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Artigos de revistas sobre o assunto "Recombinant proteins Analysis"
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Southan, Christopher. "Purification and analysis of recombinant proteins." Trends in Biotechnology 10 (1992): 226. http://dx.doi.org/10.1016/0167-7799(92)90226-l.
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Kermasha, S., and I. Alli. "Purification and analysis of recombinant proteins." Food Research International 26, no. 2 (January 1993): 158–59. http://dx.doi.org/10.1016/0963-9969(93)90072-q.
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Kaufman, Randal J. "Mammalian recombinant proteins: Structure, function and immunological analysis." Current Opinion in Biotechnology 1, no. 2 (December 1990): 141–50. http://dx.doi.org/10.1016/0958-1669(90)90023-e.
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Senear, Donald F., Robert A. Mendelson, Deborah B. Stone, Linda A. Luck, Elena Rusinova, and J. B. Alexander Ross. "Quantitative Analysis of Tryptophan Analogue Incorporation in Recombinant Proteins." Analytical Biochemistry 300, no. 1 (January 2002): 77–86. http://dx.doi.org/10.1006/abio.2001.5441.
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Kollárovič, G., D. Majera, K. Luciaková, and P. Baráth. "Expression and purification of recombinant NFI proteins for functional analysis." General Physiology and Biophysics 28, no. 4 (2009): 331–39. http://dx.doi.org/10.4149/gpb_2009_04_331.
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Coutinho, M., K. S. Aulak, and A. E. Davis. "Functional analysis of the serpin domain of C1 inhibitor." Journal of Immunology 153, no. 8 (October 15, 1994): 3648–54. http://dx.doi.org/10.4049/jimmunol.153.8.3648.
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Abstract To analyze the role of the heavily glycosylated amino-terminal domain of C1 inhibitor in protease inhibitory activity, two truncated C1 inhibitor molecules were constructed. The abilities of the recombinant truncated inhibitors to complex with target proteases were compared with that of the wild-type recombinant protein. One recombinant truncated molecule consisted of amino acid residues 76 to 478 (C-serp(76)) and the other of residues 98 to 478 (C-serp(98)). The recombinant proteins were each expressed in similar quantities. The thermal denaturation profiles of the two truncated proteins were similar to that of the wild-type protein. Identical binding of C1s, C1r, kallikrein, and beta factor XIIa was observed with the three molecules. Furthermore, the truncated molecules also effectively inhibited C1 activity in hemolytic assays. These studies therefore clearly demonstrate that the amino-terminal domain of C1 inhibitor does not influence complex formation with target proteases.
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Jerlström-Hultqvist, Jon, Britta Stadelmann, Sandra Birkestedt, Ulf Hellman, and Staffan G. Svärd. "Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome." Eukaryotic Cell 11, no. 7 (May 18, 2012): 864–73. http://dx.doi.org/10.1128/ec.00092-12.
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ABSTRACTIn recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoanGiardia intestinaliscan be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use inGiardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathioneS-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinantGiardiaproteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of theG. intestinalis26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and fromGiardia, which will allow the study of specific parasite proteins and protein complexes.
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Sims, Andrew H., Manda E. Gent, Karin Lanthaler, Nigel S. Dunn-Coleman, Stephen G. Oliver, and Geoffrey D. Robson. "Transcriptome Analysis of Recombinant Protein Secretion by Aspergillus nidulans and the Unfolded-Protein Response In Vivo." Applied and Environmental Microbiology 71, no. 5 (May 2005): 2737–47. http://dx.doi.org/10.1128/aem.71.5.2737-2747.2005.
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ABSTRACT Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.
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Zeck, Anne, Jörg Thomas Regula, Vincent Larraillet, Björn Mautz, Oliver Popp, Ulrich Göpfert, Frank Wiegeshoff, et al. "Low Level Sequence Variant Analysis of Recombinant Proteins: An Optimized Approach." PLoS ONE 7, no. 7 (July 6, 2012): e40328. http://dx.doi.org/10.1371/journal.pone.0040328.
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Viseux, N., X. Hronowski, J. Delaney, and B. Domon. "Qualitative and Quantitative Analysis of the Glycosylation Pattern of Recombinant Proteins." Analytical Chemistry 73, no. 20 (October 2001): 4755–62. http://dx.doi.org/10.1021/ac015560a.
Texto completo da fonteTeses / dissertações sobre o assunto "Recombinant proteins Analysis"
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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.
Livros sobre o assunto "Recombinant proteins Analysis"
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1952-, Seetharam Ramnath, and Sharma Satish K. 1951-, eds. Purification and analysis of recombinant proteins. New York: M. Dekker, 1991.
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W, Thorner Jeremy, Emr Scott, and Abelson John, eds. Applications of chimeric genes and hybrid proteins. San Diego, CA: Academic Press, 2000.
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Protein affinity tags: Methods and protocols. New York: Humana Press, 2014.
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Thomas, Sandra M. Patenting of recombinant proteins: An analysis of tissue plasminogen activator (t-PA) in Europe, United States and Japan. Brighton: University of Sussex, Science Policy Research Unit, 1994.
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1947-, Stein Stanley, ed. Fundamentals of protein biotechnology. New York: M. Dekker, 1990.
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Haining, Robert Luddy. Structure/function studies at the active-site region of cyanobacterial ribulose bisphosphate carboxylase/oxygenase by site-directed mutagenesis. 1993.
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Michaud, Dominique. Recombinant Protease Inhibitors in Plants (Biotechnology Intelligence Unit, 3). Landes Bioscience, 2000.
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Giannone, Richard J., and Andrew B. Dykstra. Protein Affinity Tags: Methods and Protocols. Springer New York, 2016.
Encontre o texto completo da fonteCapítulos de livros sobre o assunto "Recombinant proteins Analysis"
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Buntru, Matthias, Simon Vogel, Ricarda Finnern, and Stefan Schillberg. "Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins." In Recombinant Proteins in Plants, 113–24. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2241-4_8.
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AbstractPlant cell-free lysates contain all the cellular components of the protein biosynthesis machinery, providing an alternative to intact plant cells, tissues, and whole plants for the production of recombinant proteins. Cell-free lysates achieve rapid protein production (within hours or days) and allow the synthesis of proteins that are cytotoxic or unstable in living cells. The open nature of cell-free lysates and their homogeneous and reproducible performance is ideal for protein production, especially for screening applications, allowing the direct addition of nucleic acid templates encoding proteins of interest, as well as other components such as enzyme substrates, chaperones, artificial amino acids, or labeling molecules. Here we describe procedures for the production of recombinant proteins in the ALiCE (Almost Living Cell-free Expression) system, a lysate derived from tobacco cell suspension cultures that can be used to manufacture protein products for molecular and biochemical analysis as well as applications in the pharmaceutical industry.
