Дисертації з теми "Recombinant proteins Purification"
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Rutt, George Clifford. "Purification of recombinant proteins." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/42614.
Повний текст джерелаDitsch, Andre (Andre Paul). "Purification of recombinant proteins with magnetic nanoclusters." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/34160.
Повний текст джерелаIncludes bibliographical references.
This thesis focused on the development and analysis of a new class of magnetic fluids for recovery of recombinant proteins from fermentation broth. Magnetic fluids are colloidally stable dispersions of magnetic nanoclusters in water that do not settle gravitational and moderate magnetic fields due to their small size. The magnetic nanoclusters possess large surface area for protein adsorption without any porous structure, resulting in much faster mass transfer than in traditional separations. The magnetic nanoclusters consist of 25-200 nm clusters of 8 nm magnetite (Fe₃0₄) cores coated with poly(acrylic acid-co-styrenesulfonic acid-co-vinylsulfonic acid). For use in separation, clusters must be recoverable from solution. Individual nanoparticles are too small to be recovered efficiently, while 50nm or larger clusters of primary particles are easily recovered. Cluster size depends on polymer molecular weight and hydrophobicity as well as the amount of polymer present at nucleation. When a polymer coating with optimal molecular weight is used in limited amounts, clusters are formed. When the clusters are subsequently coated with additional polymer, the clusters are stable in high ionic strength environments (>5M NaCl), while retaining the necessary cluster size for efficient magnetic recovery.
(cont.) Models have been developed to predict the optimal molecular weight, and the cluster size obtained with limited amounts of polymer or polymers other than the optimal molecular weight. The models and methods have been verified with other polymer coatings, indicating that the methods can be used to synthesize a wide range of stable nanoclusters. Due to rapid mass transfer, the rate-limiting step of the purification scheme is recovery of the nanoclusters from solution with high gradient magnetic separation (HGMS). The nanoclusters can be recovered extremely efficiently, up to 99.9% at high flow rates, up to 10,000 cm/hr. A detailed model of HGMS has been developed to quantitatively predict capture, and simpler methods have been developed to predict the maximum capture and capacity of the column without computationally expensive simulations. The use of the nanoclusters for protein purification was studied both with model proteins the recombinant protein drosomycin from Pichia pastoris fermentation broth. The nanoclusters have high adsorptive capacities of up to 900 mg protein/mL adsorbent, nearly an order of magnitude higher than the best commercially available porous adsorbents. Adsorption can be performed both by ion exchange and hydrophobic interactions, allowing nearly pure drosomycin to be recovered from clarified fermentation broth in a single step.
(cont.) When used in whole cell broth, the nanoclusters bind to proteins on the surface of the Pichia pastoris cells at conditions where drosomycin is bound, limiting the effectiveness of the separation. When proteins are bound at conditions where nanoclusters do not bind to cells, cell clarification and protein purification can be performed in one fast step. A simple model of the cell binding has been developed, providing guidelines for use of magnetic nanoparticles in the presence of cells.
by Andre Ditsch.
Ph.D.
Lee, Jae-Yong. "Expression, purification and interaction analysis of recombinant SRB proteins." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407809.
Повний текст джерелаGaztambide, Danielle A. "Production and Purification of Synthetic Minor Ampullate Silk Proteins." DigitalCommons@USU, 2018. https://digitalcommons.usu.edu/etd/7306.
Повний текст джерелаBiedendieck, Rebekka Katrin Johanna. "Bacillus megaterium versatile tools for production, secretion and purification of recombinant proteins /." kostenfrei, 2007. http://www.digibib.tu-bs.de/?docid=00018998.
Повний текст джерелаMiozzi, Jackelyn. "Column-free Purification Method for Recombinant Proteins Using a Self-Cleaving Aggregating tag." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu15229401048581.
Повний текст джерелаRamey, Aaron Thomson. "Optimizing production methods for artificial silk proteins through bioreactor and purification studies of recombinant proteins expressed from Pichia pastoris." Connect to this title online, 2006. http://etd.lib.clemson.edu/documents/1175185569/.
Повний текст джерелаRiddle, Suzette Renee. "Purification and characterization of two recombinant proteins: Annexin III and phosphatidyl inositol specific-phospholipase C /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487943610782957.
Повний текст джерелаKavoosi, Mojgan. "The CBM9 fusion tag : a new technology for inexpensive production and affinity purification of recombinant proteins." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31363.
