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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.
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Kotlarski, Nicholas. "Process-scale renaturation of recombinant proteins from inclusion bodies /." Title page, contents and summary only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phk87.pdf.
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Kepple, Kevin V. "Analysis of the binding mechanisms and cellular targets of peptide inhibitors that block site-specific recombination in vitro /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3208620.
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Castilho, Alexandra Marina Machado Ferreira. "Molecular cytogenic analysis of recombinant chromosomes in wheat - Aegilops umbellulata lines." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296341.
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Rauf, Femina. "Chimeric and Recombinant Protein Reagents for Cellular Analysis and Immunoassays." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/145441.
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Development of chimeric, recombinant peptides, proteins and enzymes expands the availability of protein/enzyme–based tools for cellular analysis and new assay platforms. Ideal protein reagents for cellular analysis must translocate into a variety of cells with minimum cell damage, retain stability and biological activity within the cell during analysis, and provide a reliable, measurable signal. This work focused on development, characterization and utilization of chimeric recombinant peptide, protein and enzyme reagents for cellular analysis and immunoassays. A cell-penetrating, fluorescent protein substrate (PKAS) was developed to monitor intracellular protein kinase A activity in cells without the need for cellular transfection. PKAS translocated into HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity. Upon cellular loading, glucose dependent phosphorylation of PKAS was observed in both βTC-3 and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6 %) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4 %) in βTC-3 cells was with 3 mM glucose indicating a left-shifted glucose sensitivity. A cell-penetrating luciferase chimera (Luc-TAT) and a cell-penetrating phospholipid nanoshell entrapped luciferase (Luc-PPN) was constructed to monitor dynamic changes in intracellular ATP levels in mammalian cells. Upon cellular loading, the activity of Luc-TAT and Luc-PPN was monitored with time. Luc-TAT lost approximately 50% activity within one hour, and decreased rapidly over time. In contrast Luc-PPNs retain approximately 95% activity in 1 hour and 77% after 12 hours showing longer biological lifetime. Luc-PPNs were able to detect dynamic ATP changes in intact HeLa cells in the presence of KCN and NaN3. The bioluminescence returned to background levels within 8-10 minutes after treatment with KCN, whereas NaN₃ showed ~ 40% reduction. Two novel recombinant human parathyroid hormone (hPTH) analogs hPTHEGFP and hPTH-Cys were prepared to develop immunoassays for PTH detection in clinical samples. Initial experiments show promise for these analogs for use in CZELIF based immunoassays. The analogs present a number of distinctive advantages for clinical assays and can be used to develop several immunoassay platforms.
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Larsen, Sasha Ellen Marie. "Characterization of components that increase secretion of recombinant proteins in pichia pastoris." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/769.
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Pichia pastoris is a methylotrophic yeast that is commonly used for its ability to express and secrete heterologous proteins. However, some proteins are not readily secreted in P pastoris and so adjustments in the secretion pathway must be made in order to achieve secretion. The Lin-Cereghino lab previously developed mutant strains using restriction enzyme-mediated integration that enabled P pastoris to secrete Pgalactosidase at higher levels than the wild type strain. This study focuses on characterizing the random pREMI-Z mutations in the genomic DNA and examining their secretory phenotype, in hopes of creating a super secretor strain. The ah3 mutant was specifically chosen and characterized for its ability to secrete HRP and SLPI proteins and the effect of the pREMI-Z mutation on the morphology of the cells using transmission electron microscopy. An examination into the AH3 protein yielded a comparative B-galactosidase secretion study between the ah3 mutant and ah3 mutant cells transformed with the pKANB-AH3 rescue construct. Lastly, a cell localization experiment was done to examine where the AH3 protein may be found. These experiments help to increase the current understanding of the secretion pathway in Pichia pastoris and serves as an outline of how to characterize other pREMI -Z mutant strains.
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Carre, Heather Emily. "Expression and analysis of recombinant mycoplasma hyponeumoniae proteins as potential subunit vaccine candidates." Thesis, Royal Veterinary College (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522182.
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Moore, Shona. "An analysis of the structure and function of malarial Duffy-binding-like protein domains using recombinant fusion proteins." Thesis, University of Warwick, 2016. http://wrap.warwick.ac.uk/86933/.
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Duffy-binding-like domains are present in two potential malaria vaccine candidates. Located on the merozoite surface, MSPDBL1 and MSPDBL2 have been implicated in erythrocyte invasion and identified as targets of natural immunity. Merozoite DBL domains have been shown to bind the Fc region of natural IgM. This is characteristic of several PfEMP1s, and is also well documented in bacteria, viruses and other parasites, where it is thought to prevent specific binding of the more deadly IgG antibodies. We have developed a mammalian expression system to produce merozoite DBL domains as Fc fusion proteins, facilitating investigation into their adhesive properties. Fc-fusion proteins are composed of the Fc region of IgG fused to a peptide and are a rapidly expanding field of bio-engineering. They have been successful in drug delivery due to their ability to increase serum half-life of the fused protein by the interaction of the IgG Fc with the neonatal Fc receptor (FcRn). Engineering of the Fc scaffold has shown improved receptor binding, allowing cross-linking of Fc receptors for improved vaccine design. The expression of homodimeric DBL-Fc fusions is di cult, evidenced by incorrect folding and low protein yield. A flexible ,extended hinge region was designed to increase the distance between the Fc and the fused DBL domain, and improved protein folding and IgM binding. Further work may optimise this hinge region for the development of malarial vaccines, or therapeutics for IgM-mediated diseases. The structural analysis of all known IgM-binding DBL domains and residues on the merozoite DBL surface predict the involvement of helix 2a in IgM binding. This contradicts a recent homology model of the IgM-binding interaction, and suggests that the model needs revision. An improved DBL-Fc fusion could be used to identify critical binding residues located in this helix using the more focused approach of site-directed mutagenesis.
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Prasad, Alpana. "Immune function and structural analysis of recombinant bovine conglutinin and human lung surfactant protein-D." Thesis, University of Oxford, 2000. http://ora.ox.ac.uk/objects/uuid:f9a5ae66-4ed0-4bdf-90eb-c873ca44147d.
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Recognition of sugar moieties on the surface of microorganisms is one of the ways the body distinguishes potential pathogens from self-cells. The sugarbinding proteins, lectins, mediate this recognition role of the first line of defence against infections, preceding the antibody-mediated (adaptive) immune response. Collectins are calcium-dependent carbohydrate-binding proteins that have been implicated in innate immunity. Bovine conglutinin (BC) and lung surfactant protein-D (SP-D), belong to the family of 'collectins' which are characterised by four domains: an N-terminal cysteine-rich region, a collagenlike region linked with the carbohydrate recognition domain (CRD) via an ahelical neck region. BC and SP-D show remarkable similarity in their amino acid sequence (79% identity), function and biological characteristics. They have been shown to mediate microbial clearance either by directly binding to bacteria leading to phagocytosis or interacting with complement system components. The present study aims to elucidate the biological function of these proteins more precisely. Recombinant fragments (r) of BC and SP-D consisting of their CRDs and neck regions have been cloned in pET-21a and pMal-c2 vectors respectively, for expression in Escherichia coli. Recombinant conglutinin was expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturing procedure. Binding of rBC(N/CRD) to mannan and complement component, iC3b, was assessed in real-time by BIAcore. The dissociation constants were calculated by Scatchard analysis. The carbohydrate structures present on the surface of the microorganisms play an important role in mediating the interactions with the immune cells. The recombinant molecules showed calcium-dependent binding to lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited in presence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed by ELISA and retained its capacity to recognise various complement system components and the carbohydrate moieties on the surface of various pathogenic microorganisms. The recombinant protein retained its ability to bind various sugar residues, although with lower affinity than that of the native molecule. rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination of bacterial cell suspensions. A novel model has been used to describe the interactions of the collectins at the molecular level based on specificity of carbohydrate-recognition by the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeae has sequential deletions of the terminal sugars in their lipooligosaccharides (LOS). Conglutinin showed a preferential high affinity binding to 1291a mutant that expresses GlcNAc as the terminal hexose, in comparison to other mutants. This provides a unique system to understand the specific cell-surface interactions in relevance to a particular lectin. Further elucidation of the function of CRD and neck region at a structural level is in progress, using X-ray crystallography. Since the submission of the thesis, the structure of the monomeric CRD has been solved, which revealed a remarkable similarity to the SP-D and MBL structure. Trials are underway to get the structure of the trimeric CRDs. These studies aim to provide a better understanding of the collectinpathogen interaction at the biological and structural levels. The ultimate aim is to determine if the recombinant forms of these proteins can be used therapeutically to enhance the uptake and killing of pathogens.