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Thangaraj, Harry. "Freedom to Operate Analysis of Molecular Farming Projects." In Recombinant Proteins in Plants, 335–42. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2241-4_18.
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Fitchette-Lainé, Anne-Catherine, Lise-Anne Denmat, Patrice Lerouge, and Loïc Faye. "Analysis of N- and O-Glycosylation of Plant Proteins." In Recombinant Proteins from Plants, 271–90. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1007/978-1-60327-260-5_19.
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Tong, Kit I., Masayuki Yamamoto, and Toshiyuki Tanaka. "Selective Isotope Labeling of Recombinant Proteins in Escherichia coli." In Intrinsically Disordered Protein Analysis, 439–48. New York, NY: Springer New York, 2012. http://dx.doi.org/10.1007/978-1-4614-3704-8_30.
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Sominskaya, Irina, and Kaspars Tars. "Immunological Methods for Analysis of Recombinant Proteins." In Basic Cloning Procedures, 135–44. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-71965-3_7.
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Arakawa, Tsutomu, and John S. Philo. "Biophysical and Biochemical Analysis of Recombinant Proteins." In Pharmaceutical Biotechnology, 19–45. New York, NY: Springer New York, 2013. http://dx.doi.org/10.1007/978-1-4614-6486-0_2.
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Tolkatchev, Dmitri, Josee Plamondon, Richard Gingras, Zhengding Su, and Feng Ni. "Recombinant Production of Intrinsically Disordered Proteins for Biophysical and Structural Characterization." In Instrumental Analysis of Intrinsically Disordered Proteins, 653–70. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470602614.ch22.
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Micsonai, András, Éva Bulyáki, and József Kardos. "BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy." In Methods in Molecular Biology, 175–89. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0892-0_11.
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Abstract Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold. The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.
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Mattaliano, R. J., J. J. Rosa, C. Foeller, J. P. Woodard, and M. J. Bertolini. "Analysis of Recombinant Proteins — Current Trends and Practical Limits in Analytical Stringency." In Methods in Protein Sequence Analysis · 1986, 79–95. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-59259-480-1_6.
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Chen, Zeya, and Jingjing Huang. "In Vitro Biochemical Analysis of Recombinant Plant Proteins Under Oxidation." In Methods in Molecular Biology, 143–60. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2469-2_11.
Texto completo da fonteTrabalhos de conferências sobre o assunto "Recombinant proteins Analysis"
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Furis, B. C., M. J. Jorgensem, M. J. Rabiet, A. B. Contor, C. L. Brown, C. B. Shoemaker, and B. Furie. "RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643992.
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Factor IX and prothrombin vitamin K-dependent proteins that participate in blood coagulation undergo post-translationalmodification in which glutamic acid residues in the amino terminus of the protein are converted to gamma-carboxyglutamic acid residues. This modification confers divalent metal ion binding ability upon the proteins.As a consequence of binding divalent metal ions these proteins undergoconformational changes necessary for biological function.The vitamin K-dependent proteins are synthesized with an NH2-terminal extension. The region distal to the NH2-terminus of the mature protein is a prototypic signal sequence while the proximal region is a propeptide with homology among the vitamin K-dependent proteins. The boundary between the pre and pro sequences has been established for factor IX by analysis of three naturally occurring factor IX mutants factor IX Cambridge factor IX Oxford-3 and factor IX San Dimas, in which processing is incomplete.For human factor IX the propeptide extends from residue -18 to -1. The homology among the propeptides of vitamin K-dependent proteins suggests that the propeptide may designate adjacent gamma-carboxyglutamic acids for carboxylation. To test this hypothesis alterations in sequence were introduced into the propeptide region of human factor IX cDNA by oligonucleotide directed site specific mutagenesis.Mutated genes were expressed in Chinese hamster ovary cells. Rapid and efficient isolationof the mutant proteins by immunoaffinity chromatography permitted detailed analysis of the mutants on quantities of protein easily obtainable at low expression levels. The extent of gamma-carboxylation was assessed by the ability of the mutant proteins to interact with conformation specific antibodies directed against the gamma-carboxyglutamic acid-dependent metal stabilized native structure of factor IX as well as by direct amino acid analysis. Unmodified recombinant factor IX contained, on average, 9 gamma-carboxyglutamic acid residues, as compared to 12 for plasma factor IX. About 70% of the recombinant wild type factor IX bound to the conformation specific antibodies. Deletion of the propiece or point mutations at residues -10 or -16 led to secretion of uncarboxylated factor IX unreaotive with antibodies specific for the native structure but with the NH2-terminus of mature factor IX. In order to assess the universality of these observations we have recently cloned human prothrombin cDNA and expressed the gene in the same Chinese hamster ovary cell system used for factor IX. In contrast to factor IX, at low levels ofexpressionof the prothrombin gene, the prothrombin is fully carboxylated relative to a plasma prothrombin standard.The recombinant prothrombin exhibits the same specific clotting activity as plasma derivedprothrombin and is fully native as evaluated by conformation specific antibodies. At high levels of expression the capacityof the cells to carboxylate prothrombin can be exceeded leading to secretion of under carboxylated prothrombin. However, the absolute amount of fully carboxylated prothrombin that can be produced in this system appears to be a least fivefold greater that the absolute amount of highly carboxylated factor IX that can be synthesized.The elimination of carboxylation observed upon mutation of the propiece of factor IX suggest that the propiece contains a recognition element required for carboxylation of the protein. Assignment of a functional role to the propiece of factor IX represents the first determination of function for any pro sequence. It is anticipated that extension of these studies to prothrombin will demonstrate that this recognition signal is used by all the members of this class of proteins. In order to determine if the propiece is sufficient to designate a protein for gamma-carboxylation we are currently constructing chimeric proteins incorporating the propieceof prothrombin into the cDNA of normally uncarboxylated proteins.
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Ashok Kumar, A., Margaret Insley, Jay Gambee, Sharon J. Busby, and Kathleen L. Berkner. "SITE SPECIFIC MUTAGENESIS WITHIN THE GLA-DOMAIN OF HUMAN FACTOR IX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644079.
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Factor IX, a plasma protein, plays a critical role in blood coagulation. The biological activity of factor IX as well as several other plasma proteins depends on the presence of gamma-carboxy glutamic acid (Gla) residues in their amino terminal region. In vitro mutagenesis has been used to selectively replace Gla residues of factor IX with aspartic acid (Asp) residues in order to establish the contribution of individual as well as paired Gla residues to the normal functioning of the protein. These substitutions were made at positions 7, 15, 20 and 26 in human factor IX. In addition, residue number 18, a cysteine has been changed to serine in an attempt to disrupt the highly conserved disulfide bond in the gla-domain. The gla-domain mutants will be produced in mammalian cells and compared with native recombinant factor IX. A rapid immunoaffinity purification procedure, which has been used to obtain recombinant factor IX produced in the presence or absence of vitamin K, is being used to purify the mutants. Protein sequence analysis has been used to confirm complete processing and proper gamma-carboxylation of recombinant factor IX. The properties of these mutants as compared to human factor IX will be discussed.