Повний текст джерелаApplied Science, Faculty of
Chemical and Biological Engineering, Department of
Graduate
Bandmann, Nina. "Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification." Doctoral thesis, Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4318.
Повний текст джерелаZhou, Yinhan. "Expression and functional characterization of the recombinant spider protein GW2 in yeast Pichia pastoris." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/193.
Повний текст джерелаGrewal, Tarlochan Singh. "Expression and purification of recombinant human proopiomelanocortin proteins in Escherichia coli : a combined biological and immunological approach." Thesis, University of Reading, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306366.
Повний текст джерелаKaul, Anita. "Rationale for the selection of aqueous two-phase systems for the purification of recombinant proteins expressed in Escherichia coli." Thesis, University of Reading, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307797.
Повний текст джерелаBelchior, Gustavo Gross. "Geração de clones de células HEK293 superprodutores de isoformas recombinantes de VEGF-A (Fator de Crescimento Endotelial Vascular A) humano visando à produção de biofármacos para terapia molecular e engenharia tecidual." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-01102014-145602/.
Повний текст джерелаThe first blood vessels of the vertebrate embryo are formed de novo from mesoderm-derived cells and give rise to lymph vessels in a process termed vasculogenesis. In the adult, new blood vessels are formed mainly through angiogenesis (or lymphangiogenesis) from the pre-existing vasculature. In healthy individuals, the vascular architecture is fairly static, and both the excess and the insufficiency of vessels comprise a pathological angiogenic state, to which is credited the onset and/or progression of several diseases such as cancer, age-related macular degeneration, limb ischemia, and many others. Therefore, locally controlling the blood vessel density becomes interesting for the treatment of pathological conditions aiming at prognosis improvement and cure. Among the various known growth factors, the vascular endothelial growth factor, VEGF, stands out as the major regulator of the angiogenic process. This process is mediated through the action of pro- (VEGFxxx) and antiangiogenic (VEGFxxxb) isoforms, which are derived from the VEGF-A gene. Consequently, the proteins encoded by this gene are potential therapeutic targets. In this work, we set out to produce the recombinant protein isoforms rhVEGF165, rhVEGF165b, and rhVEGF121, which originate from the human VEGF-A gene, with the aim of generating biopharmaceuticals to be used for molecular therapy and tissue engineering. The rhVEGF165 and rhVEGF121 coding sequences were amplified from total cDNA sythesized from human lung total RNA. Conversely, the rhVEGF165b coding sequence was generated by site-directed mutagenesis of the rhVEGF165 sequence. The sequences were cloned into the pGEM®-T Easy cloning vector. These cDNAs were then subcloned into pLV-eGFP, a plasmid lentiviral transfer vector that allows for expression of transgenes and the eGFP reporter protein in mammalian cells. Human HEK293 cells cultivated under adherent conditions were independently co-transfected with each of the obtained constructs (pLV-rhVEGF165, pLV-rhVEGF165b, and pLV-rhVEGF121) and the pTK-Hyg vector at a proportion of 40:1 (m/m), enabling for the selection of transfectants with hygromycin B, apart from the detection of eGFP. The cell clones overexpressing the proteins of interest were evaluated for expression kinetics in serum-deprived conditioned media and adapted to static suspension culture in medium free of animal-derived components, demonstrating that expression of the protein isoforms was possible in these culture conditions. To the best of our knowledge, this is the first work that describes the expression of VEGF-A isoforms in HEK293 cells in suspension culture. The rhVEGF165 and rhVEGF165b isoforms were purified by affinity chromatography from media previously conditioned by the overexpressing cell clones. The biological activity of rhVEGF165 was demonstrated in vitro, by the AngioPhaseTM Kit assay, and in vivo with the CAM (chorioallantoic membrane) assay, both of which are suitable for evaluating the pro- and antiangiogenic activity of different compounds. The rhVEGF165b did not show the expected antiangiogenic activity. These isoforms were tested in a model of murine tissue-engineered small intestine, indicating a possible contribution to therapeutic use in this context. The purification of rhVEGF121, as well as the structural analysis of all three proteins, are in the process of being optimized.
Silva, Marcelo da. "Clonagem, expressão e purificação das proteínas de superfície, PsaA e fragmentos de PspA de Streptococcus pneumoniae." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-27102005-163558/.