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Alodailah, Sattam Sonitan. "The Generation of Recombinant Zea mays Spastin and Katanin Proteins for In Vitro Analysis." Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc1062897/.
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Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning after traditional cloning methods were not successful. The constructs were expressed in E. coli, then the recombinant proteins were purified. To determine if the GST-tagged proteins are functional, ATPase activity and tubulin polymerization assays were performed. While both GST-katanin-p60 and GST-spastin hydrolyzed ATP indicating that the ATPase domains are functional, the results of the tubulin polymerization assays were less clear and further experimentation is necessary.
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Wu, Xiaoqiu. "Functional genomics at the interface of protein expression and biophysical analysis /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-772-X.
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Wangsa-Wirawan, Norbertus Djajasantosa. "Physicochemical properties of protein inclusion bodies." Title page, contents and introduction only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phw2465.pdf.
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Bibliography: leaves 182-198. Improvements in the current production system of inclusion bodies and the downstream processing sequence are essential to maintain a competitive advantage in the market place. Optimisation of fermentation is considered to improve production yield; then flotation as a possible inclusion body recovery method.
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Porter, Alison J. "Analysis of the efficiency of selecting GS-CHO cell lines for cGMP manufacture of recombinant proteins." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.692544.
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The market size for biotherapeutics is growing rapidly and the hunt is always on for the next blockbuster drug. A wide range of cell phenotypes are obtained during the process of generating mammalian cell lines suitable for the production of a biotherapeutic such as a monoclonal antibody. A strategy is therefore required for the selection of a cell line with ’desirable’ characteristics, from a population of closely related cell lines, for the successful manufacture of the biotherapeutic. As this process is included in the sequence of activities which determines the minimum time to start cGMP manufacturing of the biotherapeutic, companies are actively seeking to improve its efficiency. Stringent examination of a typical selection strategy revealed that it was capable of identifying high producing cell lines. However, it only isolated a fraction of the highest producing cell lines and ranking positions between assessment stages that form the selection strategy were not consistent. These results suggested that there was potential to improve selection strategies. Models of alternative selection strategies, built by in silico analysis of data typically collected during cell line construction, were not able to identify ’better’ cell lines. Alternative markers for cell lines capable of achieving high product concentrations could be used to improve selection strategies. In this study, parameters that showed a relationship with recombinant product concentration were identified, suggesting the possibility for development of more predictive assessment criteria. However, results also showed that it was unlikely that a single predictive parameter would be identified. The study also highlighted the affect the environment, which may change between assessment stages, had on cell lines. Closer matching of the cellular environment was therefore also suggested as a means to improve selection strategies. This study has highlighted the importance of designing a selection strategy that is highly predictive of the final production process. In the future, cell line selection strategies are likely to require the incorporation of closer matching of the cellular environment and the use of multiple predictive markers which form profiles of cell lines capable of producing the highest product concentrations.
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Choi, Leo. "Analysis of the effects of spatial localisation of transgenes on expression of recombinant proteins in CHO-DG44 cells." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/analysis-of-the-effects-of-spatial-localisation-of-transgenes-on-expression-of-recombinant-proteins-in-chodg44-cells(76d0a965-9c12-4745-a6a9-ada226dfe2a0).html.
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Chinese hamster ovary cell lines are commonly used as host for production of recombinant protein both in research and in the biotech industry. Recombinant cell lines are generated through random integration of multi-cistronic plasmid vector containing the genes of interest and selection marker gene into the host genome. The recombinant cell lines require several rounds of limiting clonal dilution to isolate stable high expressing clones. Stable high expression of the genes of interest is a rare and desired trait in recombinant clonal cell lines. Despite being clonal, cell lines eventually become heterogeneous and lose productivity. The Mammalian genome resides as packaged chromatin in the nucleus, which is a highly organized structure with specialized spatial and functional compartments. Increasingly, evidence is pointing to the interaction between plasmid and nuclear architecture as a factor that affects expression and stability. This project is based on the hypothesis that certain locations within the three-dimensional structure of the nucleus are more favourable for stable high-level expression of recombinant genes, integration of transgene into these specific location will produce high expressing stable cell linesTo prove the hypothesis a set of far-red reporter gene expressing recombinant clonal cell lines were generated to use as model cell lines. These cell lines were characterized over 80 days of continuous culture to determine their expression and stability. Flow cytometry results showed that all cell lines showed heterogeneity and gradual loss of expression of the far-red gene. Majority of the cell lines loss 80% of their gene copy number by day 25 of continuous culture. The possibility of using telomeric repeat sequences and nuclear bodies as nuclear landmarks was explored in order to develop tools that can be used to study the localization and interactions of the integrated recombinant genes and the CHO genome in the nuclear environment. Telomeric repeat sequences were far too numerous and scattered to use as nuclear landmark. While PML nuclear bodies and Cajal bodies appeared to be randomly positioned. The possibility of cross-species chromosome painting using FACS sorted Chinese hamster chromosome as paints was investigated, results showed that two copies of chromosome 1 and a copy of chromosome 4 along with segments of other chromosomes 7 and X&Y are conserved in the CHO genome. This finding allowed us to further explore the localization of chromosomes in the interphase nuclei. Results showed that the CHO cell equivalent of hamster chromosome 4 does not have a preferred radial localization in the interphase nuclei. FISH data also showed that the site of reporter gene integration is variable and that the integrated plasmid does not have a preferred radial position within the nuclei. These findings indicated that the CHO nuclei in our cell lines do not have a common nuclear organization.
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Greenham, Trevor. "Pointillism in Plant Systems Biology: I. Proteomic Analysis of Plant Exosome-like Particles II. Amyloplast-binding Puroindoline Fusion Proteins for Recombinant Protein Expression." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39647.
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Expanding upon our understanding of plant defense is critical, particularly with the perilous threats of climate change and overpopulation to our food security, health and well-being. In this study, we focused on plant defense using two distinct approaches. First, we performed a proteomic analysis of plant exosome-like nanoparticles in order to elucidate their defense related protein cargo. Secondly, we used a wheat antimicrobial protein, puroindoline, as a fusion partner for the expression of recombinant proteins in rice endosperm.
Plant exosome-like nanoparticles (ELP) were isolated from fresh tomato and subjected to mass spectrometry (MS) analysis. The ELPs were compared to fresh pressed tomato juice, and the proteins that were significantly upregulated in the ELPs were analyzed for their defensive properties. Bioinformatic analysis identified 30 proteins upregulated in the ELPs, with a majority of these being involved in plant defense.
Puroindoline is a protein found in soft wheat varieties. A unique feature of this protein is the presence of a tryptophan-rich domain, which causes it to localize and tether onto starch granule surfaces; a property we are seeking to exploit for recombinant protein isolation. We hypothesized that when expressed in a pin-null crop, such as rice, puroindoline along with its fusion partner will localize and adhere to starch granule surfaces. PIN fusions were expressed in rice, and their subcellular localization was determined by immunolocalization. It was observed that PIN localizes to rice starch
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granules in vitro and in planta, and retains its starch granule binding abilities as a fusion partner.
To identify other possible starch granule binding fusion partners, an anhydrous cleavage method was developed that can scan dry biological materials for associated proteins, in this case the starch granule surface. Incubation of our cleavage reagent with isolated rice starch granules yielded several cleavage products as determined through SDS-PAGE. These cleavage products were compared with previous proteomic data of trypsin digested rice starch granules.
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Pakkanen, O. (Outi). "Assembly and secretion of recombinant human collagens and gelatins in the yeast Pichia pastoris, and generation and analysis of knock-out mice for collagen prolyl 4-hydroxylase type I." Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514281047.
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Collagen molecules consist of three polypeptide chains that are coiled around each other to form a triple-helical structure. The formation of stable collagen triple helices requires the hydroxylation of proline residues catalyzed by collagen prolyl 4-hydroxylases (C-P4H). Vertebrate C-P4H is an ER-resident enzyme that consists of two catalytically active α subunits and two β subunits. Production of recombinant human collagen and gelatin could have numerous medical and industrial applications, but most recombinant systems lack the C-P4H activity. The yeast Pichia pastoris has been successfully engineered to produce stable human collagens and gelatins by co-expression of the collagen polypeptide chains with the two C-P4H subunits.
This study examined the effect of deletion of the C-propeptide, or its replacement by a trimerizing foldon domain, on the assembly of type I and III collagen triple helices in P. pastoris. It was observed that the absence of the C-propeptide leads to inefficient collagen chain assembly whereas the replacement of C-propeptide with a foldon domain increased the assembly up to 3-fold. Moreover, the co-expression of α1(I) and α2(I) chains fused with foldon yielded heterotrimeric type I collagen molecules with a typical chain ratio of 2:1. As the foldon domain contains no information for collagen chain recognition, the present data indicate that the chain assembly is defined not only by the C-propeptides but also by other determinants present in the α chains.