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Higgins, Deborah L., and William E. Holmes. "CHARACTERIZATION OF RECOMBINANT HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR MISSING THE FINGER DOMAIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643842.
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Site-specific mutagenesis was used to produce a mutant form of tissue-type plasminogen activator (t-PA) which was missing the first 44 amino acids. This domain has sequence homology with the type 1 regions in proteins such as fibronectin and is commonly called the finger domain. The mutant protein (des 1-44 t-PA) was expressed in Chinese Hampster Ovary cells, and was purified using chromatography on Zn-chelate sepharose and lysine-sepharose. Sequence analysis indicated that the resulting protein was homogeneous and started at amino acid 45 in the sequence of the normal protein. The two-chain forms of both des 1-44 t-PA and normal sequence t-PA exhibited similar kinetic constants with a small synthetic substrate (H-D-Isoleucyl-L- prolyl-L-arginyl-p-nitroani 1 ide). The ability of des 1-44 t-PA to activate plasminogen was decreased to 70% of the rate of normal t-PA. The rate of plasminogen activation by normal t-PA was stimulated 51-fold in the presence of fibrin, whereas with des 1-44 t-PA it was stimulated only 40-fold. Although des 1-44 t-PA bound to lysine-agarose, little (if any) binding was observed to either intact or degraded fibrin indicating that fibrin stimulation is due in part to the ability of t-PA to recognize plasminogen bound to fibrin as a preferable substrate. The mutant t-PA was capable of forming complexes in vitro with all of the inhibitors in blood which react with normal sequence t-PA. The rate of reaction with α2-macroglobulin, however, was slower with des 1-44 t-PA than with normal sequence t-PA. The similar resistance of des 1-44 t-PA and normal sequence t-PA to proteolysis and the ability to react with a battery of monoclonal antibodies suggests that the deletion did not cause perturbed folding, but rather that alterations in function of des 1-44 t-PA were due to the lack of the finger domain.
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Al-Mohannadi, Anjud Khamis, Sara Deola, and Ahmed Malki. "Visualization of Factor Viii with Flow-Cytometry as a tool for Novel Gene Therapy Approach in Hemophilia A." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0164.
Texto completo da fonteResumo:
Haemophilia A is a genetic X-linked disorder, characterized by coagulation Factor VIII (FVIII) deficiency and leading to pathological bleedings. The disease occurs at a rate of 1 in 5000 males’ births. The treatment is the administration of plasma-derived or recombinant Factor VIII, which is expensive and leads to the development of inhibitory antibodies in around 40% of patients affected by the severe form of the disease. The disease becomes for these patients as life threatening. In new approaches to treat Haemophilia include gene therapy (GT), cells corrected through genetic modifications are used to produce in Haemophilia A patients FVIII protein in a sustained manner, as long-term treatment for this disorder. The cells of choice should be persistent and equipped with themachinery for large protein assembly and secretion. So far, target cells for Haemophilia gene correction are mostly liver cells, although they are highly immunogenic and exposed to immune-mediated destruction after GT. Based on literature evidences, bone marrow transplantation can correct Haemophilia A in mice, providing evidence that Hematopoietic stem cells (HSC) or their progeny are able to produce FVIII. We chose the approach of correcting HSC with lentiviral vectors carrying the FVIII gene cassette. Whereas classically FVIII protein is visualized on adherent cells through immunohistochemistry staining, flow-cytometry (FC) literature publications are very scarce. FC analysis is an attractive method for analysing hematopoietic cells, and in general, a versatile method for protein visualization. However, large proteins as FVIII are difficult to be carefully analysed, and the method requires several steps of optimization. This joint project with Dr. Muhammad Elnaggar, aims to optimize a method to characterize large proteins as FVIII with a reliable FC staining protocol. To this aim, we used cell lines to evaluate the expression and secretion pathways of FVIII, the intracellular requirements to fold and secrete large proteins, and the toxicities of protein accumulation, in case of GT mediated protein overexpression. For this purpose, the FC experiments were performed to optimise the FC protocol for FVIII visualization, by improving blocking efficacy, antibody-labelling efficacy and to ensure accuracy and validity through qPCR and FC double staining. This FC protocol proved its validity and usefulness in visualizing and studying functionally FVIII.
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Busby, S., K. Berkner, L. Halfpap, J. Gambee, and A. Kumar. "ALTERATION OF PROPTIDE SEQUENCE IMPAIRS BIOLOGICAL ACTIVITY OF HUMAN FACTOR VII." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643784.
Texto completo da fonteResumo:
We have investigated the effect of altering the leader sequence of human factor VII on its biological activity. Factor VII is a vitamin K-dependent blood coagulation protein whose activity depends on the presence of gamma-carboxyglutamic acid (gla) residues in its amino terminal region. Since factor VII and other vitamin K-dependent proteins exhibit structural homology in the propeptide, it has been suggested that the propeptide is involved in gamma-carboxylation. Recently, two factor IX patients were identified with point mutations which prevented the processing of the propeptide and generated a factor IX with greatly reduced biological activity (Diuguid et al., PNAS 83; 5803; Bentley et al., Cell 45: 343). To examine this question using recombinant DNA technology, we altered the sequence of the factor VII propeptide by in vitro mutagenesis of the factor VII cDNA and then expressed the altered genes in baby hamster kidney (BHK) cells. For the 60 and 38 aa leader forms of factor VII, the arg (R) at -1 was changed to ser (S), yielding the sequence HRRRS before the +1 ala. In addition, for the 60 aa leader form, a ser was inserted after the arg at -1, resulting in the sequence HRRRRS before the +1 ala. As determined by ELISA, the mutant proteins were synthesized and secreted by BHK cells at levels comparable to the wild-type forms of factor VII. Analysis by radioimmune precipitation and SDS-PAGE indicated that substitution of arg by ser at -1 prohibits processing of the factor VII propeptide, whereas, insertion of a ser after the four arg's does not. However, all three proteins have reduced biological activity by approximately 5-fold when compared to the wild-type forms with the one-stage clotting assay. All three proteins are also quantitatively precipitated by Ba citrate, indicating they are at least partially gamma-carboxylated. These results suggest that the correct sequence of the propeptide, not just cleavage of the propeptide, is necessary for generating a biologically active molecule. The effect of these sequence alterations on gamma-carboxylation will be evaluated further by analysis of the amino acid sequence and composition of the mutant proteins.
