidic recombinant protein from transgenic tobacco." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/32379.
Full textAbstract:
Tobacco has been studied as a host for producing recombinant therapeutic proteins on a large-scale, commercial basis. However, the proteins expressed in tobacco usually need to be purified to high yield and purity from large amounts of biomass in order for their production to be commercially viable. The methods needed to purify proteins from tobacco are very challenging and not well studied. The objective of this research was to develop a process for the purification of the acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco leaf tissue to high yield and purity.
Polyelectrolyte precipitation with polyethyleneimine (PEI) was identified as an initial purification step for purifying acidic recombinant proteins from tobacco. Polyethyleneimine precipitation allowed for high recovery and concentration of the target protein while removing large amounts of impurities from the initial extract. At dosages of 700-800 mg PEI/g total protein, nearly 100% of the rGUS activity was precipitated with generally more than 90% recovered from the pellet. In addition, more than 60% of the native tobacco proteins were removed in the process, resulting in a purification factor near 4.
Recombinant GUS was further purified by a step of hydrophobic interaction chromatography (HIC) followed by a step of hydroxyapatite chromatography (HAC). The HIC step served to remove PEI and other contaminants such as nucleic acids that were accumulated during the precipitation step, while the HAC step served to separate rGUS from the remaining native tobacco proteins, most notably ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). Nearly 40% of the initial rGUS activity was recovered as a near homogeneous fraction based on SDS-PAGE analysis after the three step process.
The main steps used in this process are anticipated to be scalable and do not rely on affinity separations, making the process potentially applicable to a wide variety of acidic recombinant proteins expressed in tobacco as well as other leafy crops.
Master of Science
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Brestrich, Nina [Verfasser], and J. [Akademischer Betreuer] Hubbuch. "Development of process analytical technologies for chromatography based protein purification / Nina Brestrich. Betreuer: J. Hubbuch." Karlsruhe : KIT-Bibliothek, 2016. http://d-nb.info/1108450806/34.
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Lan, Chi-Wei. "Protein purification using fluidised bed chromatography : physical and biochemical characterisation of a simple adsorbent contactor." Thesis, University of Birmingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368474.
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