Another aspect studied here was the expression and secretion of gelatin fragments of varying size and conformation in P. pastoris. It was discovered that gelatin fragment size affects its secretion as the 90 kDa fragment was less efficiently secreted than the 45 kDa fragment. Secretion was also dependent on the fragment conformation as induction of the triple helix formation by either C-propeptide or foldon led to the accumulation of the fragments inside the yeast cells despite the presence of an efficient secretory signal.
C-P4H was long assumed to exist as one type only but the cloning of several C-P4H α subunits raised questions concerning the specific roles of the C-P4H isoenzymes. The generation of mice lacking the type I C-P4H, which is regarded as the major C-P4H isoenzyme, indicated that this isoenzyme is essential for the embryonic development of the mouse. The embryos lacking type I C-P4H died at an early stage of their development due to the disruption of basement membranes. It was found that the basement membranes of the homozygous null embryos lacked type IV collagen whereas the fibrillar collagens were synthesized, although with altered morphology. The data reported here also demonstrate that the other C-P4H isoenzymes cannot compensate for the lack of type I isoenzyme.
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Suzuki, Noriaki. "Applications of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and x-ray photoelectron spectroscopy (XPS) to study interactions of genetically engineered proteins with noble metal films /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/10618.
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Coelho, Tatiane Maldonado. "Comparação da atividade biológica e da glicosilação da gonadotrofia coriônica equina recombinante (reCGβα) expressa em duas linhagens celulares de mamíferos visando à geração de um biofármaco." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-20072015-133508/.
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Atualmente, o Brasil encontra-se na privilegiada posição de maior produtor e exportador mundial de carne bovina, tornando a pecuária uma das atividades nacionais mais importantes e rentáveis. Este dado enfatiza a importância de pesquisa e desenvolvimento em reprodução bovina, especialmente em hormônios estimuladores da ovulação, tais como a gonadotrofina coriônica equina (eCG). Os produtos comerciais à base de eCG comercialmente disponíveis são purificados a partir do sangue de éguas gestantes, apresentando variabilidade de lote para lote e presença de contaminantes. Estes fatos, juntamente com a limitação do material de partida (sangue equino), enfatizam a necessidade de haver um sistema de expressão de eCG recombinante passível de ser explorado comercialmente. Neste quesito, as células de mamíferos se mostram um sistema robusto para tal finalidade, visto que são capazes de adicionar modificações pós-traducionais às cadeias polipeptídicas, tais como a glicosilação, o que é essencial para o correto dobramento, maturação e montagem das duas subunidades, além de interferir diretamente com a meia-vida, o reconhecimento do receptor, a solubilidade e a atividade biológica das proteínas. No entanto, mesmo entre os sistemas de expressão heteróloga em células de mamífero, encontra-se muita variabilidade nos padrões de glicosilação adicionado. No presente trabalho, foi realizado um estudo comparativo através da clonagem e expressão de uma forma fusionada de eCG (reCGβα) em duas linhagens celulares diferentes: (1) CHO-DG44, um dos sistemas de expressão mais utilizados pelas indústrias farmacêuticas, capaz de adicionar N-glicanos complexos; e (2) 293T, uma linhagem humana capaz de produzir glicoproteínas carreando oligossacarídeos complexos e sialilados. Os resultados de atividade biológica (in vitro e in vivo) apontam uma maior atividade de reCG produzido por células CHO-DG44. O perfil de N-glicosilação de reCG produzido pelas células CHOD-G44 assemelhou-se mais à eCG selvagem, quando comparado a reCG produzido por células 293T. Por fim, estudos clínicos foram realizados com reCG produzido em meio livre de soro fetal bovino e parcialmente purificado, onde atividade específica de reCG produzido por células CHO-DG44 mostrou-se similar ao produto comercial selvagem.<br>Brazil is currently the major beef producer and exporter, rendering to livestock one of the country´s most economically relevant activities. This emphasizes the importance of research and development in bovine reproduction, especially at ovulation-stimulatory hormones, such as equine gonadotropin (eCG). The commercially available eCG-based products are purified from blood of pregnant heifers, presenting batch-to-batch variability and the presence of contaminants. These facts, together with the limitation of the bulk material (equine blood), emphasize the need of an eCG expression system able to be commercially explored. In this aspect, mammalian cells are a robust system, capable of add post-translational modifications to polypeptide chains, such as glycosylation, which is essential for the correct folding, maturation and assembly of both eCG subunits. In addition, glycosylation directly interferes with the protein half-life, receptor recognition, solubility and biological activity. In the present work, a comparative study was carried out by cloning and expressing a fusion form of eCG (reCGβα) in two different mammalian cell lines: (1) CHO-DG44, one of the most used by pharmaceutical companies expression systems, capable of add complex-type N-glycans; and (2) 293T, a human cell line capable of produce glycoproteins carrying complex and sialylated oligosaccharides. The in vitro and in vivo biological activity results show a higher potency of reCG produced by CHO-DG44 cells. The N-glycosylation pattern produced by CHO-DG44 cells was more similar to native eCG in comparison to the N-glycosylation produced by 293T cells. Finally, clinical studies were performed with serum absent media produced and partially purified reCG, showing that the specific activity of reCG produced by CHO cells was similar to the commercial wild type product.
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Murakaeva, Asiya. "Structure, evolution and expression of the duplicated growth hormone genes of common carp (Cyprinus carpio)." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15982.
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Der Karpfen, Cyprinus carpio, ist eine tetraploide Fischart aus der Familie Cyprinidae, die vor 20-50 Mio Jahren entstanden ist. Das Ziel der vorliegenden Arbeit war der Versuch, die funktionelle Rolle der duplizierten GH Gene des Karpfens durch das Studium ihrer Struktur, Evolution und Expression zu verstehen. Die Introns des zweiten GH Gens des Karpfens wurden erstmalig sequenziert und Sequenzvergleiche der kodierenden und nicht-kodierenden Bereiche von Allelen beider GH Gene wurden vorgenommen. Eine phylogenetische Analyse wurde durchgefuhrt, um die Beziehungen der GH Gene des Karpfens zu denen des tetraploiden Goldfischs und anderer diploider Cypriniden zu untersuchen. Zusatzlich wurden weitere duplizierte Gene des Karpfens, von denen einige auch fur das Wachstum von Bedeutung sind, phylogenetisch analysiert. Der Test der relativen Evolutionsrate nach Tajima (1993) zeigte einen statistisch signifikanten Anstieg der Evolutionsrate des GH I Gens beim Karpfen. Es wurden in der vorliegenden Arbeit einige weitere duplizierte Genpaare des Karpfens und Goldfischs gefunden, die ebenfalls eine Lockerung funktioneller Zwange oder sogar Beweise fur positive Darwin?sche Selektion bei einem der beiden Duplikate zeigen. Der Expressionstest hat gezeigt, dass die GH I und GH II Gene auf identischen Niveaus bei Karpfenbrut exprimiert werden, wahrend bei ein Jahr alten Karpfen, drei Jahre alten Mannchen und Weibchen sowie den 10 Monate alten, an kalte Temperaturen (2°C) angepassten Fischen die Expression von GH II statistisch signifikant geringer war als die von GH I. Es wurde eine neue und einfache Methode zur Herstellung von rekombinanten, biologisch aktiven GH-Proteinen ohne Notwendigkeit des Refolding entwickelt. Sie ermoglicht spatere Tests, ob die Aktivitat von unterschiedlichen GH-Varianten des Karpfens gleich oder unterschiedlich ist.<br>The common carp, Cyprinus carpio, is a tetraploid fish species from the family Cyprinidae that arose about 20-50 Myr ago. The aim of the present work was attempting to understand the functional role of the duplicated common carp GH genes by studying their structure, evolution and expression. The introns of the second GH gene of common carp were sequenced for the first time and sequence comparisons of coding and non-coding regions of alleles of both GH genes were carried out. A phylogenetic analysis was done to examine the relationships of common carp GH genes with GH genes of the tetraploid goldfish and other diploid Cyprinids. In addition, phylogenetic analyses were done with other duplicated genes of common carp, some of which also important for growth. The relative rate test of Tajima (1993) showed a statistically significant increase in the evolution rate of the common carp GH I gene. In addition, some other duplicated gene pairs in common carp and goldfish with relaxation of functional constraints or even evidence of positive Darwinian selection in one of the two gene duplicates were found in the present study. The test of expression rates of the two GH genes has shown that the GH I and GH II genes were expressed at similar levels in carp fry. In contrast, the expression of GH II was statistically significantly lower than that of GH I in one year old carp, three years old males and females as well as in 10 months old fish adapted to cold temperature (2°C). To enable testing the hypothesis if activity of GH diverged between different GH variants of common carp a new and simple method for production of recombinant, biologically active GH proteins without the necessity of refolding was developed.