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Jorgensen, M. J., MJ Rabiet, A. B. Cantor, B. Furie, C. L. Brown, C. B. Shoemaker та B. C. Furie. "VITAMIN K-DEPENDENT γ-CARBOXYLATION OF FACTOR IX REQUIRES A RECOGNITION SITE CONTAINED WITHIN THE PROPEPTIDE". У XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643564.
Texto completo da fonteResumo:
The vitamin K-dependent proteins, including Factor IX (FIX), are calcium-binding proteins that undergo vitamin K-dependent post-translational modification to convert amino terminal glutamic aoid residues to Gla residues. Sequence homology among the propeptides of these proteins suggests a role for this region in designating the adjacent glutamic acid-rich domain for γ-carboxylation during intraoellular processing. Mutations vere made in the propeptide (residues -1 to -18) of FIX, and the effects on γ-carboxylation were assessed. The human FIX cDNA coding sequenoe was modified using oligonucleotide-directed site-specific mutagenesis and was expressed in Chinese hamster ovary cells. The extent of γ-carboxylation of secreted FIX was determined by (1) ability to interact with conformation-specific antibodies directed against the Gla-dependent, metal-stabilized, native structure of FIX, and (2) direct Gla analysis of the alkaline hydrolysate. Using the unmodified coding sequence, 64 ± 17 % of recombinant Factor IX bound to the conformation-specific antibodies, and 9.4 ± 0.7 Gla residues were found (compared with 12 Gla in plasma FIX). When the 18-residue propeptide was deleted, secreted FIX contained no detectable native FIX antigen and no detectable Gla. Similarly, point mutations leading to substitution of Ala for Phe at residue -16 or Glu for Ala at residue -10 led to secretion of FIX containing 2% and 6% native antigen, respectively, and approximately 1-2 Gla residues. The molecular weight of each of the reoombinant FIX species, as estimated by SDS-PAGE, was identical to that of plasma FIX. NH2-terminal sequence analysis of the mutant FIX speoies yielded the NH2-terminal sequence of plasma FIX. These data indicate that the mutations made in the propeptide did not interfere with intracellular proteolytic prooessing of FIX. We conolude that the FIX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for γ-carboxylation. The association of the propeptide with the γ-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in post-translational protein processing.
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Quertermous, T., J. M. Schnee, M. S. Runge, G. R. Matsueda, N. W. Hudson, J. G. Seidman, and E. Haber. "EXPRESSION OF A RECOMBINANT ANTIBODY-TARGETED THROMBOLYTIC MOLECULE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644616.
Texto completo da fonteResumo:
We have recently shown that targeting tissue-type plasminogen activator (t-PA) by covalent linkage to a fibrin-specific monoclonal antibody (59D8) produces a more potent thrombolytic agent which also induces less fibrinogenolysis. A recombinant molecule encoding a t-PA-59D8 fusion protein was constructed to provide a ready source of this agent for further study, and to allow tailoring of the active moities for maximal activity. DNA sequence coding for the 59D8 heavy chain (HC) antigen combining site was cloned from a lambdaphage library by selection with a joining region probe. Gene segments coding for this cloned HC rearrangement, the amino portion of the mouse gamma 2b HC constant region, and the catalytic B chain of t-PA were joined in the pSV2-gpt expression vector. The desired coding sequence was confirmed by nucleotide sequence analysis. The construct was transfected by electroporation into 59D8 hybridoma HC loss variants. Transfectants were screened for antifibrin antibody activity. Positive clones were shown to produce mRNA which hybridized to the human t-PA gene in Northern blot analysis. Supernatants from 5 of these clones were subjected to affinity chromatography on a synthetic fibrin-like peptide-Sepharose column followed by a benzamidine-Sepharose column. Western blots of SDS polyacrylamide gels run under reducing conditions revealed binding to a 60 kd band by a monoclonal antihuman t-PA antibody, consistent with a 59D8 HC-t-PA fusion protein. Also, binding to a 25 kd band by goat anti-mouse Fab indicated the presence of 59D8 light chain. Affinity purified protein was shown to have amidolytic activity of similar potency to t-PA in a chromogenic substrate assay utilizing S-2288. Bifunctionality of the purified protein was demonstrated first by an assay which requires the protein to bind to immobilized fibrin and simultaneously exhibit activity in the S2288 assay, and second by simultaneous fibrin and iodinated anti-t-PA binding.
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LeDuc, Philip R., and Michael J. Betenbaugh. "Implementation of a Pharmocokinetic Approach to a Baculovirus System for Analytic Solutions to Virus and Cell Interactions." In ASME 1997 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 1997. http://dx.doi.org/10.1115/imece1997-0282.
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Abstract The baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), expression system can be employed for a variety of different cellular applications. In recent years, this baculovirus system has been manipulated to serve as a recombinant system for the expression of heterologous proteins and as a possible retrovirus for gene therapy. A quantitative understanding of the cellular mechanics of virus trafficking would be useful in developing viral expression systems, understanding gene therapy and maximizing recombinant protein production. An analytic solution is presented which incorporates a pharmocokinetic system in order to analyze this problem of viral and cellular mechanics. The multiple stages of viral infection systems, specifically for the baculovirus system, include attachment, internalization, endosomal fusion, cytosol transportation, and nuclear accumulation. The effects of the rate parameters are investigated to determine the parameter sensitivity of the viral infection model in relation to accumulation of surface virus, internalized virus and nuclear virus. The concepts from this model can be used to design infection regimens for various cell lines and to analyze the inherent inefficiency of current baculovirus infecting systems. This approach can also be used to determine the effects of the rate-limiting behaviors exhibited by these types of cellular mechanics and can be further implemented to examine other types of infection applications including viral gene therapy [1].
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Giannelli, B. F. "MOLECULAR GENETICS OF HAEMOPHILIA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643981.