Повний текст джерелаStreptococcus pneumoniae is the main causative agent of bacterial pneumonia. The current vaccines available contain capsular polysaccharide conjugated or not with carrier proteins. However these are either too expensive or do not protect the high-risk groups. Surface proteins of S. pneumoniae, such as PsaA and PspA, are considered strong vaccine candidates. With the aim of developing a broad-coverage and low-cost vaccine against pneumococcus, the psaA and pspA genes were cloned in E. coli expression vectors, pAE and pET and the expressed proteins were purified through affinity and anion exchange chromatography. The yield of the recombinant protein obtained with the construction based in pET was 3-fold higher than that obtained with pAE. Culture conditions were established using defined media with IPTG and/or lactose induction. The recombinant strains are now ready to undergo studies for scale-up of production in bioreactors.
Fonseca, Marisa Cristina da. "Produção de estreptavidina recombinante pela levedura Pichia pastoris." Universidade Federal de Viçosa, 2006. http://locus.ufv.br/handle/123456789/5386.
Повний текст джерелаCoordenação de Aperfeiçoamento de Pessoal de Nível Superior
Streptavidin has been exploited as affinity tag to isolate protein in biotinilated columns. Recombinant Pichia pastoris KM71/Stp strains containing the streptavidin core gene were cultured in fed-batch generating 150 g L-1 biomass. This biomass was achieved with a simpler and shorter process that has never been reported. The yeast was pre-cultured into 50 mL minimum medium with glycerol as only carbon source at 25ºC and 250 rpm. After 12 hours incubation, the culture was transferred to a bioreactor with 200 mL of the same initial A600 0,2. After 24 hours incubation the culture was fed-batch with glycerol and basal salts medium to reach 400 mL. The glycerol concentration of 2.0 moles. L-1 combined with a flow of 0.11 mL. min-1 and aeration by air injection dispersed with a porous stone and magnetic stirring of 500 rpm were the set of conditions to yield maximum biomass. The streptavidin concentration at the supernatant of the free cell culture at 96 hours, the maximum induction period, has achieved 4.0 g. L-1, reducing to 3.2 and 0.87 g. L-1 with two reutilizations. At the same time period the immobilized culture yield 75 %, 50 % and 80% less at the first, second and third culture utilization, respectively. The immobilization and recycling of recombinant P. pastoris biomass can prove to be a potential strategy to improve volumetric productivity.
Com o intuito de utilizar estreptavidina como alvo para isolar proteínas de interesse numa coluna biotinilada, P. pastoris KM71 recombinante contendo o gene do core da estreptavidina, foi cultivada em regime de batelada alimentada na fase de produção de biomassa, alcançando uma concentração de 150 g L-1. Essa biomassa foi alcançada com um novo protocolo, que além de reduzir os passos, introduziu um aparato simples, mas eficiente de dispersão de ar. Um reator com capacidade para 1 L, com 200 mL de meio mínimo com glicerol, foi inoculado com uma pré-cultura para uma A600 inicial de 0,2. Após 24 horas de incubação, a 25 ºC, 500 rpm e injeção e dispersão de ar através de pedra porosa, a alimentação foi iniciada com meio de sais basais e glicerol até atingir um volume de 400 mL. A concentração de 2,0 moles L-1 de glicerol e fluxo de 0,11 mL min-1 utilizados na alimentação, permitiram obter máxima biomassa. Na fase de indução de estreptavidina, foi estudada a reutilização da biomassa na produção de estreptavidina em duas condições: livres em suspensão e imobilizadas em partículas de alginato de cálcio. Em ambos os casos a proteína produzida apresentou-se biologicamente funcional, exibindo ligação esperada à biotina. A concentração de estreptavidina no sobrenadante da cultura de células livres no período de máxima indução (96 horas) atingiu 4,0 g L-1, reduzindo para 3,2 e 0,87 g L-1 respectivamente em duas reutilizações. Quando comparada às concentrações de estreptavidina obtidas pelas células livres em cada utilização, a imobilização resultou na produção de 75% na primeira utilização, 50% na segunda, mas alcançando quase 80% na terceira utilização. A imobilização e a reutilização da biomassa de P. pastoris recombinante ainda não haviam sido reportadas e a produção de estreptavidina nessas condições demonstrou ser uma técnica em potencial.
Robic, Goran. "Soja como biorreator : estudo de extração e purificação de proteina recombinante utilizando 'beta'-glucuronidase." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/267421.