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Reid, Helen Isobel. "Molecular and immunological analysis of recombinant Bacteroides forsythus heat shock protein 60." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321982.
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Riley, Aidan. "Analysis and exploitation of GPI anchors in the expression of recombinant protein biopharmaceuticals." Thesis, University of Sheffield, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578056.
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In this thesis we show the generation and characteriation of specific GPI-anchored cell surface markers. The creation of these constructs has also allowed the quantification of levels of GPI linked protein shedding into the cell-culture media. Experiments presented in the thesis also show that the addition of a GPI anchor to Growth hormone (GH) appears to reduce expression in the media and that these molecules retain the GPI moiety. We speculated that anchoring cytokine ligands to the cell surface might also modulate receptor mediated signalling. To test the effect of GPI modification on cytokine ligands we fused the GH, and Ob mRNA sequences to the GPI anchor signal sequence of the naturally GPI-anchored protein, Thy-l. To our surprise we found that endogenously expressed GPI anchored cytokines acted as potent non-competitive cytokine receptor antagonists. Furthermore, we demonstrate that GPI anchored GH from a stable CHO cell-line can be purified and reinserted into mammalian cell membranes where they are able to induce receptor mediated signalling. Mammalian cell expression technologies are reliant on methods for obtaining high-yielding stable clones. This thesis describes the development of a high-throughput screening procedure based on the direct pull-down and enrichment of stably transfected cells using magnetic activared cell sorting (MACS). Results presented in this thesis demonstrate that it may be possible to use this procedure to isolate transfected cells from a heterogeneous population and to preferentially recover those rare cell populations which express higher levels of recombinant biopharmaceutical. Thus, GPI anchored markers may be used to facilitating facile cloning and identify population heterogeniety.
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Bruggeman, Andrew. "Generation of fluorescent recombinant listeriolysin O toxin for analysis of interactions with host protein." Connect to resource, 2008. http://hdl.handle.net/1811/32222.
Full text
23
Perry, William B. "Global transcriptional analysis of an Escherichia coli recombinant protein process during hypoxia and hyperoxia." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28666.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004.<br>Includes bibliographical references (p. 289-302).<br>(cont.) The effects of recombinant protein production were observed through expression analysis of induced, uninduced, and Empty-Vector cultures. As expected, recombinant α₁AT production led to increased expression of heat-shock genes, including proteases and chaperones that are known to be involved in α₁AT degradation. Based on expression analysis data, production of recombinant α₁AT also resulted in catabolite repression and decreased amino acid biosynthesis. This work demonstrates the utility of DNA microarrays in analyzing and improving microbial fermentations. Global expression studies have suggested several strategies for increasing the resistance of bioprocesses to the damaging effects of oxygen and recombinant protein production.<br>Both exposure to oxygen and recombinant protein production are known to have adverse effects on microbial fermentation, including increased proteolytic and oxidative damage to the product. In an effort to characterize the effects of these stresses on the cell, DNA microarrays were used to monitor global gene expression of E. coli producing recombinant human αl-antitrypsin (α₁AT) during exposure to defined aeration conditions. Recombinant α₁AT has been shown to undergo oxygen-dependent degradation during production in E. coli, due in part to activation of the heat-shock response. The goal of this work is to better understand the effects of oxygen in order to improve this recombinant protein production process. In order to study the effects of oxygen extremes, global expression analysis was performed on α₁AT-producing cultures exposed to pure nitrogen, air, and pure oxygen. The most notable effects of oxygen exposure were those of superoxide. This reactive oxygen species is generated upon oxygen exposure and is known to oxidize iron-sulfur clusters. In response to hyperoxic conditions, the SoxRS stress response was activated, as were genes involved in iron uptake and the Isc and Suf repair systems for Fe-S clusters. Supplementation of iron in the growth medium resulted in expression changes consistent with improved formation of Fe-S clusters. Iron supplementation also decreased superoxide stress at the expense of a short-term increase in the peroxide (OxyR) stress response. In addition, iron supplementation dramatically reduced the oxygen dependence of recombinant α₁AT degradation. Regeneration of Fe-S clusters is proposed to improve protein folding and clusters is proposed to improve protein folding and limit activation of the heat-shock response.<br>by William Bryon Perry.<br>Ph.D.
24
Yan, Junhong. "DNA-Assisted Immunoassays for High-Performance Protein Analyses." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-236591.
Full textAbstract:
Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.
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Cuccinello, Sarah Elizabeth. "Analysis of Ahr Expression and Stability in a Recombinant Yeast Model System." Scholar Commons, 2011. http://scholarcommons.usf.edu/etd/3053.
Full textAbstract:
The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr*Arnt transcription factor complex. Previous studies have implicated involvement of the 26S proteasome complex in the degradation process, but the exact identity of the intermediary proteins and/or ligases remains to be defined. Identification and characterization of the protein(s) involved in degrading the receptor is essential for understanding the signaling pathway in its entirety including the mechanism for regulating the genetic response to Ahr ligands.
The model organism, Saccharomyces cerevisiae, was used in order to characterize the Ahr signaling pathway and degradation mechanism in a more simplified cellular setting in which the major processes required for growth and development are conserved. First, the AHR and ARNT cDNAs were stably inserted into the yeast genome such that protein expression was inducible. A time course of induction demonstrated detectable levels of Ahr and Arnt proteins via western blotting while protein function was confirmed by detection of ligand-dependent reporter activity in an expressor strain carrying the pLXRE5-Z beta-galactosidase reporter plasmid. Additionally, a rapid reduction in protein levels was observed upon turning off the inducible GAL1 promoter located upstream of both AHR and ARNT cDNAs.
Studies in mammalian cell culture have demonstrated that disrupting receptor chaperoning results in rapid Ahr protein turnover, as demonstrated by treatment with Hsp90 inhibitors. In order to determine if reduced Ahr protein expression in the yeast system was attributed to improper chaperoning of the exogenous protein; human heat shock proteins were constitutively expressed from yeast expression vectors in the Ahr and Arnt expressing strains, but did not confer any effect on Ahr stability when protein levels were evaluated by western blotting. Additionally, a strain of yeast was constructed such that the gene encoding the cell-wall protein, ERG6, was deleted from the yeast genome to allow for permeation of proteasome inhibitors. Treatment of this strain with proteasome inhibitors blocked the receptor degradation, therefore implicating the 26S proteasome in Ahr degradation when expressed exogenously in yeast.
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Celik, Akdur Eda. "Bioprocess Development For Therapeutical Protein Production." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/3/12610236/index.pdf.
Full textAbstract:
In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models.
Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion.
Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h.
Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.
27
Grosse-Holz, Friederike. "Proteases and inhibitors in the interaction between Nicotiana benthamiana and Agrobacterium tumefaciens : systematic analysis and emerging solutions for molecular farming." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:6146918c-3749-4604-88fa-01d426e4a817.
Full textAbstract:
Nicotiana benthamiana is now an established platform for molecular farming, the production of biopharmaceuticals in plants. Infiltration with Agrobacterium tumefaciens (agroinfiltration) is commonly used to transiently express one or multiple transgenes in N. benthamiana leaves. Agroinfiltrated N. benthamiana is a flexible and scalable recombinant protein (RP) production platform, but is impeded by low RP yields. Plant proteases can degrade RPs and thus limit RP accumulation. To inform, design and implement strategies for enhancing RP accumulation, I present four papers about proteases and protease inhibitors in agroinfiltrated N. benthamiana. First, I investigated the transcriptome, extracellular proteome and active secretome to understand the plant response to agroinfiltration and investigate the expressed proteases. I show that an extracellular immune response is mounted at the expense of photosynthesis. Comprehensive annotation and monitoring uncover a large, diverse repertoire of proteases in agroinfiltrated leaves, indicating that broad-range depletion of protease activity may be required to enhance RP accumulation. Second, I reviewed the literature on multifunctional plant protease inhibitors (PIs) and grouped them into three types of multifunctional PIs that evolved independently. Third, I screened candidate PIs and discovered that three new, unrelated PIs enhance RP accumulation. I present universal elements of the RP degradation machinery, uncovering new questions on our understanding of the protease network that degrades RPs. Fourth, I identified targets of SlCYS8, a PI that enhances RP accumulation. The target proteases of SlCYS8 are implicated in RP degradation and the high specificity of SlCYS8 can be used to study their role in other processes. By elucidating the immune response to agroinfiltration, by uncovering the N. benthamiana protease repertoire and by providing new tools to deplete the activity of specific proteases, this thesis makes a relevant contribution to both basic plant research and molecular farming.
28
Simila, Henry Allan. "Recombinant production and in silico analysis of the Androgen receptor ligand binding domain." Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16349/1/Henry_Simila_Thesis.pdf.