Texto completo da fonteResumo:
Haemophilia B, an X-linked recessive disease with an incidence of 1/30,000 newborn males, is due to defects in the gene for coagulation factor IX, which is on the long am of the X chromosome at band Xq27.1. This gene consists of approximately 34 Kb and contains 8 exons which specify a mRtfc of 2803 residues coding for a protein of 415 aa preceded by a prepro signal peptide of 46 aa. Coripanson of the functional domains of the factor IX protein with the exon structure of the gene supports the exon/protein domain hypothesis of gene evolution. The factor IX gene seems to be formed by a number of functionally and evolutionally independent modules. The signal peptide and the gla (γcarboxy-glutamic) region encoded in the first three exons are homologous to those of factor X, protein C and prothrombin. Thevfourth and fifth exons which code for the connecting peptide are homologous to one another and to the epidermal growth factor, a module that has been used in the construction of a great variety of proteins including different members of the coagulation and fibrinolytic pathways. The sixth exon encodes the activation peptide region, while the catalytic region of factor IX is coded by the seventh and eighth exon. This is at variance with other serine protease genes that have different exons for the segments containing the cardinal ami no-acids of the active centre (histidine, aspartic acid and serine).Natural selection acts against detrimental mutations of the factor IX gene and at each generation a proportion of haemophilia B genes is eliminated, as a significant number of patients does not reproduce. There appears to be no selective advantage to the heterozygote and therefore haemophilia B is maintained in the population by new mutations. Consequently, a significant proportion of patients should be born to non-carrier mothers, and unrelated patients should carry different gene defects, as recently verified by detailed analysis of individual haemophilia B genes.The defects of factor IX described so far comprise both point mutations and gene deletions. The latter affect either part or the whole of the gene and are often associated with the development of antibodies against therapeutically adninistered factor IX (the inhibitor complication). Since gene deletions may result in the complete absenceof factor IX synthesis or in the production of an extremely abnormal product, it has been suggested that mutationspreventing the synthesis of a factor IX gene product capable of inducing immune tolerance to normal factor IX is important in predisposing to the inhibitor complication.Among the point mutations described so far, those affecting the signal peptide are of particular interest. Substitutions of the arginine at positions -4 and -1 cause failure of propeptide cleavage. Thus they indicate that the propeptide consists of 18 aa an(lthat lts excision is necessary for factor IX function. It appears also that the propeptide contains a signal for γcarboxylation which has been conserved during the evolution of different γcarboxylated proteins.In spite of coagulant treatment, haemophilia B is a serious disease and one for which genetic counselling is required. Paramount for this is the detection of carriers and the diagnosis ofaffected male fetuses. DNA probes derived from the cloned factor IX gene have been used for this purpose. Carrier and first or second trimester prenatal diagnoses have been done using factors IX gene markers to follow the transmission of haemophilia B genes. Six sequence variations causing restriction fragment length polymorphisms (RFLP) in the factor IX gene have been detected and used as markers for such indirect diagnoses The efficiency of the above markers is reduced by linkage disequilibrium but, nevertheless, they offer definite carrier and nremtal diagnoses in 75-80% of the relatives of familial cases of haemophilia B.The indirect detection of gene defects is of modest help in the counselling of individuals from the families of isolated patients, but new methods for the direct detection of gene mutations promise better results in such families and also the attainment of % diagnostic success in relatives of familial cases.Finally the successful expression of recombinant factor IX genes in tissue culture and transgenic mammals raises hopes of therapeutic advances.
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Zhang, Y., W. McKeand, T. Yuraszeck, W. Seifert, A. Feussner, E. Santagostino, and J. Sidhu. "Population Pharmacokinetic Analysis of Recombinant Fusion Protein Linking Coagulation Factor IX with Recombinant Albumin (rIX-FP) in Adult and Pediatric Patients with Severe Hemophilia B." In 63rd Annual Meeting of the Society of Thrombosis and Haemostasis Research. Georg Thieme Verlag KG, 2019. http://dx.doi.org/10.1055/s-0039-1680223.
Texto completo da fonteRelatórios de organizações sobre o assunto "Recombinant proteins Analysis"
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Morrison, Mark, Joshuah Miron, Edward A. Bayer, and Raphael Lamed. Molecular Analysis of Cellulosome Organization in Ruminococcus Albus and Fibrobacter Intestinalis for Optimization of Fiber Digestibility in Ruminants. United States Department of Agriculture, March 2004. http://dx.doi.org/10.32747/2004.7586475.bard.
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Improving plant cell wall (fiber) degradation remains one of the highest priority research goals for all ruminant enterprises dependent on forages, hay, silage, or other fibrous byproducts as energy sources, because it governs the provision of energy-yielding nutrients to the host animal. Although the predominant species of microbes responsible for ruminal fiber degradation are culturable, the enzymology and genetics underpinning the process are poorly defined. In that context, there were two broad objectives for this proposal. The first objective was to identify the key cellulosomal components in Ruminococcus albus and to characterize their structural features as well as regulation of their expression, in response to polysaccharides and (or) P AA/PPA. The second objective was to evaluate the similarities in the structure and architecture of cellulosomal components between R. albus and other ruminal and non-ruminal cellulolytic bacteria. The cooperation among the investigators resulted in the identification of two glycoside hydrolases rate-limiting to cellulose degradation by Ruminococcus albus (Cel48A and CeI9B) and our demonstration that these enzymes possess a novel modular architecture specific to this bacterium (Devillard et al. 2004). We have now shown that the novel X-domains in Cel48A and Cel9B represent a new type of carbohydrate binding module, and the enzymes are not part of a ceiluiosome-like complex (CBM37, Xu et al. 2004). Both Cel48A and Cel9B are conditionally expressed in response to P AA/PPA, explaining why cellulose degradation in this bacterium is affected by the availability of these compounds, but additional studies have shown for the first time that neither PAA nor PPA influence xylan degradation by R. albus (Reveneau et al. 2003). Additionally, the R. albus genome sequencing project, led by the PI. Morrison, has supported our identification of many dockerin containing proteins. However, the identification of gene(s) encoding a scaffoldin has been more elusive, and recombinant proteins encoding candidate cohesin modules are now being used in Israel to verify the existence of dockerin-cohesin interactions and cellulosome production by R. albus. The Israeli partners have also conducted virtually all of the studies specific to the second Objective of the proposal. Comparative blotting studies have been conducted using specific antibodies prepare against purified recombinant cohesins and X-domains, derived from cellulosomal scaffoldins of R. flavefaciens 17, a Clostridium thermocellum mutant-preabsorbed antibody preparation, or against CbpC (fimbrial protein) of R. albus 8. The data also suggest that additional cellulolytic bacteria including Fibrobacter succinogenes S85, F. intestinalis DR7 and Butyrivibrio fibrisolvens Dl may also employ cellulosomal modules similar to those of R. flavefaciens 17. Collectively, our work during the grant period has shown that R. albus and other ruminal bacteria employ several novel mechanisms for their adhesion to plant surfaces, and produce both cellulosomal and non-cellulosomal forms of glycoside hydrolases underpinning plant fiber degradation. These improvements in our mechanistic understanding of bacterial adhesion and enzyme regulation now offers the potential to: i) optimize ruminal and hindgut conditions by dietary additives to maximize fiber degradation (e.g. by the addition of select enzymes or PAA/PPA); ii) identify plant-borne influences on adhesion and fiber-degradation, which might be overcome (or improved) by conventional breeding or transgenic plant technologies and; iii) engineer or select microbes with improved adhesion capabilities, cellulosome assembly and fiber degradation. The potential benefits associated with this research proposal are likely to be realized in the medium term (5-10 years).