Повний текст джерелаDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
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Resumo: Os trabalhos realizados utilizando plantas transgênicas como biorreatores para produção de proteínas recombinantes indicam que a cano Ia, o milho e a soja são candidatos de grande potencial. Apesar da semente de soja e suas proteínas serem sistemas bem estudados, não existem estudos sistemáticos e comparativos sobre da utilização da soja como um biorreator, no tocante à extração e purificação de proteínas recombinantes. Até hoje, segundo a literatura consultada, só existe uma tentativa de usar soja como biorreator (Russel et al., 2005), mas por causa da baixa expressão da proteína recombinante (hormônio de crescimento humano), este estudo aparentemente não teve continuidade. O objetivo deste trabalho foi avaliar, sob o ponto de vista de recuperação (extração) e purificação, sementes de soja como biorreatores para produção de proteínas recombinantes, usando b-glucuronidase recombinante (rGUS) como proteína modelo
Abstract: The work done on the field of using trasgenic plants as bioreactors indicates the soybean, canola and corn as a plants of choice. Although the extraction and purification of soybean seed proteins is well studied and the plant is relatively easy to transform, there is practically no study done with transgenic soybean seeds expressing recombinant proteins in terms of downstream processing. To our knowledge, soybean was used to produce human growth hormone (Russel et al., 2005), but the study of purification was not done due to the low expression level of that recombinant protein. The objective of this work to evaluate the soybean seeds, in terms of recuperation (extraction) and purification, as a bioreactor for production of recombinant proteins using b-glucuronidase (rGUS) as a model protein
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química
Abdullah, N. "Strategies for expanded bed purification of recombinant protein." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595323.
Повний текст джерелаHoller, Christopher J. "Purification of an acidic recombinant protein from transgenic tobacco." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/32379.
Повний текст джерелаPolyelectrolyte 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
Dwivedi, Gaurav Dutta. "Cloning and Expression of Streptococcal Recombinant Protein G." Thesis, Umeå universitet, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-106723.
Повний текст джерела
Luther, Kelvin B. "Expression and purification of recombinant HIV-1 BH10 Tat protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ62787.pdf.
Повний текст джерелаGanne, Géraldine. "Etudes structurales et fonctionnelles de lectines et d'adhésines chez Pseudomonas aeruginosa." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00949168.
Повний текст джерелаSloane, Rhona Patricia. "Construction, expression, and purification of a histidine-tailed bacteriophage T4 lysozyme." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243912.
Повний текст джерелаMattsson, Johanna. "Purification of the recombinant SAD-C protein from Pisum sativum (pea)." Thesis, Örebro University, Institutionen för naturvetenskap Department of Natural Sciences, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-2201.
Повний текст джерелаSAD-C, a gene belonging to the small short-chain alcohol dehydrogenase-like protein (SAD) gene family, is up-regulated in Pisum sativum (pea) when the plant is exposed to UV-B (280-320 nm) radiation. SAD-C has a molecular weight of about 28 kDa and adopts a tetrameric structure. The aim of this work was to purify the protein SAD-C from Pisum sativum when overexpressed in E. coli strain BL21 StarTM (DE3) One Shot®.
The purification was facilitated by the presence of a His-tag consisting of six histidine residues at the C-terminal end of the protein. The purification trials of SAD-C were faced with problems since the sample fractions contained several other proteins as well. Several purification steps seem to be necessary for future trials. A crystallization trial was still set up and crystals were formed, but the crystals formed were probably not of SAD-C.
Lai, Wen Bin. "Purification and refolding of recombinant viral protein expressed in Escherichia coli." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619606.
Повний текст джерелаHan, Tzu-Chiang. "Control of Intein-Mediated Self-Cleaving Tag for Recombinant Protein Purification." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1453206997.
Повний текст джерелаNguyen, Tien Cuong, Thi Tuyen Do, Thi Hien Trang Nguyen, and Dinh Thi Quyen. "Expression, purification and evaluation of recombinant L-asparaginase inmehthylotrophic yeast Pichia pastoris: Expression, purification and evaluation of recombinant L-asparaginase in mehthylotrophic yeast Pichia pastoris: Research article." Technische Universität Dresden, 2014. https://tud.qucosa.de/id/qucosa%3A29113.
Повний текст джерелаL-asparaginase (EC 3.5.1.1), một loại enzyme được sử dụng trong điều trị bệng ung thư bạch cầu mãn tính ở trẻ em. Mục tiêu của nghiên cứu này là biểu hiện và đánh giá hoạt tính thủy phân của L-asparaginase mã hóa bởi đoạn gene (X12746) tương ứng từ Erwinia chrysanthemi NCPBB1125 được biểu hiện trong nấm men Pichia pastoris. Gene đã được cắt signal peptide và biểu hiện trong P. pastoris SMD1168 and X33. Qua phân tích kết quả điện di SDS-PAGE của môi trường sau lên men, L-asparaginase tái tổ hợp được tìm thấy trong dịch ngoại bào của P. pastoris. Với khả năng sản xuất protein có hoạt tính cao hơn so với chủng P. pastoris X33, SMD1168 được lựa chọn để biểu hiện L-asparaginase tái tổ hợp. Sau khi tinh sạch, sự xuất hiện của một băng có kích khối lượng phân tử xấp xỉ 45 kDa trên điện di SDS-PAGE cho thấy protein tái tổ hợp đã bị glycosyl hóa với hoạt tính riêng 6.251 Umg-1 và đạt độ sạch 3.471 lần.
Miladi, Baligh. "Développement d’outils moléculaires de production et de purification de protéines recombinantes par suivi en temps réel." Thesis, Cergy-Pontoise, 2011. http://www.theses.fr/2011CERG0533/document.
Повний текст джерелаIn recent years, the need for recombinant proteins has substantially increased in various bio-industry activities. However, actual recombinant processes are still limited by the lack of markers allowing real-time expression and purification monitoring of target proteins, by inclusion bodies formation and by low quality of purity of the products. To overcome these difficulties, we have developed a new process for production and purification of recombinant proteins in Escherichia coli. The method combines the use of a multifunctional expression cassette, termed Multitags and an immobilized modified TEV protease on a streptavidin matrix. The Multitags contains, its N-terminus, two affinity purification tags (10xHis and SBP) and as a marker tag, the heme-binding domain of cytochrome b5 followed by the TEV cleavage site. Using two model proteins (MyRIP and Pfu DNA polymerase), we have demonstrated the visual and the quantitative monitoring capability of the cytochrome b5 during the expression and purification steps. When expressed in E. coli KRX more than 90% of both fusion proteins were produced in a soluble form. In addition, high purity (99%) of Multitags-MyRIP and Multitags-Pfu was achieved after two consecutive affinity purification steps using the dual affinity tag. We also produced the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence and realized their affinity immobilization on a streptavidin-agarose matrix. The characterization of the proteolytic columns and their application to the recombinant model proteins demonstrated the advantage of this immobilization method in terms of retaining activities, enzyme stabilities, possibility of reuse and simplification of the cleavage downstream steps.In conclusion, this study allowed the development and the validation of innovative tools for expression and purification of recombinant proteins
Nguyen, Tien Cuong, Thi Tuyen Do, Thi Hien Trang Nguyen, and Dinh Thi Quyen. "Expression, purification and evaluation of recombinant L-asparaginase in mehthylotrophic yeast Pichia pastoris." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-190837.
Повний текст джерелаL-asparaginase (EC 3.5.1.1), một loại enzyme được sử dụng trong điều trị bệng ung thư bạch cầu mãn tính ở trẻ em. Mục tiêu của nghiên cứu này là biểu hiện và đánh giá hoạt tính thủy phân của L-asparaginase mã hóa bởi đoạn gene (X12746) tương ứng từ Erwinia chrysanthemi NCPBB1125 được biểu hiện trong nấm men Pichia pastoris. Gene đã được cắt signal peptide và biểu hiện trong P. pastoris SMD1168 and X33. Qua phân tích kết quả điện di SDS-PAGE của môi trường sau lên men, L-asparaginase tái tổ hợp được tìm thấy trong dịch ngoại bào của P. pastoris. Với khả năng sản xuất protein có hoạt tính cao hơn so với chủng P. pastoris X33, SMD1168 được lựa chọn để biểu hiện L-asparaginase tái tổ hợp. Sau khi tinh sạch, sự xuất hiện của một băng có kích khối lượng phân tử xấp xỉ 45 kDa trên điện di SDS-PAGE cho thấy protein tái tổ hợp đã bị glycosyl hóa với hoạt tính riêng 6.251 Umg-1 và đạt độ sạch 3.471 lần
Granovski, Vladimir. "Purificação de fatores de coagulação VIII e VII recombinantes para o tratamento das hemofilias A e B produzidos a partir de células humanas." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17156/tde-26042018-173449/.
Повний текст джерелаIn this work, several chromatographic methods were studied for the purification of recombinant clotting factors VII (FVIIr) and VIII (FVIIIr) derived from human SK-Hep cell lines. The FVIIIr is used for the treatment of Hemophilia A, while the FVIIr is used for the treatment of Hemophilia B and Hemophilia A. Producing these factors in human cell lines results in glycosylation, sulphation and folding patterns similar to the endogenous factors produced in the human organism. Purification of FVIIIr by multimodal chromatography techniques using CaptoMMC resin, affinity using FVIIISelect resin and ion exchange (SP-Sepharose) yielded a fairly homogeneous and well-defined band profile (by SDS-PAGE) which demonstrated the expected presence of the light and heavy chains, Westen-Blott indicated that commercial antibodies recognized the heavy chain of the studied molecule. The techniques allowed a high reproducibility of the process where purification sequences indicated the same behavior of chromatographic profiles and the process eliminated 99.5% ± 0.5% nonspecific proteins and recovering up to 64% FVIIIr. FVIIr was purified with only a single chromatographic technique using the FVIISelect resin which isolated the protein by removing about 99% impurities and recovering virtually the entire product. The affinity chromatography eluate was dialyzed on 5 kDa membranes which resulted in the autoactivation process of the FVIIr molecule resulting in a signal increase of up to 5 fold over the initial amount. The SDS-PAGE gel and Westen-Blott demonstrated the auto-activation process where a migration of 50 kDa to 30 kDa band was observed and the commercial antibodies against FVII were able to detect the band. The purification method was also quite reproducible and the band profile very similar compared to the commercial products. Thus, it was possible to obtain purification platforms for the FVIIr and FVIIIr proteins.
Selamoglu, Hande. "High-level Expression Of Hepatitis B Surface Antigen In Pichia Pastoris, Its Purification And Immunological Characterization." Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12611390/index.pdf.
Повний текст джерелаDias, Ana Margarida Gonçalves Carvalho. "Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification." Master's thesis, Faculdade de Ciências e Tecnologia, 2010. http://hdl.handle.net/10362/10924.
Повний текст джерелаThe main objective of this work was the development of an affinity pair for the purification of recombinant proteins. In this work, ligands based on the Ugi Reaction and the 1,3,5-Triazine scaffold were synthesised in solid-phase and screened for binding to an affinity tag, an hexapeptide constituted by asparagine aminoacid (N). The ligands were tested against pure solutions of the hexapeptide and Green Fluorescence Protein (GFP), used as a model protein. The ligands that had the highest affinity for the hexapeptide and lowest affinity for the protein were selected for further studies with cellular extracts. The cellular extracts were produced in HEK 293T cells transfected with two designed vectors: one containing the GFP tagged with the affinity tag, and the other containing GFP without tag. The efficient expression of a recombinant GFP fused with the designed affinity tag was demonstrated. The cellular extracts were then loaded onto chromatographic columns containing the lead ligands immobilised onto agarose, and the amount of total protein and GFP bound and eluted noted. The results demonstrated that the Ugi ligands were less selective than the Triazine ligands for the hexapeptide. The triazine ligand 7,4 has been considered as the most selective for the designed affinity tag. In addition, preselected lead ligands for another hexapeptide (RW) of interest were studied. Mammalian cells HEK 293T were transfected with a vector expressing for GFP tagged with this peptide. The ligands immobilized onto agarose were loaded with cellular extracts, being noted that the lead A6C3 showed a high selectivity for the tag tested.
Stimple, Samuel Douglas. "Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1514494485801145.
Повний текст джерелаTheron, Louwrens Wiid. "Expression and purification of recombinant extracellular proteases originating from non-Saccharomyces yeasts." Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/85704.
Повний текст джерелаENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine properties. While the extracellular proteins of Saccharomyces cerevisiae have been characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound molecules. Another category of enzymes of oenological interest is represented by acid proteases that are able to prevent or reduce protein haze, as reported in literature, while simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino acids from peptides and proteins that serve as aroma precursors may also have an indirect effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology (IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384, respectively. However, no further characterization was undertaken. This study aimed to clone these two genes into a recombinant bacterial host for expression and purify the corresponding enzymes as a first step toward characterizing their kinetic properties. Considering that some non-Saccharomyces species have been shown to produce more than one acid protease, an additional aim was to identify novel acid proteases within M. pulcherrima IWBT Y1123. Cloning of the genes and transformation of the expression vectors into E. coli were achieved. Optimal conditions for induced expression were established following extensive optimization. Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity. Purification was followed by refolding experiments, but yielded poor recovery of active enzymes. Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1. Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola IWBT Y1384 revealed that both possess at least one additional protease other than those previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH. Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small groups of proteins thereof did not allow the identification of these enzymes.
AFRIKAANSE OPSOMMING: Gedurende fermentasie van druiwe sap skei gis ekstrasellulêre ensieme af wat ‘n aanmerklike impak op wyn eienskappe het. Terwyl die ekstrasellulêre proteïene vanaf Saccharomyces cerevisiae al gekarakteriseer is, bly die van nie-Saccharomyces spesies grootliks onbekend. Meeste van hierdie ensieme breek suiker polimere af of kataliseer die vrystelling van glikosiediese verbonde molekules. ‘n Ander klas van ensieme wat van belang is vir oenologie word voorgestel deur proteases wat in staat is daartoe om proteïenewaas te verminder, soos voorheen geraporteer is in literatuur, terwyl dit terselfde tyd die assimileerbare stikstof inhoud kan vermeerder. Die vrystelling van aminosure vanaf peptiede en/of proteïene wat as aroma voorlopers dien mag ook ‘n indirekte effek op die wyn se aroma profiel hê. In ‘n onlangse studie wat uitgevoer is by die Instituut vir Wynbiotegnologie (IWBT) was die volgordes van twee aspartiese proteases bepaal vanaf twee nie-Saccharomyces gis spesies wat geisoleer was uit Suid-Afrikaanse wyne. Die gene MpAPr1 en CaAPr1, was afsonderlik geisoleer vanuit twee nie- Saccharomyces giste, Metschnikowia pulcherrima IWBT Y1123 en Candida apicola IWBT Y1384. Egter was daar geen verder karakterisering van hierdie ensieme nie. Die doel van hierdie studie is om die bogenoemde gene in ‘n rekombinante bakteriese gasheer te kloneer vir uitdrukking en suiwering as ‘n eerste stap tot karakterisering van hul kinetiese eienskappe. Om in ag te neem dat sommige nie-Saccharomyces spesies meer as een protease produseer was ‘n aditionele mikpunt om vir nuwe suur proteases te soek binne M. pulcherrima IWBT Y1123. Klonering van hierdie gene en transformasie van die uitdrukkings vektore in E. coli was suksesvol. Optimale kondisies vir die induksie van ekspressie was bevestig na omvattende optimalisering. Verder, terwyl inheemse ekstraksie van die rekombinante proteïene onsuksesvol was, het denatureerende kondisies toegelaat vir suksesvolle ekstraksie, wat voorgestel het dat die rekombinante proteïene geinkapsileer word in inklusie liggame. Rekombinante MpAPr1 was gesuiwer deur gebruik te maak van ‘n niekel gebaseerde kolom sisteem en massa petied fingerafdrukke van die gesuiwerde ensiem (MpAPr1) het die identiteit bevestig. Suiwering was gevolg deur hervouing eksperimente, maar het swak opbrengste gelewer van die aktiewe ensiem. Ongelukkig kon die rekombinante ekspressie van CaAPr1 nie gevisualiseer word nie vir redes wat nog bevestig moet word, maar wat mag behels dat daar groot volgorde veskille tussen MpAPr1 en CaAPr1 kan wees. Uiteindelik was Southern blot hibridiseering analises uitgevoer op die genome van albei M. pulcherrima IWBT Y1123 en C. apicola IWBT Y1384 wat voorgestel het dat albei ten minste een addisionele protease, anders as die wat voorheen geidentifiseer was, bevat. Verder analiese van die ekstrasellulêre proteoom van M. pulcherrima IWBT Y1123 het ook die teenwoordigheid van ten minste een ensiem bevestig wat die vermoë het om BSA te hidroliseer by ‘n lae pH. Ongelukkig het massa peptied vingerafdrukbepaling wat uitgevoer was op die hele ekstrasellulêre proteoom en op klein groepe protein nie identifikasie van hierdie ensieme bevestig nie.
Rochereau, Nicolas. "Rye cell wall β-glucosidase: subcloning, expression and purification of recombinant protein from E.coli". Thesis, Södertörn University College, School of Life Sciences, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-1329.
Повний текст джерелаSeveral plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.
OH, JOONGSEOK. "DESIGN OF RECOMBINANT TENEBRIO MOLITOR ANTIFREEZE PROTEIN FOR PURIFICATION USING ELASTIN-LIKE POLYPEPTIDE TAG." Cleveland State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1409230499.
Повний текст джерелаHo, Christine Kuo. "Expression, purification and characterization of the structural properties of recombinant Pysp1 and Pysp2 spidroins." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/846.
Повний текст джерелаWoolston, Peter William. "A physicochemical database for an expert system for the selection of recombinant protein purification processes." Thesis, University of Reading, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307004.
Повний текст джерелаRoss, Kristin Coby. "Separation of Recombinant β-Glucuronidase from Transgenic Tobacco by Aqueous Two-Phase Extraction". Thesis, Virginia Tech, 2008. http://hdl.handle.net/10919/43471.
Повний текст джерелаMaster of Science
Ho, Christine Kuo. "Expression, purification and characterization of the structural properties of recombinant Pysp1 and Pysp2 spidroins : a thesis." Scholarly Commons, 2001. https://scholarlycommons.pacific.edu/uop_etds/846.
Повний текст джерелаSonnendecker, Christian, Ren Wei, Elisabeth Kurze, Jinpeng Wang, Thorsten Oeser, and Wolfgang Zimmermann. "Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli." Unievrsität Leipzig, 2017. https://ul.qucosa.de/id/qucosa%3A21101.
Повний текст джерелаAzhari, Ala. "Cloning of the Gene, Purification as Recombinant Protein and Functional Characterization of Leishmania mexicana Cytochrome b5 Reductase." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4282.
Повний текст джерелаSouabni, Hager. "Modulation de l’activité du flavocytochrome b₅₅₈ : étude fonctionnelle." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112036/document.
Повний текст джерелаNADPH oxidase complex is a major actor of both antimicrobial host defense and inflammation by generating highly regulated superoxide anion, rapidly converted into reactive oxygen species (ROS). The NADPH oxidase complex consists of a heterodimeric integral membrane flavocytochrome b₅₅₈ and three cytosolic components p67phox, p47phox and p40phox, and the small GTP binding protein Rac. In response to a cellular stimulus, cytosolic proteins are recruited to the phagosomal membrane where they are assembled with the Cytb₅₅₈ to form the active NADPH oxidase. The aim of the work was to better understand the modulation of superoxide anion production by this enzyme. For this purpose, we performed experiments with both bovine neutrophil membranes and yeast membranes expressing the bovine recombinant Cytb₅₅₈. We first investigated the effect of the trans-isomerization of the cis-arachidonic acid, the activator of NADPH oxidase in vitro and showed that specific geometry of the activator plays an important role in the activation of the complex. We also studied the role of the membrane environment on the functioning of NADPH oxidase and determined the kinetics and thermodynamics of NADPH oxidase activity depending on the lipid composition of Cytb₅₅₈ proteoliposomes. Comparison with these properties obtained with recombinant Cytb₅₅₈ embedded into endoplasmic reticulum and plasma membranes, we showed that the NADPH oxidase activity is highly temperature dependent and can be modulated by the lipid environment and the physic state of the membrane
Cedergren, Linda. "Expression of recombinant protein including an His-tag to facilitate purification for diagnosis of CCHF and Lassa Viruses." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7064.
Повний текст джерелаAbstract
Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.
Parisien, Albert. "Large-Scale Production in 'Escherichia coli' TG1 and Purification of Llama Single Domain Antibody ToxA5.1 Against 'Clostridium difficile' Toxin A." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26242.
Повний текст джерелаLiu, Jianyun. "Plant-derived Murine IL-12 and Ricin B-Murine IL-12 Fusions." Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/30190.
Повний текст джерелаPh. D.
Andersson, Christin. "Production and delivery of recombinant subunit vaccines." Doctoral thesis, KTH, Biotechnology, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3027.
Повний текст джерелаRecombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated.
Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived fromStaphylococcus aureusprotein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of thePlasmodium falciparumblood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies forin vivoorin vitrolipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived fromToxoplasma gondii. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation.
Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product.
Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy.
We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against theP. falciparumPf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein.
Keywords: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,Staphylococcus aureusprotein A, Subunit vaccine,Toxoplasma gondii
Ferreira, Francisco C. "Periplasmic binding protein FhuD of Escherichia coli K-12 : overexpression in Bacillus subtilis, purification, and renaturation of the recombinant FhuD." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0005/MQ44166.pdf.
Повний текст джерелаOLIVEIRA, JOAO E. de. "Purificacao de hormonio de crescimento humano recombinante obtido no espaco periplasmico de ESCHERICHIA COLI, visando sua aplicacao clinica." reponame:Repositório Institucional do IPEN, 1999. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10755.
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Dissertacao (Mestrado)
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Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
Milne, Trudy Jane. "Purification and characterisation of Tex31, a conotoxin precursor processing protease, isolated from the venom duct of Conus textile." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/16960/1/Trudy_J_Milne_Thesis.pdf.
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