Full textAbstract:
The androgen receptor (AR) fulfils important roles for both sexes. By mediating the biological function of androgens, the AR has remained the target for endocrine therapies treating prostate cancer. The AR also determines the effectiveness of medroxyprogesterone acetate (MPA) in treating AR positive breast cancer.
Every man will be affected by prostate cancer if he lives long enough. Prostate cancer continues to be a leading cause of death for males despite research into this cancer covering more than 60 years since Huggins' seminal 1941 study showing that androgens play a key role in this cancer. Unfortunately, significant advances have not been forthcoming and the effect of treatment has remained largely the same over past decades, whereby initial treatment provides temporary remission but eventually advanced cases become refractory to further intervention and the disease recurs in a more aggressive form.
A plethora of factors are exquisitely sensitive to minute changes in the AR's structural profile, which can be altered by a single mutation, resulting in aberrant activity. A principal feature of these variant ARs associated with prostate cancer, is enhanced capacity to bind a number of molecules other than its cognate ligand, dihydrotestosterone (DHT). The promiscuous activity of this receptor leads to continued AR signalling and stimulus for the cancer despite low androgen levels induced by treatment regimes.
A key question is whether mutations occurring within the AR occur as a result of cancer or contribute to the propagation of the cancer. Recent research has demonstrated that treatments incorporating anti-androgens such as flutamide, which are designed to impede prostate cancer progression by inhibiting AR activity, may actually provide selective pressure favouring somatic mutation of the receptor to take place.
The specific changes to the AR which are responsible for gains of function have not been resolved as their crystal structures, which are used to provide conformational analysis of proteins, are tremendously problematic to produce with little success found in literature. Generating representative crystals of the AR protein involves producing soluble recombinant protein. Unfortunately the AR is prone to aggregation and is highly unstable, especially in the presence of antagonistic molecules or absence of a stabilising ligand, preventing the protein from being maintained in the soluble state required for crystallization.
In order to produce sufficient quantities of soluble material for crystallization, the androgen receptor's ligand binding domain (LBD) was produced as a recombinant protein in Escherichia coli bacteria strain BL21 (DE3) in the presence of DHT, flutamide, as well as in the absence of ligand. Since soluble unbound AR-LBD has not been produced until now, the bacterial culture containing no ligand was further processed to the stage of cleaving the purification tag from the recombinant protein and represents considerable progress into producing soluble material for crystallizing the troublesome yet considerably important AR in the absence of ligand.
Although distinct from prostate cancer in males, AR activity in breast tissue is also a factor determining the action of drugs, such as MPA, included in therapies aimed at breast cancer. The use of MPA has declined primarily due to its adverse effects including unsuccessful generation of a biological response, as well as the advent of other drugs administered for hormonal therapies treating breast cancer. Alternative drugs are needed when breast cancer therapies fail as tumours develop resistance to primary drugs. Although there are a number of drugs on the market, success would be maximised if the determined therapy is matched with the patient, based for example, on their genetic makeup. There is a conundrum whereby some patients with an AR do not respond to MPA, a drug normally recognised by the receptor.
In clinical trials it was discovered that an AR with threonine instead of methionine at residue 780 (M780T) fails to activate in response to MPA, but the exact mechanism has remained elusive and needs to be answered at the molecular level. The X-ray crystallographic studies that generate 3D images of macromolecules and wet chemistry, which have traditionally been used to provide insight into science in these dimensions, are incorporated with computer based molecular simulation. This is both complementary and distinct to traditional scientific methodologies, enabling further elucidation of protein-protein interactions, and the influence applied to such inter-relations by natural and drug ligands. This approach has been used, and is continually developed, to understand the binding mechanisms of current drugs as well as designing new drugs.
In order to produce a receptor representing the M780T variant, the crystal structure representing the AR-LBD was mutated in silico, into which MPA was then docked. It was found that MPA binds into the M780T AR-LBD with considerably more spatial displacement compared to the position of DHT in the crystal structure, and is predicted to be the primary reason for the inability of MPA to activate this variant AR.
The clarification of MPA binding and failure to elicit a response from the variant AR is significant for a cohort of breast cancer patients, as not only does the presence of an AR in the tumour determine the effectiveness of MPA, but correct composition of the AR, specifically, the absence of a M780T mutation. In the absence of this AR mutation, MPA could effectively be used either as an alternative to primary drugs, or in secondary therapies when primary therapies fail. Aberrant activity of variant ARs in response to MPA should also be taken into consideration when analysing drug studies about the effectiveness of MPA.
The findings on the loss of response to MPA by the M780T variant AR have been included in the journal article "Decreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate" appearing in the September 2005 issue of Cancer Research journal.
29
Simila, Henry Allan. "Recombinant production and in silico analysis of the Androgen receptor ligand binding domain." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16349/.
Full textAbstract:
The androgen receptor (AR) fulfils important roles for both sexes. By mediating the biological function of androgens, the AR has remained the target for endocrine therapies treating prostate cancer. The AR also determines the effectiveness of medroxyprogesterone acetate (MPA) in treating AR positive breast cancer. Every man will be affected by prostate cancer if he lives long enough. Prostate cancer continues to be a leading cause of death for males despite research into this cancer covering more than 60 years since Huggins' seminal 1941 study showing that androgens play a key role in this cancer. Unfortunately, significant advances have not been forthcoming and the effect of treatment has remained largely the same over past decades, whereby initial treatment provides temporary remission but eventually advanced cases become refractory to further intervention and the disease recurs in a more aggressive form. A plethora of factors are exquisitely sensitive to minute changes in the AR's structural profile, which can be altered by a single mutation, resulting in aberrant activity. A principal feature of these variant ARs associated with prostate cancer, is enhanced capacity to bind a number of molecules other than its cognate ligand, dihydrotestosterone (DHT). The promiscuous activity of this receptor leads to continued AR signalling and stimulus for the cancer despite low androgen levels induced by treatment regimes. A key question is whether mutations occurring within the AR occur as a result of cancer or contribute to the propagation of the cancer. Recent research has demonstrated that treatments incorporating anti-androgens such as flutamide, which are designed to impede prostate cancer progression by inhibiting AR activity, may actually provide selective pressure favouring somatic mutation of the receptor to take place. The specific changes to the AR which are responsible for gains of function have not been resolved as their crystal structures, which are used to provide conformational analysis of proteins, are tremendously problematic to produce with little success found in literature. Generating representative crystals of the AR protein involves producing soluble recombinant protein. Unfortunately the AR is prone to aggregation and is highly unstable, especially in the presence of antagonistic molecules or absence of a stabilising ligand, preventing the protein from being maintained in the soluble state required for crystallization. In order to produce sufficient quantities of soluble material for crystallization, the androgen receptor's ligand binding domain (LBD) was produced as a recombinant protein in Escherichia coli bacteria strain BL21 (DE3) in the presence of DHT, flutamide, as well as in the absence of ligand. Since soluble unbound AR-LBD has not been produced until now, the bacterial culture containing no ligand was further processed to the stage of cleaving the purification tag from the recombinant protein and represents considerable progress into producing soluble material for crystallizing the troublesome yet considerably important AR in the absence of ligand. Although distinct from prostate cancer in males, AR activity in breast tissue is also a factor determining the action of drugs, such as MPA, included in therapies aimed at breast cancer. The use of MPA has declined primarily due to its adverse effects including unsuccessful generation of a biological response, as well as the advent of other drugs administered for hormonal therapies treating breast cancer. Alternative drugs are needed when breast cancer therapies fail as tumours develop resistance to primary drugs. Although there are a number of drugs on the market, success would be maximised if the determined therapy is matched with the patient, based for example, on their genetic makeup. There is a conundrum whereby some patients with an AR do not respond to MPA, a drug normally recognised by the receptor. In clinical trials it was discovered that an AR with threonine instead of methionine at residue 780 (M780T) fails to activate in response to MPA, but the exact mechanism has remained elusive and needs to be answered at the molecular level. The X-ray crystallographic studies that generate 3D images of macromolecules and wet chemistry, which have traditionally been used to provide insight into science in these dimensions, are incorporated with computer based molecular simulation. This is both complementary and distinct to traditional scientific methodologies, enabling further elucidation of protein-protein interactions, and the influence applied to such inter-relations by natural and drug ligands. This approach has been used, and is continually developed, to understand the binding mechanisms of current drugs as well as designing new drugs. In order to produce a receptor representing the M780T variant, the crystal structure representing the AR-LBD was mutated in silico, into which MPA was then docked. It was found that MPA binds into the M780T AR-LBD with considerably more spatial displacement compared to the position of DHT in the crystal structure, and is predicted to be the primary reason for the inability of MPA to activate this variant AR. The clarification of MPA binding and failure to elicit a response from the variant AR is significant for a cohort of breast cancer patients, as not only does the presence of an AR in the tumour determine the effectiveness of MPA, but correct composition of the AR, specifically, the absence of a M780T mutation. In the absence of this AR mutation, MPA could effectively be used either as an alternative to primary drugs, or in secondary therapies when primary therapies fail. Aberrant activity of variant ARs in response to MPA should also be taken into consideration when analysing drug studies about the effectiveness of MPA. The findings on the loss of response to MPA by the M780T variant AR have been included in the journal article "Decreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate" appearing in the September 2005 issue of Cancer Research journal.