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Fluhr, Robert, and Maor Bar-Peled. Novel Lectin Controls Wound-responses in Arabidopsis. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7697123.bard.
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Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.
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Sadot, Einat, Christopher Staiger, and Mohamad Abu-Abied. Studies of Novel Cytoskeletal Regulatory Proteins that are Involved in Abiotic Stress Signaling. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7592652.bard.
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In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells. Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional myosins: myosin XI and myosin VIII, both closely related to myosin V (17-19). The Arabidopsis myosin family contains 17 members: 13 myosin XI and four myosin VIII (19, 20). The data obtained from our research of myosins was published in two papers acknowledging BARD funding. To address whether specific myosins are involved with the motility of specific organelles, we cloned the cDNAs from neck to tail of all 17 Arabidopsis myosins. These were fused to GFP and used as dominant negative mutants that interact with their cargo but are unable to walk along actin filaments. Therefore arrested organelle movement in the presence of such a construct shows that a particular myosin is involved with the movement of that particular organelle. While no mutually exclusive connections between specific myosins and organelles were found, based on overexpression of dominant negative tail constructs, a group of six myosins (XIC, XIE, XIK, XI-I, MYA1 and MYA2) were found to be more important for the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum (8). Further deep and thorough analysis of myosin XIK revealed a potential regulation by head and tail interaction (Avisar et al., 2011). A similar regulatory mechanism has been reported for animal myosin V and VIIa (21, 22). In was shown that myosin V in the inhibited state is in a folded conformation such that the tail domain interacts with the head domain, inhibiting its ATPase and actinbinding activities. Cargo binding, high Ca2+, and/or phosphorylation may reduce the interaction between the head and tail domains, thus restoring its activity (23). Our collaborative work focuses on the characterization of the head tail interaction of myosin XIK. For this purpose the Israeli group built yeast expression vectors encoding the myosin XIK head. In addition, GST fusions of the wild-type tail as well as a tail mutated in the amino acids that mediate head to tail interaction. These were sent to the US group who is working on the isolation of recombinant proteins and performing the in vitro assays. While stress signals involve changes in Ca2+ levels in plants cells, the cytoplasmic streaming is sensitive to Ca2+. Therefore plant myosin activity is possibly regulated by stress. This finding is directly related to the goal of the original proposal.
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Dolja, Valerian V., Amit Gal-On, and Victor Gaba. Suppression of Potyvirus Infection by a Closterovirus Protein. United States Department of Agriculture, March 2002. http://dx.doi.org/10.32747/2002.7580682.bard.
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The plant virus family Polyviridae is the largest and most destructive of all plant viruses. Despite the continuous effort to develop resistant plant varieties, there is a desperate need for novel approaches conferring wide-range potyvirus resistance. Based on experiments with the tobacco etch potyvirus (TEV)-derived gene expression vector, we suggested approach for screening of the candidate resistance genes. This approach relies on insertion of the genes into a virus vector and evaluation of the phenotypes of the resulting recombinant viruses. The genes which suppress infection by the recombinant virus are selected as candidates for engineering transgenic resistance. Our analysis of the TEV variants expressing proteins of the beet yellows closterovirus (BYV) revealed that one of those, the leader proteinase (L-Pro), strongly and specifically interfered with the hybrid TEV infection. Since closterovirus L-Pro is evolutionary related to potyviral helper component-proteinase (HC-Pro), we suggested that the L-Pro interfered with HC-Pro function via a trans-dominant inhibitory effect. Based on these findings, we proposed to test two major hypotheses. First, we suggested that L-Pro-mediated suppression of potyvirus infection is a general phenomenon effective against a range of potyviruses. The second hypothesis stated that the suppression effect can be reproduced in transgenic plants expressing L-Pro, and can be utilized for generation of resistance to potyviruses. In accord with these hypotheses, we developed two original objectives of our proposal: A) to determine the range of the closterovirus-derived suppression of potyviral infection, and B) to try and utilize the L-Pro-mediated suppression for the development of transgenic resistance to potyviruses. In the first phase of the project, we have developed all major tools and technologies required for successful completion of the proposed research. These included TEV and ZYMV vectors engineered to express several closteroviral L-Pro variants, and generation of the large collection of transgenic plants. To our satisfaction, characterization of the infection phenotypes exhibited by chimeric TEV and ZYMV variants confirmed our first hypothesis. For instance, similar to TEV-L- Pro(BYV) chimera, ZYMV-L-Pro(LIYV) chimera was debilitated in its systemic spread. In contrast, ZYMV-GUS chimera (positive control) was competent in establishing vigorous systemic infection. These and other results with chimeric viruses indicated that several closteroviral proteinases inhibit long-distance movement of the potyviruses upon co-expression in infected plants. In order to complete the second objective, we have generated ~90 tobacco lines transformed with closteroviral L-Pro variants, as well as ~100 lines transformed with BYV Hsp70-homolog (Hsp70h; a negative control). The presence and expression of the trans gene in each line was initially confirmed using RT-PCR and RNA preparations isolated from plants. However, since detection of the trans gene-specific RNA can not guarantee production of the corresponding protein, we have also generated L-Pro- and Hsp70h-specific antisera using corresponding synthetic peptides. These antisera allowed us to confirm that the transgenic plant lines produced detectable, although highly variable levels of the closterovirus antigens. In a final phase of the project, we tested susceptibility of the transgenic lines to TEV infection. To this end, we determined that the minimal dilution of the TEV inoculum that is still capable of infecting 100% of nontransgenic plants was 1:20, and used 10 plants per line (in total, ~2,000 plants). Unfortunately, none of the lines exhibited statistically significant reduction in susceptibility. Although discouraging, this outcome prompted us to expand our experimental plan and conduct additional experiments. Our aim was to test if closteroviral proteinases are capable of functioning in trans. We have developed agroinfection protocol for BYV, and tested if co- expression of the L-Pro is capable of rescuing corresponding null-mutant. The clear-cut, negative results of these experiments demonstrated that L-Pro acts only in cis, thus explaining the lack of resistance in our transgenic plants. We have also characterized a collection of the L-Pro alanine- scanning mutants and found direct genetic evidence of the requirement for L-Pro in virus systemic spread. To conclude, our research supported by BARD confirmed one but not another of our original hypotheses. Moreover, it provided an important insight into functional specialization of the viral proteinases and generated set of tools and data with which we will be able to address the molecular mechanisms by which these proteins provide a variety of critical functions during virus life cycle.
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Eyal, Yoram, and Sheila McCormick. Molecular Mechanisms of Pollen-Pistil Interactions in Interspecific Crossing Barriers in the Tomato Family. United States Department of Agriculture, May 2000. http://dx.doi.org/10.32747/2000.7573076.bard.
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During the evolutionary process of speciation in plants, naturally occurring barriers to reproduction have developed that affect the transfer of genes within and between related species. These barriers can occur at several different levels beginning with pollination-barriers and ending with hybrid-breakdown. The interaction between pollen and pistils presents one of the major barriers to intra- and inter-specific crosses and is the focus of this research project. Our long-term goal in this research proposal was defined to resolve questions on recognition and communication during pollen-pistil interactions in the extended tomato family. In this context, this work was initiated and planned to study the potential involvement of tomato pollen-specific receptor-like kinases (RLK's) in the interaction between pollen and pistils. By special permission from BARD the objectives of this research were extended to include studies on pollen-pistil interactions and pollination barriers in horticultural crops with an emphasis on citrus. Functional characterization of 2 pollen-specific RLK's from tomato was carried out. The data shows that both encode functional kinases that were active as recombinant proteins. One of the kinases was shown to accumulate mainly after pollen germination and to be phosphorylated in-vitro in pollen membranes as well as in-vivo. The presence of style extract resulted in dephosphorylation of the RLK, although no species specificity was observed. This data implies a role for at least one RLK in pollination events following pollen germination. However, a transgenic plant analysis of the RLK's comprising overexpression, dominant-negative and anti-sense constructs failed to provide answers on their role in pollination. While genetic effects on some of the plants were observed in both the Israeli and American labs, no clear functional answers were obtained. An alternative approach to addressing function was pursued by screening for an artificial ligand for the receptor domain using a peptide phage display library. An enriched peptide sequence was obtained and will be used to design a peptide-ligand to be tested for its effect o pollen germination and tube growth. Self-incompatibility (SI) in citrus was studied on 3 varieties of pummelo. SI was observed using fluorescence microscopy in each of the 3 varieties and compatibility relations between varieties was determined. An initial screen for an S-RNase SI mechanism yielded only a cDNA homologous to the group of S-like RNases, suggesting that SI results from an as yet unknown mechanism. 2D gel electrophoresis was applied to compare pollen and style profiles of different compatibility groups. A "polymorphic" protein band from style extracts was observed, isolated and micro-sequenced. Degenerate primers designed based on the peptide sequence date will be used to isolate the relevant genes i order to study their potential involvement in SI. A study on SI in the apple cultivar Top red was initiated. SI was found, as previously shown, to be complete thus requiring a compatible pollinator variety. A new S-RNase allele was discovered fro Top red styles and was found to be highly homologous to pear S-RNases, suggesting that evolution of these genes pre-dated speciation into apples and pears but not to other Rosaceae species. The new allele provides molecular-genetic tools to determine potential pollinators for the variety Top red as well as a tool to break-down SI in this important variety.
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Eldar, Avigdor, and Donald L. Evans. Streptococcus iniae Infections in Trout and Tilapia: Host-Pathogen Interactions, the Immune Response Toward the Pathogen and Vaccine Formulation. United States Department of Agriculture, December 2000. http://dx.doi.org/10.32747/2000.7575286.bard.
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In Israel and in the U.S., Streptococcus iniae is responsible for considerable losses in various fish species. Poor understanding of its virulence factors and limited know-how-to of vaccine formulation and administration are the main reasons for the limited efficacy of vaccines. Our strategy was that in order to Improve control measures, both aspects should be equally addressed. Our proposal included the following objectives: (i) construction of host-pathogen interaction models; (ii) characterization of virulence factors and immunodominant antigens, with assessment of their relative importance in terms of protection and (iii) genetic identification of virulence factors and genes, with evaluation of the protective effect of recombinant proteins. We have shown that two different serotypes are involved. Their capsular polysaccharides (CPS) were characterized, and proved to play an important role in immune evasion and in other consequences of the infection. This is an innovative finding in fish bacteriology and resembles what, in other fields, has become apparent in the recent years: S. iniae alters surface antigens. By so doing, the pathogen escapes immune destruction. Immunological assays (agar-gel immunodiffusion and antibody titers) confirmed that only limited cross recognition between the two types occurs and that capsular polysaccharides are immunodominant. Vaccination with purified CPS (as an acellular vaccine) results in protection. In vitro and ex-vivo models have allowed us to unravel additional insights of the host-pathogen interactions. S. iniae 173 (type II) produced DNA fragmentation of TMB-8 cells characteristic of cellular necrosis; the same isolate also prevented the development of apoptosis in NCC. This was determined by finding reduced expression of phosphotidylserine (PS) on the outer membrane leaflet of NCC. NCC treated with this isolate had very high levels of cellular necrosis compared to all other isolates. This cellular pathology was confirmed by observing reduced DNA laddering in these same treated cells. Transmission EM also showed characteristic necrotic cellular changes in treated cells. To determine if the (in vitro) PCD/apoptosis protective effects of #173 correlated with any in vivo activity, tilapia were injected IV with #173 and #164 (an Israeli type I strain). Following injection, purified NCC were tested (in vitro) for cytotoxicity against HL-60 target cells. Four significant observations were made : (i) fish injected with #173 had 100-400% increased cytotoxicity compared to #164 (ii) in vivo activation occurred within 5 minutes of injection; (iii) activation occurred only within the peripheral blood compartment; and (iv) the isolate that protected NCC from apoptosis in vitro caused in vivo activation of cytotoxicity. The levels of in vivo cytotoxicity responses are associated with certain pathogens (pathogen associated molecular patterns/PAMP) and with the tissue of origin of NCC. NCC from different tissue (i.e. PBL, anterior kidney, spleen) exist in different states of differentiation. Random amplified polymorphic DNA (RAPD) analysis revealed the "adaptation" of the bacterium to the vaccinated environment, suggesting a "Darwinian-like" evolution of any bacterium. Due to the selective pressure which has occurred in the vaccinated environment, type II strains, able to evade the protective response elicited by the vaccine, have evolved from type I strains. The increased virulence through the appropriation of a novel antigenic composition conforms with pathogenic mechanisms described for other streptococci. Vaccine efficacy was improved: water-in-oil formulations were found effective in inducing protection that lasted for a period of (at least) 6 months. Protection was evaluated by functional tests - the protective effect, and immunological parameters - elicitation of T- and B-cells proliferation. Vaccinated fish were found to be resistant to the disease for (at least) six months; protection was accompanied by activation of the cellular and the humoral branches.
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Raghothama, Kashchandra G., Avner Silber, and Avraham Levy. Biotechnology approaches to enhance phosphorus acquisition of tomato plants. United States Department of Agriculture, January 2006. http://dx.doi.org/10.32747/2006.7586546.bard.