30
Khan, Obaid Yusuf. "Construction of recombinant adenoviruses encoding skeletal troponin C protein and expression analyses in transduced cardiac myocytes." Thesis, University of Glasgow, 1998. http://theses.gla.ac.uk/5438/.
Full textAbstract:
Troponin C is a regulatory protein of the myofilament which binds to calcium to trigger the process of contraction. This protein exists in two isoforms, skeletal and cardiac, which are spatially and temporally regulated. Work in this project builds the primary stage of a long-term project, for using the gene transfer method to overexpress the skeletal isoform of Troponin C in cardiomyocytes. The long-term aim is to achieve complete or partial substitution of the native cardiac isoform and study the effects on contractile force produced, in normal and ischemic cardiomyocytes, both in vitro and in vivo. This project has involved designing, constructing and analyzing expression of adenoviral gene transfer vectors overexpressing the sTnC isoform. Several adenoviral vectors were generated with the wild type sTnC gene under the control of muscle-specific promoters. To facilitate analysis of protein expression and its subcellular localization, the sTnC protein was tagged with epitope tags and adenovirus generated, with this gene under the control of constitutive (CMV) and cardiac-specific (HCA) promoters. Epitope-tagged adenoviruses were expressed in vitro using mouse fibroblast (NIH3T3) cells and analyzed by western blot analysis, showing successful constitutive expression. Recombinant adenoviruses containing epitope-tagged-sTnC under the control of the human cardiac actin promoter showed cardiac-specific expression in cultured cardiomyocytes, in situ, using immunocytochemistry. The constitutively-expressing sTnC adenoviral vector showed successful expression in cardiomyocytes in culture, using northern blot analysis. A range of adenoviral vectors have been successfully generated, and constitutive and tissue-specific expression has been established for some of these vectors. Successes attained in this project have established the initial requirements to achieve the long-term goal to alter calcium sensitivity of myofilaments, by overexpression of sTnC isoform in cardiomyocytes, both in vitro and in vivo.
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Sun, Qian. "Molecular analysis of factors involved in regulation of protein expression by recombinant Chinese hamster ovary (CHO) cells." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505481.
Full textAbstract:
The demand for approved biopharmaceuticals products from animal cell culture have been rapidly increasing since last few decades. Many efforts have been made on process refining in the past, and now, works are focusing on the selection or generation of high producer cell lines. It was therefore very important to understanding the molecular mechanisms underlying the existing high producing cell lines and to identify cellular regulators responsible for enhanced productivity.
32
Pereira, Luiz Augusto. "Identificação proteômica, seqüência de nucleotídeos, expressão heteróloga e reatividade imunológica da triose fosfato isomerase de Paracoccidioides brasiliensis." Universidade Federal de Goiás, 2004. http://repositorio.bc.ufg.br/tede/handle/tede/3778.
Full textAbstract:
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Previous issue date: 2004-05-03<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES<br>An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and
characterized. Endoproteinase Lys-C digested peptides of the purified protein which
presented, molecular mass of 29-kDa and pI of 5.8, were subjected to sequence analysis of its
amino acids. Search at databases comparing the sequence of amino acids from the three
peptides of the native protein, revealed strong homology to triosephosphate isomerase (TPI:
E.C. 5.3.1.1) from several sources. The complete cDNA and gene encoding PbTPI were
obtained and both contained an open reading frame predicted to encode a 249 amino acid
protein that presented all the peptides characterized in the native PbTPI. The Pbtpi gene
contained 6 exons interrupted by 5 introns. Analysis performed with the deduced PbTPI
suggested its usefulness in providing phylogenetic relatedness, as well as evidenced the
correlation between the phylogeny provided by the deduced proteins and introns positions in
the cognate genes. The immunological reactivity of PbTPI was examined. The complete
coding cDNA of PbTPI was over expressed in an Escherichia coli host to produce high levels
of recombinant fusion protein with glutathione S-transferase (GST) that has been purified by
affinity chromatography. The purified recombinant TPI was recognized by sera of patients
with confirmed Paracoccidioidomycosis and not by sera of healthy individuals. Thus,
recombinant PbTPI can be a valuable addition to the still small arsenal of P. brasiliensis
immunoreactive proteins, which could be tested for incorporation in assays for serodiagnosis
of the disease.<br>Um antígeno de Paracoccidioides brasiliensis (Pb) foi isolado do gel e
caracterizado. Os peptídeos digeridos por Endoproteinase Lys-C da proteína purificada, que
apresentou uma massa molecular de 29-kDA e pI de 5.8, foram submetidos à análise da
seqüência de aminoácidos. Uma busca em bancos de dados de seqüências de aminoácidos
comparados com os três peptídeos da proteína nativa revelou forte homologia com triose
fosfato isomerase (TPI: E.C. 5.3.1.1) de vários organismos. O cDNA e o gene completos que
codificam para PbTPI foram obtidos, e ambos contém uma provável ORF que codifica para
uma proteína com 249 aminoácidos que apresenta todos os peptídeos caracterizados na PbTPI
nativa. O gene Pbtpi apresentou 6 exons interrompidos por 5 introns. Análises realizadas com
a PbTPI deduzida sugerem sua utilidade em prover relações filogenéticas, como também
evidenciou a correlação entre a filogenia gerada pelas proteínas deduzidas e as posições dos
introns nos genes cognatos. A reatividade imunológica da PbTPI foi examinada. O cDNA
completo que codifica para PbTPI foi altamente expresso no hospedeiro Escherichia coli,
produzindo altos níveis de uma proteína recombinante fundida a glutathione S-transferase
(GST), que foi purificada por cromatografia de afinidade. A TPI recombinante purificada foi
reconhecida por soros de pacientes com paracoccidioidomicose confirmada e não por soros
de indivíduos saudáveis. Assim, PbTPI recombinante pode ser uma adição valiosa para o
pequeno arsenal de proteínas imunoreativas de P. brasiliensis, que poderiam ser testadas por
incorporação em ensaios de sorodiagnóstico da doença.
33
Neubauer, A. (Antje). "Expression and analysis of recombinant human collagen prolyl 4-hydroxylase in E. coli and optimization of expression." Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514281063.
Full textAbstract:
Abstract
Collagen prolyl 4-hydroxylase (C-P4H) plays a central role in the biosynthesis of collagens by hydroxylating proline residues. The enzyme has been a subject of intense interest as a target enzyme for drug development. The recombinant expression of human C-P4H in prokaryotes has not yet been described. This work reports on the development of an expression system for human C-P4H in E. coli.
The vertebrate C-P4H enzymes are α2β2 tetramers, consisting of two β subunits which are identical to protein disulphide isomerase (PDI), aside from the two α subunits which have the catalytic activity. The function of PDI is to keep the α subunit in a soluble and active state. Therefore, the expression system should assure the expression of the β subunit in the cell before the α subunit by using two different promoters. An active C-P4H tetramer was obtained in the periplasm of E. coli. However, further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human C-P4H tetramer. The exchange of four rare E. coli codons of the pdi gene and the optimized distance between ribosome binding site and translation initiation, resulted in 50-fold P4H-activity and 25 mg/l purified enzyme.
Comparison of the expression level of mRNA from the α and β subunits by Sandwich hybridization identified single induction with anhydrotetracycline in fed-batch fermentations as a limiting parameter. This caused an insufficient expression level of mRNA and thereby a low yield of C-P4H. A maximum yield was obtained by repeated addition of anhydrotetracycline that led to higher mRNA levels and increased productivity.
A newly developed stochastic simulation model of translational ribosome traffic in bacteria assesses the effect of codon usage to ribosome traffic and to the overall translation rate and mRNA stability. Using human PDI, it was shown that substitution of four 5' codons of the human PDI sequence that are rare in E. coli sequences, by synonymous codons preferred in E. coli led to a 2-fold increase of total PDI amount and even to a 10-fold increase of soluble PDI amount.
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Wandrey, Georg Benjamin [Verfasser], Jochen [Akademischer Betreuer] Büchs, and Jörg [Akademischer Betreuer] Pietruszka. "Light-mediated control and analysis of recombinant protein production in microscale cultivations / Georg Benjamin Wandrey ; Jochen Büchs, Jörg Pietruszka." Aachen : Universitätsbibliothek der RWTH Aachen, 2017. http://d-nb.info/1161809112/34.
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Myers, Andrew Ross. "Cloning, Expression, and Sequence Analysis of Camelysin, a Zinc Metalloprotease from Bacillus anthracis and B. cereus." [Tampa, Fla.] : University of South Florida, 2005. http://purl.fcla.edu/fcla/etd/SFE0001218.
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Dolata, Katarzyna [Verfasser], Katharina [Gutachter] Riedel, and Wolfgang [Gutachter] Liebl. "Proteomics-based analysis of stress responses during recombinant protein production in Escherichia coli / Katarzyna Dolata ; Gutachter: Katharina Riedel, Wolfgang Liebl." Greifswald : Universität Greifswald, 2019. http://d-nb.info/1194162835/34.
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Dolata, Katarzyna Verfasser], Katharina [Gutachter] Riedel, and Wolfgang [Gutachter] [Liebl. "Proteomics-based analysis of stress responses during recombinant protein production in Escherichia coli / Katarzyna Dolata ; Gutachter: Katharina Riedel, Wolfgang Liebl." Greifswald : Universität Greifswald, 2019. http://nbn-resolving.de/urn:nbn:de:gbv:9-opus-29819.
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Jokela, M. (Maarit). "Replicative DNA polymerase associated B-subunits." Doctoral thesis, University of Oulu, 2004. http://urn.fi/urn:isbn:9514274814.
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Abstract
Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels.
By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases.
At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors.
To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.
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Danh, Tu Thien. "ANALYSIS OF CHROMATIN ACCESSIBILITY OF THE HUMAN C-MYC REPLICATION ORIGIN." Wright State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=wright1449845546.
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Mao, Liangyan. "Assessment of Bone Regeneration in a Rat Femur Defect Model Following Recombinant Human Bone Morphogenetic Protein 2 Delivery from Keratin Hydrogels with Tunable Rates of Degradation: Micro-CT Analysis and Histology." Miami University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=miami148068386349526.
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St, Pierre Liam Daniel. "Identification and comparative analysis of novel factors from the venom gland of the coastal taipan (Oxyuranus scutellatus) and related species." Thesis, Queensland University of Technology, 2005. https://eprints.qut.edu.au/16677/1/Liam_St_Pierre_Thesis.pdf.
Full textAbstract:
Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Amongst the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family. Their venoms posses a number of unique properties by which they target the mammalian cardiovascular and neuromuscular systems and are the focus for the identification of novel pharmacologically interesting compounds which may be of diagnostic or therapeutic benefit. Although much is known about the biochemical properties of Australia snake venoms as a whole, little research attention has focused upon individual components at the molecular level. This thesis describes the cloning, characterisation and comparative analysis of a number of unique toxins from the venom gland of the coastal taipan (Oxyuranus scutellatus) and a total of seven other related Australian snakes. These include the factor X- and factor V-like components of a prothrombin activator that causes a highly coagulable state in mammals. Comparative analysis of the sequences identified in this study, along with recombinant expression of an active form of the factor X-like component, provides important information on the structural, functional and evolutionary relationships of these molecules. Numerous other toxins were similarly identified and characterised including a pseudechetoxin-like protein, multiple phospholipase A2 enzymes and neurotoxin isoforms as well as vasoactive venom natriuretic peptides. Identified transcripts included not only toxin sequences but also other cellular peptides implicated in toxin processing, including a calglandulin-like protein. This thesis is the first description of the majority of these molecules at either the cDNA or protein level, and provides a means to study the activity of individual components from snake venoms and probe their function within the systems they specifically target. This study represents the most detailed and comprehensive description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.
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St, Pierre Liam Daniel. "Identification and comparative analysis of novel factors from the venom gland of the coastal taipan (Oxyuranus scutellatus) and related species." Queensland University of Technology, 2005. http://eprints.qut.edu.au/16677/.
Full textAbstract:
Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Amongst the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family. Their venoms posses a number of unique properties by which they target the mammalian cardiovascular and neuromuscular systems and are the focus for the identification of novel pharmacologically interesting compounds which may be of diagnostic or therapeutic benefit. Although much is known about the biochemical properties of Australia snake venoms as a whole, little research attention has focused upon individual components at the molecular level. This thesis describes the cloning, characterisation and comparative analysis of a number of unique toxins from the venom gland of the coastal taipan (Oxyuranus scutellatus) and a total of seven other related Australian snakes. These include the factor X- and factor V-like components of a prothrombin activator that causes a highly coagulable state in mammals. Comparative analysis of the sequences identified in this study, along with recombinant expression of an active form of the factor X-like component, provides important information on the structural, functional and evolutionary relationships of these molecules. Numerous other toxins were similarly identified and characterised including a pseudechetoxin-like protein, multiple phospholipase A2 enzymes and neurotoxin isoforms as well as vasoactive venom natriuretic peptides. Identified transcripts included not only toxin sequences but also other cellular peptides implicated in toxin processing, including a calglandulin-like protein. This thesis is the first description of the majority of these molecules at either the cDNA or protein level, and provides a means to study the activity of individual components from snake venoms and probe their function within the systems they specifically target. This study represents the most detailed and comprehensive description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.
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Nadkarni, Aditi A. "Functional analysis of the Rad51d (E233G) breast cancer associated polymorphism and a pharmacogenetic evaluation of RAD51D status." Connect to full text in OhioLINK ETD Center, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1222877984.
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Dissertation (Ph.D.)--University of Toledo, 2008.<br>"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: pages 73-77, 93-95, 109-111, 145-172.
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Rayon, Catherine. "La N-glycosylation chez les plantes. Etude d'une glycoprotéine modèle : la phytohémagglutinine." Rouen, 1998. http://www.theses.fr/1998ROUES007.
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La production de protéines d'intérêt thérapeutique dans des plantes transgéniques suppose que la cellule végétale soit capable d'effectuer les modifications post-traductionnelles, en particulier la N-glycosylation, indispensables à la biosynthèse d'une protéine biologiquement active et stable. Les données actuelles relatives à la N-glycosylation des protéines végétales étant encore parcellaires, nous avons cherché à réaliser une analyse comparée de la biosynthèse et la maturation des N-glycannes dans différents systèmes constitutifs du règne végétal et au sein de différents organes d'une même plante. Nous avons pour cela exprimé de façon recombinante une glycoprotéine modèle végétale, la phytohémagglutinine de haricot (PHa) dans différents systèmes végétaux. Cette étude a nécessité préalablement la mise au point d'un ensemble de stratégies permettant d'établir de façon qualitative et quantitative la N-glycosylation d'une glycoprotéine végétale. Ainsi, après expression de la PHa dans un système végétal transformé (plante, cellules en culture) via Agrobacterium tumefaciens, la PHa recombinante est isolée par chromatographie d'affinité, puis analysée sur empreinte au moyen de sondes spécifiques. Ensuite les N-glycannes sont libérés et analysés par chromatographie et spectrométrie. Cette étude a permis d'établir que les processus de glycosylation d'une glycoprotéine vacuolaire sont conservés dans les différents systèmes végétaux étudiés et au sein des différents organes d'une plante. Par ailleurs, cette stratégie d'analyse a été appliquée à l'ensemble des N-glycannes associés aux protéines d'Arabidopsis thaliana, plante de référence dans le domaine végétal et chez un mutant d'A. Thaliana affecté dans les processus de glycosylation. Cette étude a permis de démontrer les potentialités des stratégies développées au cours de cette thèse pour l'étude de mutants végétaux.
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Abdallah, Bassim Violla. "Structural and functional analysis on GacS homodimeric histidine kinase reveals a non-canonical autokinase activity and new insights into the heterodimer partnership with RetS." Thesis, Aix-Marseille, 2020. http://www.theses.fr/2020AIXM0256.
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Pseudomonas aeruginosa (PA) est un pathogène qui infecte particulièrement les patients atteints de la mucoviscidose. PA provoque des infections aiguës et chroniques et alterne entre des modes de vie planctonique et sédentaire. Cette transition est principalement régulée par les systèmes à deux composants (TCS). Durant ma thèse, je me suis focalisée sur le TCS GacS/GacA, impliqué dans un réseau de signalisation multikinase. GacS est une histidine kinase (HK) et GacA est son régulateur de réponse (RR). La région cytoplasmique de GacS est composée des domaines HAMP, S-Hélice, H1, D1 et H2. Des tests fonctionnels ont montré que la région HAMP/S-Hélice est nécessaire pour la transduction du signal et contribue à son activité. L’analyse structurale a montré une boucle de fixation de l’ATP structurée contrairement à ce qui est observé dans d'autres HKs. De plus, un nouveau domaine (ND) a été identifié et s’est révélé essentiel pour assurer une activité autokinase. Nous avons proposé que le ND contribue à des changements conformationnels pour placer GacS dans une configuration active. Par ailleurs, RetS HK inhibe GacS par des interactions directes. Des tests de spectrométrie de masse native et des tests d’interaction ont montré que le domaine HAMP est nécessaire pour assurer l'interaction entre ces HKs. De plus, des anticorps à domaine unique contre GacS nous ont permis d’identifier des sites potentiels d'inhibition de la voie GacS/GacA et de la formation de biofilm. Dans cette étude, nous avons dévoilé une activité autokinase non canonique de GacS et de nouvelles perspectives sur son interaction avec RetS qui contribuent au processus décisionnel pendant l'infection par PA<br>Pseudomonas aeruginosa (PA) is a pathogen that particularly infects patients with cystic fibrosis. PA causes acute and chronic infections and can switch from a planktonic to a sedentary lifestyle. This transition is mainly coordinated by Two-Component Systems (TCS). During my thesis, I focused on GacS/GacA TCS, a master regulator of the acute-to-chronic transition. In fact, GacS is a membrane-bound histidine kinase (HK) and GacA is the response regulator (RR). GacS cytoplasmic region is composed by the HAMP, S-Helix coiled-coil region. This helical part is followed by the H1, the D1 and H2 domains. GacS/GacA TCS is involved in a multikinase regulatory network and its activity is modulated by three HKs. Mutagenesis and functional assays showed that the HAMP and S-Helix orchestrate the signal transduction prior to its autokinase activity. In addition, GacS presented a folded ATP lid in contrast to what is observed in other HKs. Furthermore, we unveiled a new domain (ND) in GacS sequence. Functional assays showed that the ND domain is essential to insure a full autokinase activity of GacS. We proposed that this domain contributes in conformational rearrangements in order to shift GacS to its active state. RetS HK is known to inhibit GacS/GacA via direct interactions. Native mass spectrometry and microscale thermophoresis assays showed that the HAMP domain is essential for this interaction. Furthermore, nanobodies generated against the cytoplasmic region of GacS revealed potential sites of inhibition of GacS activity. In this study, we unveiled a non-canonical autokinase activity in GacS and new insights into its interaction with RetS that underlie the decision-making of PA
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Lin, Han-Yun, and 林涵芸. "Immunogenic Analysis of the Recombinant Proteins of Mannheimia haemolytica." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/11556401006999604581.
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碩士<br>國立屏東科技大學<br>動物疫苗科技研究所<br>97<br>Mannheimia (Pasteurella) haemolytica is a member of the Pasteurellaceae family, which is the major cause of acute fibrinous pneumonia in ruminants. The mortality is high and vaccination is the major strategy for controlling this disease. There is no effect vaccine to protect pneumonic pasteurellosis. It is urgency to develop new vaccine to prevent M. haemolytica infection and reduce the economical loss. The previous studies indicated that outer membrane proteins GS60 are the most important antigens and the leukotoxin (LKT) plays a major role in lung injury. Our objectives are to clone and express GS60 and LKT of M. haemolytica by the prokaryotic expression system and characterize their antigenicities. The vaccines (M. h, M. h+rGS60, rGS60, M. h+rLKT, rLKT) made of inactivated wild-type M. haemolytica and recombinant proteins (rGS60, rLKT) with w/o/w adjuvant. The survival rate of protection efficacy in immunized mice was 60% to 80%, while control mice were all dead. The antibody titer of immunized mice was significantly higher than control group (p&lt;0.01) and the major subtype was IgG2a. The T-cell proliferation index of immunized groups was significantly higher than control group (p&lt;0.01) in mice and goat. Moreover, the antibody titer of immunized rGS60 goat was significantly higher than control group (p&lt;0.01) and continued at the 12th week. The IFN-γ gene expression was detected in vaccinated (rLKT and rGS60) goats. Furthermore the rLKT antibody titer and T-cell proliferation index was significantly higher than rGS60 group (p&lt;0.01) at 4 weeks. In summary, these expressed proteins could induce humoral and cellular immune response used as a potential immunogen or adjuvant to develop new vaccines against M. haemolytica.
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Yeh, Po-Hsien, and 葉伯賢. "Antigenic Analysis of Actinobacillus pleuropeumoninae Recombinant Apx Toxin Proteins." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/20921799649786339545.
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碩士<br>國立屏東科技大學<br>動物疫苗科技研究所<br>98<br>Actinobacillus pleuropeumoninae (A.p.) is a type of hemolytic Gram-negative bacteria, which includes a total of 15 serotypes, and can cause fibrinonecrotic and hemorrhagic pleuropneumonia, resulting in serious economic loss in farms. The pathogenic factors of A.p. are capsular polysaccharides, lipopolysaccharides, outer membrane proteins (OMP), adsorption factors, and exotoxins. Actinobacillus pleuropneumoniae toxins (Apxs) belong to RTX (repeat in the structural toxin) family, are exotoxins and important virulence factors with immunogenicity, is more efficient than others to induce immune response. Due to weak cross reactivity among different serotypes, the protective ability of traditional vaccines is still limited. Therefore, it is important to develop a new effective vaccine. The results in this study showed successfully expressed these recombinant (Apx I, Apx II, Apx IV and OMP) proteins and can be recognized by antiserum form infected pigs. The ELISA antibodies of immunized mice with these recombinant proteins combined with adjuvant higher than those from control mice. When challenged with A.p. virulent serotype 1 and 5 (1×108 CFU/mL), the survival rate of immunized mice was 66.6% compared to 0% of control and commercial vaccine group. After immunization the pigs challenged with A.p. virulent serotype 1 (8×107 CFU/mL), control group showed the clinical signs including fever, anorexia, and dyspnea . In anatomy result showed the immunized pig was reduce the pathological levels 63% than control. It is demonstrated the rApx could protect pigs suffered from A.p invade. Futhermore, the flow cytometry results in vaccine group CD8+ percentage is 35.6% higher than control group (19%) . The results indicated rApx may have potential as the subunit vaccine candidate.
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Kim, Hyun-Eui. "Biochemical analysis of apoptosome formation." 2006. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=215.
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Kotlarski, Nicholas. "Process-scale renaturation of recombinant proteins from inclusion bodies / by Nicholas Kotlarski." Thesis, 1998. http://hdl.handle.net/2440/19259.
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Bibliography: leaves 215-236.<br>x, 249 leaves : ill. ; 30 cm.<br>Scale-up of a biochemical process involving expression of an Insulin-like Growth Factor-I analogue (LongR3IGF-I) as inclusion bodies within the bacterium Escherichia coli has been investigated. The principal focus was directed to the operation of refolding wherein the biological potency of the protein is imparted.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1998?
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Zhang, Yuan Heidi. "Mass spectrometric analysis of proteins and peptides : elucidation of the folding pathways of recombinant human macrophage colony stimulating factor beta." Thesis, 2002. http://hdl.handle.net/1957/37223.
Full textAbstract:
Recombinant human macrophage colony stimulating factor beta
(rhm-CSFβ) is a glycoprotein that stimulates the proliferation, differentiation
and survival of cells belonging to the monocyte-macrophage lineage. It
contains nine inter-subunit and intra-subunit disulfide bonds and represents
an excellent model system for studying disulfide bond formation during
protein folding because the assembly of its monomeric subunits and the
maturation of its biological activity depend on the progressive formation of
the correct disulfide structure during in vitro folding. Knowledge obtained
from these studies can be potentially useful in understanding the roles of
disulfide bond formation during protein folding in general.
rhm-CSF8 was modified by partial reduction of disulfide bonds,
yielding CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ. The modification did not affect the
biological activity, stability, or the overall conformation of the protein.
However, the C-terminal regions near the modification sites were shown to
exhibit faster deuterium exchange behavior as a result of the chemical
modification, indicating that the C-terminal regions became more flexible.
Folding kinetics of rhm-CSFβ and CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ were shown
to be essentially the same, suggesting that the modification did not affect
the folding kinetics of the oxidized rhm-CSFβ.
The denatured and reduced rhm-CSFβ was refolded with the aid of a
chemical oxidant. The data indicated that the in vitro folding rhm-CSFβ
proceeded via multiple pathways involving monomeric and dimeric
intermediates. Disulfide bond shuffling catalyzed by GSH/GSSG
represented an important isomerization step in folding. A dimeric
intermediate, D-SS8-cam2, was isolated and identified as a kinetic trap,
perhaps requiring significant structural arrangement to convert to the native
protein. The heterogeneous folding mixture detected by both disulfide bond
quenching and H/D pulsed labeling indicate that rhm -CSFβ folding is a
diffusion like process as described by the folding funnel model.<br>Graduation date: 2003