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Abstract: Phosphorus is one of the least available macronutrient in the soil. The high affinity phosphate transporters are known to be associated with phosphate acquisition under natural conditions. Due to unique interactions of phosphate with soil particles, up to 80% of the applied phosphates may be fixed forcing the farmers to apply 4 to 5 times the fertilizers necessary for crop production. Efficient uptake and utilization of this essential nutrient is essential for sustainability and profitability of agriculture. Many predictions point to utilization/exhaustion of high quality phosphate rocks within this century. This calls for efforts to improve the ability of plants to acquire and utilize limiting sources of phosphate in the rhizosphere. Two important molecular and biochemical components associated with phosphate efficiency are phosphate transporters and phosphatases. This research project is aimed at defining molecular determinants of phosphate acquisition and utilization in addition to generating phosphate uptake efficient plants. The main objectives of the project were; Creation and analysis of transgenic tomato plants over-expressing phosphatases and transporters Characterization of the recently identified members (LePT3 and LePT4) of the Pi transporter family Generate molecular tools to study genetic responses of plants to Pi deficiency During the project period we have successfully identified and characterized a novel phosphate transporter associated with mycorrhizal symbiosis. The expression of this transporter increases with mycorrhizal symbiosis. A thorough characterization of mutant tomato lacking the expression of this gene revealed the biological significance of LePT3 and another novel gene LePT4. In addition we have isolated and characterized several phosphate starvation induced genes from tomato using a combination of differential and subtractive mRNA hybridization techniques. One of the genes, LePS2 belongs to the family of phospho-protein phosphatase. The functionality of the recombinant protein was determined using synthetic phosphor-peptides. Over expression of this gene in tomato resulted in significant changes in growth, delay in flowering and senescence. It is anticipated that phospho-protein phosphatase may have regulatory role in phosphate deficiency responses of plants. In addition a novel phosphate starvation induced glycerol 3-phosphate permease gene family was also characterized. Two doctoral research students are continuing the characterization and functional analysis of these genes. Over expression of high affinity phosphate transporters in tobacco showed increased phosphate content under hydroponic conditions. There is growing evidence suggesting that high affinity phosphate transporters are crucial for phosphate acquisition even under phosphate sufficiency conditions. This project has helped train several postdoctoral fellows and graduate students. Further analysis of transgenic plants expressing phosphatases and transporters will not only reveal the biological function of the targeted genes but also result in phosphate uptake and utilization efficient plants.
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Funkenstein, Bruria, and Shaojun (Jim) Du. Interactions Between the GH-IGF axis and Myostatin in Regulating Muscle Growth in Sparus aurata. United States Department of Agriculture, March 2009. http://dx.doi.org/10.32747/2009.7696530.bard.
Texto completo da fonteResumo:
Growth rate of cultured fish from hatching to commercial size is a major factor in the success of aquaculture. The normal stimulus for muscle growth in growing fish is not well understood and understanding the regulation of muscle growth in fish is of particular importance for aquaculture. Fish meat constitutes mostly of skeletal muscles and provides high value proteins in most people's diet. Unlike mammals, fish continue to grow throughout their lives, although the size fish attain, as adults, is species specific. Evidence indicates that muscle growth is regulated positively and negatively by a variety of growth and transcription factors that control both muscle cell proliferation and differentiation. In particular, growth hormone (GH), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs) and transforming growth factor-13 (TGF-13) play critical roles in myogenesis during animal growth. An important advance in our understanding of muscle growth was provided by the recent discovery of the crucial functions of myostatin (MSTN) in controlling muscle growth. MSTN is a member of the TGF-13 superfamily and functions as a negative regulator of skeletal muscle growth in mammals. Studies in mammals also provided evidence for possible interactions between GH, IGFs, MSTN and the musclespecific transcription factor My oD with regards to muscle development and growth. The goal of our project was to try to clarify the role of MSTNs in Sparus aurata muscle growth and in particular determine the possible interaction between the GH-IGFaxis and MSTN in regulating muscle growth in fish. The steps to achieve this goal included: i) Determining possible relationship between changes in the expression of growth-related genes, MSTN and MyoD in muscle from slow and fast growing sea bream progeny of full-sib families and that of growth rate; ii) Testing the possible effect of over-expressing GH, IGF-I and IGF-Il on the expression of MSTN and MyoD in skeletal muscle both in vivo and in vitro; iii) Studying the regulation of the two S. aurata MSTN promoters and investigating the possible role of MyoD in this regulation. The major findings of our research can be summarized as follows: 1) Two MSTN promoters (saMSTN-1 and saMSTN-2) were isolated and characterized from S. aurata and were found to direct reporter gene activity in A204 cells. Studies were initiated to decipher the regulation of fish MSTN expression in vitro using the cloned promoters; 2) The gene coding for saMSTN-2 was cloned. Both the promoter and the first intron were found to be polymorphic. The first intron zygosity appears to be associated with growth rate; 3) Full length cDNA coding for S. aurata growth differentiation factor-l I (GDF-II), a closely related growth factor to MSTN, was cloned from S. aurata brain, and the mature peptide (C-terminal) was found to be highly conserved throughout evolution. GDF-II transcript was detected by RT -PCR analysis throughout development in S. aurata embryos and larvae, suggesting that this mRNA is the product of the embryonic genome. Transcripts for GDF-Il were detected by RT-PCR in brain, eye and spleen with highest level found in brain; 4) A novel member of the TGF-Bsuperfamily was partially cloned from S. aurata. It is highly homologous to an unidentified protein (TGF-B-like) from Tetraodon nigroviridisand is expressed in various tissues, including muscle; 5) Recombinant S. aurata GH was produced in bacteria, refolded and purified and was used in in vitro and in vivo experiments. Generally, the results of gene expression in response to GH administration in vivo depended on the nutritional state (starvation or feeding) and the time at which the fish were sacrificed after GH administration. In vitro, recombinantsaGH activated signal transduction in two fish cell lines: RTHI49 and SAFI; 6) A fibroblastic-like cell line from S. aurata (SAF-I) was characterized for its gene expression and was found to be a suitable experimental system for studies on GH-IGF and MSTN interactions; 7) The gene of the muscle-specific transcription factor Myogenin was cloned from S. aurata, its expression and promoter activity were characterized; 8) Three genes important to myofibrillogenesis were cloned from zebrafish: SmyDl, Hsp90al and skNAC. Our data suggests the existence of an interaction between the GH-IGFaxis and MSTN. This project yielded a great number of experimental tools, both DNA constructs and in vitro systems that will enable further studies on the regulation of MSTN expression and on the interactions between members of the GHIGFaxis and MSTN in regulating muscle growth in S. aurata.
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Mawassi, Munir, and Valerian Dolja. Role of RNA Silencing Suppression in the Pathogenicity and Host Specificity of the Grapevine Virus A. United States Department of Agriculture, January 2010. http://dx.doi.org/10.32747/2010.7592114.bard.
Texto completo da fonteResumo:
RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair.