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Artykuły w czasopismach na temat "Recombinase protein"
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Price, Candice, i Isabel Darcy. "Application of a skein relation to difference topology experiments". Journal of Knot Theory and Its Ramifications 28, nr 13 (listopad 2019): 1940016. http://dx.doi.org/10.1142/s0218216519400169.
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Difference topology is a technique used to study any protein that can stably bind to DNA. This technique is used to determine the conformation of DNA bound by protein. Motivated by difference topology experiments, we use the skein relation tangle model as a novel technique to study experiments using topoisomerase to study SMC proteins, a family of proteins that stably bind to DNA. The oriented skein relation involves an oriented knot, [Formula: see text], with a distinguished positive crossing; a knot [Formula: see text], obtained by changing the distinguished positive crossing of [Formula: see text] to a negative crossing; a knot, [Formula: see text], resulting from the non-orientation persevering resolution of the distinguished crossing; and a link [Formula: see text], the orientation preserving resolution of the distinguished crossing. We refer to [Formula: see text] as the skein quadruple. Topoisomerases are proteins that break one segment of DNA allowing a DNA segment to pass through before resealing the break. Recombinases are proteins that cut two segments of DNA and recombine them in some manner. They can act on direct repeat or inverted repeat sites, resulting in a link or knot, respectively. Thus, the skein quadruple is now viewed as [Formula: see text] circular DNA substrate, [Formula: see text] product of topoisomerase action, [Formula: see text] product of recombinase action on directed repeat sites, and [Formula: see text] product of recombinase action of inverted repeat sites.
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Briones, Gabriel, Dirk Hofreuter i Jorge E. Galán. "Cre Reporter System To Monitor the Translocation of Type III Secreted Proteins into Host Cells". Infection and Immunity 74, nr 2 (luty 2006): 1084–90. http://dx.doi.org/10.1128/iai.74.2.1084-1090.2006.
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ABSTRACT Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III secretion system-dependent fashion. More importantly, the SopE-Cre chimera was translocated into host cells via the type III secretion system and activated the expression of luciferase and green fluorescent protein reporters of Cre recombinase activity.
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Letunic, Ivica, Supriya Khedkar i Peer Bork. "SMART: recent updates, new developments and status in 2020". Nucleic Acids Research 49, nr D1 (26.10.2020): D458—D460. http://dx.doi.org/10.1093/nar/gkaa937.
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Abstract SMART (Simple Modular Architecture Research Tool) is a web resource (https://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 9 contains manually curated models for more than 1300 protein domains, with a topical set of 68 new models added since our last update article (1). All the new models are for diverse recombinase families and subfamilies and as a set they provide a comprehensive overview of mobile element recombinases namely transposase, integrase, relaxase, resolvase, cas1 casposase and Xer like cellular recombinase. Further updates include the synchronization of the underlying protein databases with UniProt (2), Ensembl (3) and STRING (4), greatly increasing the total number of annotated domains and other protein features available in architecture analysis mode. Furthermore, SMART’s vector-based protein display engine has been extended and updated to use the latest web technologies and the domain architecture analysis components have been optimized to handle the increased number of protein features available.
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Marshall Stark, W., Martin R. Boocock, Femi J. Olorunniji i Sally-J. Rowland. "Intermediates in serine recombinase-mediated site-specific recombination". Biochemical Society Transactions 39, nr 2 (22.03.2011): 617–22. http://dx.doi.org/10.1042/bst0390617.
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Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer–dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated.
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Orth, Peter, Petra Jekow, Juan C. Alonso i Winfried Hinrichs. "Proteolytic cleavage of Gram-positive β recombinase is required for crystallization". Protein Engineering, Design and Selection 12, nr 5 (maj 1999): 371–73. http://dx.doi.org/10.1093/protein/12.5.371.
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Chen, J. W., B. R. Evans, S. H. Yang, H. Araki, Y. Oshima i M. Jayaram. "Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine". Molecular and Cellular Biology 12, nr 9 (wrzesień 1992): 3757–65. http://dx.doi.org/10.1128/mcb.12.9.3757-3765.1992.
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The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.
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Chen, J. W., B. R. Evans, S. H. Yang, H. Araki, Y. Oshima i M. Jayaram. "Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine." Molecular and Cellular Biology 12, nr 9 (wrzesień 1992): 3757–65. http://dx.doi.org/10.1128/mcb.12.9.3757.
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The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.
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Koornneef, Lieke, Johan A. Slotman, Esther Sleddens-Linkels, Wiggert A. van Cappellen, Marco Barchi, Attila Tóth, Joost Gribnau, Adriaan B. Houtsmuller i Willy M. Baarends. "Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure". PLOS Genetics 18, nr 7 (20.07.2022): e1010046. http://dx.doi.org/10.1371/journal.pgen.1010046.
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Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.
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Meador, Kyle, Christina L. Wysoczynski, Aaron J. Norris, Jason Aoto, Michael R. Bruchas i Chandra L. Tucker. "Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent". Nucleic Acids Research 47, nr 17 (9.07.2019): e97-e97. http://dx.doi.org/10.1093/nar/gkz585.
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AbstractA common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.
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Dreyfus, David. "RAG-1 (Recombination Activating Gene-1) protein is closely related to herpes virus recombinases: Implications for the origins of the acquired immune system. (105.20)". Journal of Immunology 188, nr 1_Supplement (1.05.2012): 105.20. http://dx.doi.org/10.4049/jimmunol.188.supp.105.20.
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Abstract The acquired immune system results from recombination of germ-line DNA V(D)J signal sequences by the RAG-1 recombinase. RAG-1 is a dde magnesium binding recombinase related to both retroviral integrases and transposases. However the closest phylogenetic relative to RAG-1 appears to be a family of recombinases encoded by the herpesviridae termed the DBP (DNA binding proteins) that catalyze strand exchange and possibly other recombination events during herpes virus replication. Both the dde magnesium binding triad and the nonamer binding regions of RAG-1 are present and conserved in the DBP. These observations suggest that initially RAG-1 arose from an ancient insertion of a herpes-like infectious agent into a deuterostome genome prior to the origins of the acquired immune system. The author proposes a model in which the primordial RAG-1 recombinase was selected through its ability to promote viral immune responses through innate immune receptors in ancient deuterostomes prior to the origins of V(D)J recombination.
Rozprawy doktorskie na temat "Recombinase protein"
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Liu, Siyu. "Dynamics of Rad51 during homologous recombination in living yeast". Electronic Thesis or Diss., Université Paris sciences et lettres, 2022. http://www.theses.fr/2022UPSLS050.
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L'ADN est le principal vecteur d'information génétique et son intégrité est vitale pour la survie des cellules. Pourtant, l'ADN est sous la pression des dommages causés par des facteurs exogènes et endogènes. Les cassures double brin (CDB) sont parmi les dommages les plus toxiques puisqu’une CDB non réparée peut être léthale. Les cellules ont développé plusieurs voies pour réparer les CDB, dont la jonction d'extrémités non homologues (NHEJ) et la recombinaison homologue (RH). RH est une voie de réparation fidèle qui utilise une séquence homologue intacte comme modèle pour réparer les dommages. Cela implique d'identifier la séquence homologue parmi les mégabases du génome et dans le volume nucléaire des cellules eucaryotes. Au niveau moléculaire, la recherche d’homologie de l'ADN et l'invasion de l’ADN double brin homologue sont réalisées par un filament nucléoprotéique (NPF), formé par la recombinase, RecA chez les bactéries et Rad51 chez les eucaryotes, associée à l'ADN simple brin flanquant la CDB. Ce mécanisme a été largement étudié in vitro et in vivo par des approches génétiques et moléculaires au niveau des populations cellulaires, mais sa dynamique n'a pas pu être étudiée dans les cellules vivantes faute de version fluorescente fonctionnelle de Rad51. Ainsi, comment l'ADN brisé peut-il trouver une séquence homologue dans le volume du noyau et parmi les mégabases d'ADN reste mystérieux.Sur la base d’études structurales, en collaboration avec Raphael Guerois (I2BC, CEA, France), nous avons développé et caractérisé la première version fonctionnelle étiquetée d'une recombinase chez la levure S. cerevisiae.Suite à l'induction de DSB unique, nous observons pour la première fois dans des cellules vivantes, Rad51 formant des filaments pouvant atteindre un ou deux microns de longueur et traverser le volume du noyau pour contacter une séquence homologue. Comme prédit par des études génétiques et des données obtenues in vitro, leur formation nécessite le chargeur de recombinase Rad52 et la formation d'un long fragment d'ADNsb. De plus, les filaments Rad51 adoptent une variété de formes qui sont modulées par des facteurs auxiliaires de Rad51, apportant un nouvel éclairage sur la fonction de ces facteurs dans les cellules vivantes.Contrairement à ce qui a été rapporté pour les filaments RecA chez les bactéries, les filaments Rad51 montrent un comportement étonnamment dynamique : avec de fréquents événements de compaction suivis d’une ré-extension offrant des opportunités pour le filament d'être projeté dans une zone nucléaire différente, et ainsi d'explorer de nouvelles régions génomiques. La modélisation biophysique du processus de recherche d'homologie par notre collaborateur Leonid Mirny (MIT, USA) révèle que ces cycles de compaction/extension constituent une stratégie très robuste pour qu'une identité unique trouve sa cible dans l'espace nucléaireDNA is the major carrier of genetic information in prokaryotic and eukaryotic cells and its integrity is vital for the survival of cells. However, DNA is under pressure of damages caused by both exogenous and endogenous factors. Double strand break (DSB) is one of the most toxic DNA damages and even one unrepaired DSB is lethal to cells. Cells have evolved several pathways to repair DSBs, including non-homologous end joining (NHEJ), and homologous recombination (HR). HR is an error free repair pathway that uses an intact homologous sequence as a template to repair the damage. This involves identifying the homologous sequence among the mega bases of the genome and in the nuclear volume of eukaryotic cells. At the molecular level, DNA sampling and strand invasion of the homologous dsDNA is achieved by a nucleoprotein filament (NPF), formed by the recombinase, RecA in bacteria and Rad51 in eukaryotes, coating ssDNA. This mechanism has been extensively studied in vitro and in vivo through genetic and molecular approaches at the level of cell populations, but its dynamics could not be studied in living cells due to lack of functional fluorescent version of Rad51. Thus, how broken DNA can find a homologous sequence in the volume of the nucleus and among the megabases of DNA remains mysterious.Thanks to structural insights from our collaborator Raphael Guerois (I2BC, CEA, France), we developed and characterized the first functional, internally tagged version of a recombinase in the yeast S. cerevisiae. Following the induction of unique DSB, we observe for the first time in living cells, Rad51 forming micrometer long filaments spanning across the whole nucleus and contacting the donor sequence. As predicted from genetic and in vitro data, their formation requires the recombinase loader Rad52 and the formation of long stretch of ssDNA. Furthermore, emerging filaments adopt a variety of shapes, not reported in vitro and modulated by Rad51 ancillary factors, shedding new light on the function of these factors in living cells.In contrast to what has been reported for RecA filaments in bacteria, Rad51 filaments show a surprisingly dynamic behavior: with frequent compaction events followed by re-extension providing opportunities for the NPF to be projected into a different nuclear area, and thus explore new genomic regions. Biophysical modeling of the homology search process by our collaborator Leonid Mirny (MIT, USA) reveals that these cycles of compaction/extension constitute a very robust strategy for a unique identity to find its target in the nuclear space
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Zhekov, Ivailo. "Dissection of a functional interaction between the XerD recombinase and the DNA translocase FtsK". Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572642.
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Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsK(C), which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex. We show that a 62-amino-acid fragment of FtsK(C) interacts directly with the XerD C-terminus in order to stimulate the cleavage by XerD of BSN, a dif-DNA suicide substrate containing a nick in the 'bottom' strand. The resulting recombinase-DNA covalent complex can undergo strand exchange with intact duplex dif in the absence of ATP. FtsK(C)-mediated stimulation of BSN cleavage by XerD requires synaptic complex formation. Mutational impairment of the XerD-FtsK(C) interaction leads to reduction in the in vitro stimulation of BSN cleavage by XerD and a concomitant deficiency in the resolution of chromosomal dimers at dif in vivo, although other XerD functions are not affected.
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Corbett, Sybilla Louise. "Nanoscale patterning of complex DNA structures with the bacterial protein Recombinase A". Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15373/.
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The use of DNA as a structural material has been intensively developed since its inception in the early 1980s. The potential of DNA structures in the field of materials science is hampered by current approaches to augmentation. It is not currently possible to alter the targeting of heterogenous additional elements to structures once they have been made. The post hoc patterning of DNA architectures is therefore of great importance. The bacterial protein Recombinase A (RecA) may be able to provide this function. This thesis will discuss the patterning of DNA structures with RecA. RecA has been shown to pattern linear dsDNA strands with high levels of efficiency. To test the potential of RecA to pattern more complex DNA, novel strategies for creating DNA topologies have been explored. This work has produced DNA strands containing regions of base pair mismatching and with terminal three-way junctions. A method has also been developed for the creation of a 200 base product with unpaired branched junctions, using four synthetic oligomers in a scaffolded cycling ligation reaction with a heat stable ligase. A method to create longer DNA strands with three-way junctions at the termini has also been developed. RecA patterning of a structure with internal mismatches was carried out. Mismatches proximal to the patterning area led to an increase in patterning efficiency with an increase in mismatch length. When the mismatch was separated from the patterning region a more complex relationship was observed, with intermediate-length mismatches resulting in a decrease in pattering efficiency. The introduction of a nick in the phosphate backbone proximal to the patterning region also increased patterning efficiency. Two further DNA structures were produced on which patterning did not prove possible. The ligase chain reaction was shown to produce DNA strands that could be incorporated into a structure with central base pairing and terminal single stranded DNA regions. Attempts to create three-way junctions from these structures were not successful. A second structure was created through treatment of double stranded DNA from the polymerase chain reaction. Single strands of DNA were produced that could be annealed to produce terminal three-way junctions. Atomic force microscopy demonstrated the correct annealing of this structure. However, it did not prove possible to pattern these structures with RecA. Recombinant RecA production through bacterial induction produced soluble protein at a high yield. There was some evidence of DNA contamination and the purified protein showed low activity.
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Yu, David Sung-wen. "Role of the BRCA2 breast cancer susceptibility protein in control of RAD51 recombinase". Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620033.
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Bates, D. L. "Control of the RAD51 recombinase by the BRC repeat motifs in the breast cancer susceptibility protein BRCA2". Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596469.
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The interaction between the breast cancer susceptibility protein BRCA2 and the RAD51 DNA recombinase is essential for DNA repair via homologous recombination. Its disruption in human cells causes genomic instability and cancer predisposition. Studies to define the biochemical and biological features of the BRCA2:RAD51 interaction are described. Human BRCA2 features eight BRC repeat motifs encoded within exon 11 through which it can bind RAD51. The BRC repeat motifs are believed to mimic and disrupt RAD51 oligomerisation at its self-association interface. I defined the minimal structural determinants required for RAD51 binding by ‘humanising’ a primitive RAD51 from an Archaeon species lacking BRCA2. Surface Plasmon Resonance (SPR) technology supported by cell biology was employed to study the characteristics of RAD51 binding to each of the BRC repeat motifs, both independently and collectively within the context of the BRCA2 protein. A single point mutation within an individual BRC repeat motif impaired RAD51 binding. Further, RAD51 was unable to interact effectively with a BRC repeat motif in which the proposed interaction interface had been disrupted by mutagenesis. Kinetic data for the interaction of an individual BRC repeat motif with RAD51 were obtained. An SPR competition assay was developed, revealing that the binding affinity of each BRC repeat motif for RAD51 differs significantly, and that their organisation within the scaffold of BRCA2 contributes to efficient interaction. Thus, I propose that both the differential binding affinities of the individual BRC repeat motifs for RAD51, and their observed cooperativity, contribute to the control of RAD51 by BRCA2. The implications of this proposal for DNA repair via homologous recombination, and for the role of BRCA2 mutations in human carcinogenesis, are discussed.
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Amero, Carlos D. "Protein Function Study by NMR Spectroscopy". The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1205431343.
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Ghorbal, Mehdi. "Caractérisation biochimique, fonctionnelle et structurale de l'integrase Pf-Int de plasmodium". Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00685428.
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Plasmodium falciparum est un parasite protozoaire responsable de la forme la plus sévère de la malaria. Depuis quelques années, les cas de résistance aux antipaludiques sont devenus de plus en plus fréquents et de plus en plus répandus. En plus de sa résistance aux drogues actuellement disponibles, ce parasite reste jusqu' à aujourd'hui réfractaire aux vaccinations. L'identification de nouvelles approches basées sur l'inhibition spécifique de certaines de ses cibles moléculaires vitales est devenue une nécessité. La recombinase à site spécifique de P. falciparum (Pf-Int) est un enzyme qui a été récemment identifié dans le laboratoire à partir de PlasmoDB. Cette recombinase à site spécifique joue potentiellement un rôle clé dans le système de recombinaison nécessaire à la viabilité du parasite. Cette protéine de 490 acides aminés, soit ~57 kDa, contient une région C-terminale qui porte les résidus conservés du site catalytique des recombinases à tyrosine R-H-K-R-(H/W)-Y. La prédiction montre une région N-terminale qui ressemble à celle de l'intégrase du phage lambda avec un mélange de structures secondaires α et β.Lors de ces travaux, nous avons d'abord montré par RT-PCR que le gène (MAL13P1.42) qui code pour PF-Int est transcrit pendant le cycle intra-érythrocytaire avec un maximum pendant la phase schizont. Nous avons ensuite essayé de montrer l'implication de Pf-Int dans le cycle parasitaire. Ceci a été réalisé grâce à un parasite (KO: knock-out) dont le gène Pf-Int a été invalidé. Ces analyses montrent que Pf-Int n'a aucun impact apparent sur le cycle de développement intra-érythrocytaire du parasite, en particulier sur la durée du cycle et le taux de croissance. Au niveau moléculaire, nous avons également procédé à la production d'anticorps anti-Pf-Int en utilisant le fragment C-162 (Résidus 162-490). La comparaison des profils de marquage, par cet anticorps, des extraits protéiques du KO et du parasite sauvage par la technique de Western blot n'a pas permis d'identifier la protéine endogène dans le parasite sauvage. Dans le but de déterminer la localisation sub-cellulaire de Pf-Int, nous avons réalisé des essais de sur-expression de différentes protéines de fusion dans le parasite. Nous avons essayé de déterminer l'impact de trois codons d'initiation différents ainsi que l'impact de la présence de la région N-terminale (1-190aa) de Pf-Int sur sa localisation subcellulaire en utilisant une chimère entre la partie N-terminale et la protéine GFP. Lors de ces travaux, nous avons réussi à sur-exprimer différentes régions de Pf-Int sous forme recombinante dans E. coli. Nous l'avons d'abord caractérisé par des études biophysiques. Ainsi nous avons pu déterminer, par dichroïsme circulaire (CD), le contenu en structures secondaires de Pf-Int, qui est proche de celui des autres membres de la même famille. Nous avons également démontré sa stabilité par CD couplé à la dénaturation thermique. Le spectre RMN-1D a aussi pu être enregistré. La troisième partie de nos travaux a concerné l'identification des cibles ADN de Pf-Int. Deux stratégies de recherche de cibles par affinité ont été utilisées au laboratoire en utilisant une première bibliothèque de séquences synthétisées chimiquement et une deuxième bibliothèque formée de fragments d'ADN génomique de P. falciparum. Ces deux approches ont permis l'identification de deux séries de cibles ADN. Grace aux cibles ADN identifiées, nous avons pu démontrer l'interaction de différents fragments de Pf-Int avec ces cibles par des expériences de retard sur gel natif (EMSA). Nous avons aussi pu démontrer que les protéines recombinantes sont actives in vitro. En effet, ces dernières sont capables de former des complexes covalents en présence de l'ADN cible. La conservation de la protéine, ainsi que son expression différentielle nous laisse à penser que son rôle est certes loin d'être élucidé, mais que Pf-Int reste une cible potentielle pour P. falciparum.
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Koscky, Paier Carlos Roberto 1983. "Padronização da expressão heterologa e de modelo de ensaio de atividade para a proteina quinase humana S6K". [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314787.
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Orientador: Nilson Ivo Tonin ZanchinDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A quinase de 70 kDa da proteína ribossomal S6, isoforma 1 (S6K1), é uma fosfoproteína implicada na regulação de genes relacionados ao controle da tradução em mamíferos e possui uma forma nuclear (a1) e uma citoplasmática (a2). A fosforilação do seu principal alvo, a proteína RPS6, tem sido comumente associada ao recrutamento seletivo dos 5'-TOP (5' tract of oligopyrimidine) mRNAs pela maquinaria de tradução, embora haja estudos contrariando esta hipótese. Devido às funções de seus demais alvos, S6K1 tem sido implicada na sobrevivência celular e em diversos outros processos, como crescimento, câncer e resistência à insulina. S6K1 é ativada por um mecanismo que envolve fosforilação seqüencial através da ativação das vias mTORC1 (complexo 1 do alvo da rapamicina em mamíferos) e PI3K (fosfoinositol-3 quinase). Como uma quinase da família AGC, S6K1 deve ser fosforilada por mTORC1 no resíduo Thr389 do domínio hidrofóbico e, em seguida, por PDPK1 (proteína quinase 1 dependente de fosfoinositol) no resíduo Thr229 da alça T do domínio catalítico. Estes eventos ocorrem somente após a fosforilação em diversos sítios do domínio auto-inibitório carboxiterminal, por mTORC1. O objetivo deste trabalho foi desenvolver um ensaio modelo para análise da função da S6K1 in vitro e utilizá-lo como ferramenta na elucidação do papel de proteínas adaptadoras da via de mTOR em interações com a S6K1. Para isso foi necessário produzir as proteínas recombinantes para ensaios de interação e para realização de um ensaio de atividade para a S6K1. Foram testados vários sistemas de expressão para Escherichia coli para produção das construções GST-S6K1a1-His6, GST-S6K1a2-His6 e GST-S6K1a2T389E?CT (forma a2 de S6K1 com a substituição T389E e o carboxiterminal truncado), GST-PDPK1 e GST-CDPDPK1 (domínio catalítico de PDPK1 fusionado a GST). A expressão das formas truncadas de S6K1 e PDPK1 foi mais eficiente em E. coli. Embora o rendimento tenha ficado muito aquém do esperado, foi suficiente para os ensaios de interação in vitro. Também foi feita a expressão em E. coli da região C-terminal da proteína RPS6, que é o substrato da S6K1, em fusão com a proteína D do fago ?. Posteriormente, foram montados sistemas de expressão das construções His6-S6K1a2T389E?CT e His6-CDPDPK1 em células de inseto, a partir de vetor de baculovírus. Constatou-se que essas construções são expressas na forma de fosfoproteínas em células de inseto. Ensaios de GST pull-down com GST-S6K1a2-His6 e GST-S6K1a2T389E?CT contra as duas isoformas da subunidade catalítica da PP2AC, His6-PP2ACa(maior) e His6-PP2ACa(menor), revelaram que His6-PP2ACa(maior) não interage com GST-S6K1a2-His6, embora interaja fortemente com GST-S6K1a2T389E?CT. Já a construção His6-PP2ACa(menor) interage fracamente com as construções GST-S6K1a2-His6 e GST-S6K1a2T389E?CT. Tomados em conjunto, os resultados sugerem que a presença do C-terminal não fosforilado de S6K1a2 impede a interação com PP2ACa(maior). PP2ACa(menor) comporta-se de forma completamente diferente da isoforma maior, pois a interação entre PP2ACa(menor) e S6K1a2 parece ser independente do carboxiterminal da quinase, visto que as quantidades de S6K1a2T389E?CT e de S6K1a2 inteira que interagem com PP2ACa(menor) são semelhantes. Esses resultados necessitam ainda serem confirmados in vivo. Outros experimentos de GST pull-down confirmaram que as construções de S6K1 não interagem com a4, embora interajam com TIPRL1. Se confirmado in vivo, esse resultado compõe um novo quadro na regulação coordenada entre mTOR1 e PP2A, do qual TIPRL1 parece participar. As construções genéticas e os sistemas de expressão gerados neste trabalho possibilitaram a obtenção dos reagentes necessários para analisar o mecanismo de regulação da quinase S6K1, mediado por proteínas regulatórias. Permitem também desenvolver uma série de experimentos, como busca de inibidores específicos para a S6K1, que dependem da reconstituição de ensaios de atividade in vitro com a S6K1 ativada. Contudo, o ensaio de atividade realizado não apresentou resultados satisfatórios e precisa ser desenvolvido.
Abstract: The 70kDa ribosomal S6 protein kinase 1 (S6K1) is a phosphoprotein involved in the regulation of genes related to translational control in mammals. S6K1 shows distinct nuclear (a1) and cytoplasmic (a2) forms. Phosphorylation of the S6K1 best characterized target, the protein of the small ribosomal subunit (RPS6), has been generally associated to the selective recruitment of the 5'-TOP mRNAs (5' tract of oligopyrimidine) by the translational machinery, although there is still some controversy on this issue. Due to the function of its targets, S6K1 has been implicated in several cellular processes including cell growth, cancer and insulin resistance. S6K1 is activated by a mechanism of sequential phosphorylation following activation of the mTORC1 (mammalian target of rapamycin complex 1) and PI3K (phosphoinositide-3-kinase) pathways. As a kinase of the AGC family, S6K1 activation requires mTORC1 phosphorylation of residue Thr389 of the hydrophobic domain followed by PDPK1 (phosphoinositide dependent protein kinase 1) phosphorylation of residue Thr229 at the T loop of the catalytic domain. These take place only after phosphorylation by mTORC1 of several residues of the autoinhibitory C-terminal domain. The objective of this work was to develop an assay to analyze the function of S6K1 in vitro and use it as a tool in the discovering of the functions of regulators proteins of the mTOR cascade in interactions with S6K1. For these purposes, expression systems were constructed to produce the various recombinant proteins to be used in the interaction and activity assays. Several genetic constructions were tested in Escherichia coli for the production of GST-S6K1a1-His6, GST-S6K1a2-His6 and GST-S6K1a2T389E?CT (a2 form of S6K1 with the T389E substitution and truncated carboxiterminus), GST-PDPK1 and GST-CDPDPK1 (GST fusion protein of the catalytic domain of PDPK1). The truncated forms were expressed more efficiently in E. coli. Although the yield in E. coli was lower than expected, it was sufficient to perform interaction assays. The C-terminal domain of RPS6, a substrate for S6K1, was successfully expressed in E. coli as a fusion protein with the phage ? protein D. Subsequently, expression systems for production of His6-S6K1a2T389E?CT and His6-CDPDPK1 in insect cells were constructed using baculovirus vectors. It was found that these constructs are expressed in the form of phosphoproteins in insect cells. GST pull-down assays using GST-S6K1a2-His6 e GST-S6K1a2T389E?CT to test interaction with the PP2AC isoforms His6-PP2ACa(major) and His6-PP2ACa(minor) revealed that His6-PP2ACa(major) does not interact with GST-S6K1a2-His6, although it interacts strongly with GST-S6K1a2T389E?CT. On the other hand, His6-PP2ACa(minor) interacts weakly with both GST- S6K1a2-His6 and GST-S6K1a2T389E?CT. This finding suggests that the unphosphorylated C-terminal of S6K1a2 inhibits interaction with PP2ACa(major). His6-PP2ACa(minor) behaves differently form His6-PP2ACa(major). Its interaction with S6K1a2 seems to be independent of the C-terminal since the amounts of S6K1a2T389E?CT and S6K1a2 that interact with His6-PP2ACa(minor) are similar. Future work in vivo is required to confirm these results. GST pull-down assays confirmed that a4 does not interact with the constructions of S6K1, while TIPRL1 interacts with them. If confirmed in vivo, these results provides a new perspective for the coordinated regulation between mTOR1 and PP2A, which apparently involves also TIPRL1. The genetic constructions and expression systems established in this work allow the production of the reagents required to study the mechanism of S6K1 regulation mediated by adaptor proteins. They will also allow the development of experiments such as screening for specific S6K1 inhibitors, which depend on reconstitution of S6K1 activity assays using activated S6K1. Nevertheless, the activity assay performed did not yield satisfactory outcomes and must be improved.
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular
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Lu, Yang. "Functional studies of new protein-protein interactions potentially involved in homologous recombination in hyperthermophilic archaea : study of interactions between PCNA and Mre11-Rad50 complex & Primase and RadA". Thesis, Brest, 2018. http://www.theses.fr/2018BRES0077/document.
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Les archées hyperthermophiles ont une température optimale de croissance supérieure à 80°C.Les cellules exposées à un stress thermique subissent une augmentation de la sensibilité aux agents induisant des cassures double brin de l’ADN. Les études sur les eucaryotes et bactéries ont démontré que la recombinaison homologue joue un rôle essentiel non seulement dans la réparation de l’ADN, mais aussi dans le redémarrage des arrêts de la fourche de réplication. Les enzymes associées aux étapes initiales de la recombinaison homologue chez les archées sont homologues à celles des eucaryotes, et différentes des analogues bactériens. De plus, plusieurs études ont démontré que les protéines impliquées dans la recombinaison homologue sont essentielles chez les archées hyperthermophiles, soulignant l’importance biologique de cette voie de réparation chez ces organismes particuliers. Le rôle de la recombinaison homologue pour la stabilité génomique a été bien étudié chez les eucaryotes et les bactéries, cependant, peu de ses propriétés fonctionnelles ont été étudiées chez les archées hyperthermophiles. Pour mieux comprendre le mécanisme de recombinaison homologue impliquée au niveau de la maintenance génomique chez les archées, un réseau d’interactions protéine-protéines a été révélé précédemment au laboratoire à partir des protéines de Pyrococcus abyssi. Ces travaux ont démontré de nouvelles interactions où interviennent les protéines de la réplication et les protéines de la recombinaison de l’ADN. L’objet de cette étude de thèse est de présenter deux interactions : PCNA/Mre11-rad50 (MR) complexe et Primase/RadA. Pour la première fois chez P. furiosus, une interaction physique et fonctionnelle a été démontrée entre le PCNA et le complexe MR (l’initiateur de HR). Un motif, situé en position Cterminale de Mre11, permet l’interaction avec PCNA.PCNA stimule l’activité endonucléase du complexe MR à distance proche de l’extrémité 5’ d’une cassure double brin. Cette propriété est en accord avec l’intervention ultérieure des enzymes assurant la suite du mécanisme de réparation par recombinaison homologue. Par ailleurs, les protéines RadA, Primase et P41 ont été produites et purifiées. Leurs fonctions enzymatiques ont été confirmées. Cependant, nous n’avons pas pu caractériser la fonction de l’association de RadA/PrimaseHyperthermophilic archaea (HA) are found in high-temperature environments and grow optimally above 80°C. Usually, cells exposed to heat stress display an increased sensitivity to agents inducing double-stranded DNA breaks (DSBs). Studies in Eukaryotes and Bacteria have revealed that homologous recombination (HR) plays a crucial role not only in DNA DSBs repair, but also in the collapsed/stalled DNA replication fork restart.Recombinase and various HR-associated enzymes in archaea specifically resemble the eukaryotic homologues, rather than bacterial homologues.Furthermore, several studies have demonstrated the necessity of HR proteins in HA, suggesting that, HR is an important mechanism in HA. HR influencing genome stability has been well studied in Eukaryotes andBacteria, however, few of its functional properties have been studied in HA.To better understand how HR mechanism is involved in the archaeal genome maintenance process, a previous work proposed a protein-protein interaction network based on Pyrococcus abyssi proteins. Through the network, new interactions involving proteins from DNA replication and DNA recombination were highlighted. The targets of the study presented here for two protein interaction are: PCNA/Mre11-rad50 complex (MR complex) and Primase/RadA. For the first time in P. furiosus, we showed both physical and functional interactions between PCNA (Maestro in DNA replication) and MR complex (initiator of HR). We have identified a PCNA-interaction motif (PIP) located in the C-terminal of Mre11, and shown that PCNA stimulated MR complex endonuclease cleavage proximal to the s’ strand of DNA DSBs at physiological ionic strength. For the second interaction, we have purified the proteins PabRadA/PfuRadA, PabPrimase and PabP41, and confirmed its enzymatic functions. However, we were not able to characterize the function of Primase/RadA association
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Corgozinho, Carolina Nunes Costa. "Desenvolvimento de vacina baseada em sistema de liberação sustentada contendo proteína recombinante". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/60/60137/tde-31072009-083709/.
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No Brasil, e em outros paises de clima tropical, os carrapatos se tornaram um enorme problema economico de^$de que a industria do gado se desenvolveu. O carrapato Boophilus microplus, um dos artropodes mais importantes na veterinaria, causa efeitos direto, como suc,cao de sangue, e indireto, como a transmissao de uma grande variedade de patogenos que normalmente resulta em infec,c~es letais. As vacinas genicas contendo o antigeno Bm86, uma proteina ligada a membrana do intestino do carrapato B. microplus, representam uma alternativa atrativa aos acaricidas para controlar as infesta~oes por carrapatos em contrapartida aos inconvenientes produtos quimicos. Devido sua administra,cao ser feita em 4 doses no primeiro ano, seguida de refor,cos a cada seis meses, estas formula,coes vacinais nao s3c adequadas para paises com cria,cao extensiva de gado, como no Brasil. Visando uma libera~ao sustentada do antigeno Bm86, neste trabalho desenvolveuse uma vacina de dose unica baseada em microesferas polimericas. Para obter o padrao de liberac,ao desejavel, diferentes formula,coes e parametros de processo foram variados, como a composi,cao do polimero, a taxa entre os monomeros ^Uacido latico:acido glicolico\" e o tamanho das microparticulas. As formula,coes foram preparadas pelo metodo de emulsao multipla e evapora,cao do solvente. A formula~ao que melhor se enquadrou nos objetivos da vacina de dose unica foi preparada com PLGA 75:25, solu,cao 3% de PVA como estabilizante, agita,cao de 11000 rpm para forma,cao da emulsao primaria e de 800 rpm para forma,cao da emulsao multipla e evapora,cao do solvente. As particulas assim obtidas apresentaram um tamanho medio de 25 ,um, uma taxa de encapsula,cao maior que 90% e aproximadamente 50% da proteina foi liberada in vitro em 60 dias. Analises por SDS-PAGE e Westem Bloning revelaram que a proteina se manteve integra apos encapsula,cao. Os resultados da avalia,cao da imunogenicidade em bovinos mostraram que a formula,cao baseada em microesferas polimericas biodegradaveis e habil a conseguir, com uma unica dose, uma resposta imune similar aquela conseguida com tres doses das formula,coes convencionais da vacina de Bm86.In Brazil, and in others tropical countries, the ticks have become a huge economic problem since the industry of livestock has developed. Ticks and tick-borne diseases affect animal and human health and are the cause of significant economic losses. The cattle tick Boophilus microplus is one of the most important arthropods in veterinary. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. The gene vaccines based on Bm86 antigen, a midgut membrane-bound protein of the cattle tick B. microplus, represent a good alternative to control tick infestations, compared to chemicals. However, due to these vaccine formulations need 4 doses over the first year with booster at each 6 months to be effective, they are not suitable for countries with extensive cattle raising, like Brazil. Aiming a sustained release of Bm86 antigen, in this work we developed a single shot vaccine based on Bm86 loaded polymeric microspheres. In order to obtain desired release patterns, different formulations and processing parameters were varied, for example, the composition of the polymer, the monomer ratio lactic acid:glycolic acid and the size of the microparticles. The formulations were prepared by solvent evaporation method based on double emulsion. The formulation that presented better result as single shot vaccine was prepared with PLGA 75:25, solution 3% of PVA as stabilizer, agitation of 11000 rpm to form the primary emulsion and 800 rpm to obtain the double emulsion and solvent evaporation. The particles thus obtained presented an average size of 25 m, encapsulation ratio greater than 90% and approximately 50% of the protein was released in vitro in 60 days. Analysis by SDSPAGE and Western Blot showed that the integrity of the protein remained after encapsulation. The immunogenic studies showed that the formulation based onbiodegradable polymeric microspheres is able to elicit, with a single dose, an immune response and protection similar to that attained with 3 doses of conventional Bm86 vaccine formulations.
Książki na temat "Recombinase protein"
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Jean, Garnier, red. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1988.
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Jean, Garnier, red. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1986.
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1961-, Castro Fidel O., i Jänne Juhani, red. Mammary gland transgenesis: Therapeutic protein production. Berlin: Springer, 1998.
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Robson, Barry. Introductionto proteins and protein engineering. Amsterdam: Elsevier, 1988.
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Tuan, Rocky S. Recombinant Protein Protocols. New Jersey: Humana Press, 1997. http://dx.doi.org/10.1385/089603481x.
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Buckel, Peter, red. Recombinant Protein Drugs. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7.
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Robson, Barry. Introduction to proteins and protein engineering. Amsterdam: Elsevier, 1986.
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1947-, Stein Stanley, red. Fundamentals of protein biotechnology. New York: M. Dekker, 1990.
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Bill, Roslyn M., red. Recombinant Protein Production in Yeast. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-770-5.
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Gasser, Brigitte, i Diethard Mattanovich, red. Recombinant Protein Production in Yeast. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9024-5.
Pełny tekst źródłaCzęści książek na temat "Recombinase protein"
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Buntru, Matthias, Simon Vogel, Ricarda Finnern i Stefan Schillberg. "Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins". W 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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Maetzig, Tobias, i Axel Schambach. "Development of Inducible Molecular Switches Based on All-in-One Lentiviral Vectors Equipped with Drug Controlled FLP Recombinase". W Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools, 23–39. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3753-0_2.
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Fothergill-Gilmore, Linda A. "Recombinant Protein Technology". W Protein Biotechnology, 467–87. Totowa, NJ: Humana Press, 1993. http://dx.doi.org/10.1007/978-1-59259-438-2_13.
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Weissmann, Charles. "Recombinant interferon - the 20th anniversary". W Recombinant Protein Drugs, 3–41. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_1.
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Hofschneider, Peter Hans, i Kenneth Murray. "Combining science and business: from recombinant DNA to vaccines against hepatitis B virus". W Recombinant Protein Drugs, 43–64. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_2.
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Brownlee, George G., i Paul L. F. Giangrande. "Clotting factors VIII and IX". W Recombinant Protein Drugs, 67–88. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_3.
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Welte, Karl, i Erich Platzer. "Colony-stimulating factors: altering the practice of oncology". W Recombinant Protein Drugs, 89–106. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_4.
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Collen, Désiré, i H. Roger Lijnen. "Tissue-type plasminogen activator: helping patients with acute myocardial infarction". W Recombinant Protein Drugs, 107–26. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_5.
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Gillies, Stephen D. "Designing immunocytokines: genetically engineered fusion proteins for targeted immune therapy". W Recombinant Protein Drugs, 129–47. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_6.
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Burke, Paul A., i Scott D. Putney. "Improving protein therapeutics: the evolution of the modern pharmacopoeia". W Recombinant Protein Drugs, 151–68. Basel: Birkhäuser Basel, 2001. http://dx.doi.org/10.1007/978-3-0348-8346-7_7.
Pełny tekst źródłaStreszczenia konferencji na temat "Recombinase protein"
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Berkner, K. L., S. J. Busby, J. Gambee i A. Kumar. "EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643568.
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The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.
2
Tyumentsev, A. I., M. A. Tyumentseva i V. G. Akimkin. "DEVELOPMENT OF APPROACHES FOR ENDOTOXIN REMOVAL FROM PROTEIN PREPARATIONS ON THE EXAMPLE OF NUCLEASES OF THE CRISPR/CAS SYSTEM". W Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-113.
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Removal of bacterial endotoxins from solutions of recombinant proteins is one of the most important issues in the preparation of highly purified preparations suitable for in vivo use. An optimal technology for obtaining preparations purified from bacterial endotoxins has been proposed using purification of preparations of recombinant nucleases of the CRISPR/CAS system as an example. Efficacy of developed technology was compared with other available methods. Removal of bacterial endotoxins was carried out using Triton X-114 detergent added to a concentration of 1% to a solution containing the recombinant protein. It was shown that the content of bacterial endotoxins in solutions of purified proteins obtained according to the proposed technology is 0.3–1.5 EU/ml.
3
Neklesova, M. V., i N. Deeb. "PRODUCTION OF RECOMBINANT PROTEINS AMUC_1100 AND P9 AND ASSESSMENT OF THEIR IN VITRO ANTITUMOR ACTIVITY". W X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-106.
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The probiotic properties of Akkermansia muciniphila in the treatment of metabolic, cardiovascular and oncological diseases are mediated by the surface protein Amuc_1100 (Amuc) and secreted protein P9, but their combined effect has not been investigated. In this work, a recombinant producer E. coli BL21(DE3) of proteins Amuc and P9 was obtained. It is planned to conduct experiments on the colorectal cancer cell line using a combination of Amuc and P9.
4
Vehar, G. A. "THE PRESENT STATE OF GENE TECHNOLOGY IN THE MANUFACTURE OF HUMAN COAGULATION PROTEINS". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644755.
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The production of pharmaceuticals from human plasma that are useful in the treatment of bleeding disorders had its beginning with the development of the Cohn fractionation procedure in the 1940's. As a result of these advances, concentrates became available for the treatment of the hemophilias. Although of low purity and subject to contamination by hepatitis virus, the availability of these compounds resulted in dramatic improvements in the life expectancy and quality of life of afflicted individuals. The numerous problems associated with production of pharmaceuticals from pooled plasma made these products obvious goals for recombinant DNA technology as soon as the commercial aspects of the field became apparent. The subsequent contamination of blood products with the AIDS virus has resulted in an urgent need for a production source that is independent of human plasma. Several industrial and academic laboratories have cloned the cDNA's for human factors VIII and IX. In addition to these proteins, the utility of factor Vila in the treatment of hemophiliacs with inhibitors has shown promise. Efforts to develop a recombinant preparation of factor Vila are at a comparable stage of development as factors VIII and IX. Continuing efforts have resulted in the successful expression of these recombinant proteins in mammalian cell lines, thereby successfully completing the first steps of commercial development.Although much interest has focused upon the theoretical superiority of recombinant proteins as therapeutics, one must keep in mind that there are numerous developmental aspects of large-scale production and regulatory issues that must be addressed and solved before these drugs will be available. The coagulation proteins are complex glycoproteins that will in all probability require mammalian cell cell culture in order to produce functional proteins. The fact that these preparations will be administered over the lifetime of the patient serves to reinforce that the recombinant products be as similar to the natural proteins as possible, further supporting the concept of mammalian cell expression systems.Regulatory approval of a recombinant product are fundamentally no different than those for any other product in regards to efficacy, potency, purity, and identity. There are, however, additional considerations that must be addressed in the production of recombinant cell culture derived biologies. These relate to the possible presence in the final product of pathogenic and tumorigenic agents, and possible contamination by cell culture and cell substrate compounds. A detailed characterization of the production cell line will therefore be required, including identification and characterization of any associated viral particles. These cells must be capable of being reproducibly grown, while maintaining protein production, on ascale (tens of thousands of liters) suitable to meet the market demand of the specific protein. Apurification process must be established capable of handling the resulting large volumes of feedstock, generating a protein preparation of high purity (greater than 99% pure). Numerous assays must be developed to quantitate the purity and identity of the resulting recombinant pharmaceutical on a lot by lot basis.Studies to date have shown that recombinant forms of factors VIII and IX, produced by laboratory processes, are very similar to the plasma-derived forms as assessed by a variety of in vitro and in vivo tests. Although these results are promising, the ultimate safety and efficacy testing of these drugs will have to await the initiation of human clinical trials. Such studies will have to await the successful completion of the certain regulatory concerns. Clinical trials should begin within the near future, hopefully leading to a source of these products independent of pooled human plasma.
5
Ashok Kumar, A., Margaret Insley, Jay Gambee, Sharon J. Busby i Kathleen L. Berkner. "SITE SPECIFIC MUTAGENESIS WITHIN THE GLA-DOMAIN OF HUMAN FACTOR IX". W 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.
6
Marchenko, D. M., M. S. Bozhokin, E. R. Mikhailova, Yu V. Sopova i M. G. Khotin. "STIMULATION OF HUMAN DERMAL FIBROBLASTS CHONDROGENIC DIFFERENTIATION FOR TISSUE ENGINEERING OF HYALINE CARTILAGE". W X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-101.
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This work is devoted to the possibility of human dermal fibroblasts chondrogenic differentiation using the TGF-β3 and SOX9 recombinant proteins and lentiviral vectors carrying sequences of the Tgf-β3 and Sox9 genes. It has been shown that the use of the TGF-β3 protein and vector with the Sox9 gene makes it possible to increase the expression of associated with chondrogenesis genes in fibroblasts.
7
Furis, B. C., M. J. Jorgensem, M. J. Rabiet, A. B. Contor, C. L. Brown, C. B. Shoemaker i B. Furie. "RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN". W 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.
8
Novikova, L. I., S. S. Bochkareva, A. V. Aleshkin, S. IU Kombarova, O. E. Karpov, A. A. Pulin, O. A. Orlova, IU S. Lebedin, A. M. Vorobev i E. R. Mekhtiev. "DYNAMICS OF ANTIBODIES TO VARIOUS ANTIGENS OF THE SARS-COV-2 CORONAVIRUS IN PATIENTS WITH CONFIRMED COVID-19 INFECTION". W Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-159.
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Presence of IgG and IgM antibodies in venous blood of 76 patients with confirmed presence of SARS-CoV-2 was determined. The study was carried out by ELISA using Russian test systems. Revealed different levels of IgM antibodies to N-protein and RBD (receptor binding domain of the Spike protein). The level of IgM to RBD did not reach high values, while the level of IgM to N-protein sharply increased in a short period of time to high values by the 3rd week of the disease and decreased only by the 8th week. The dynamics of IgG antibodies to the whole virion antigen and the recombinant spikes was similar, reaching high values at 4-5 weeks of the disease, however, the dynamics of IgG to the N-protein differed, showing a slight increase by the 1st week of the disease and a low level throughout the entire period of observation. Different dynamics of production of IgG and IgM antibodies to N-protein and RBD were noted. The amount of IgM to the N-protein increased sharply and reached a high level, while the amount of IgG increased smoothly and did not show a high level. The opposite picture was observed for antibodies to RBD. The characteristic dynamics of IgG, measured using test systems withsorbed whole virion or recombinant spike proteins, suggests duration of the disease
9
Teng, Weibing, Yiding Huang, Joseph Cappello i Xiaoyi Wu. "Mechanical and In-Vitro Cell Compatibility Properties of Silk-Elastinlike Protein-Based Biomaterial". W ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13141.
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A series of genetically engineered recombinant silk-elastinlike proteins (SELPs) have been produced by combining polypeptide sequences derived from native silk of superior mechanical strength and elastin that is extremely durable and resilient. They have displayed a set of outstanding properties such as good biocompatibility and controllable biodegradation rates. In the study, we characterized the mechanical property of genetically engineered, recombinant silk-elastinlike protein copolymer, SELP-47K, under physical and chemical treatments. The biocompatibility of the SELP-47K was also evaluated by cell culture. The ultimate goal of this study is to explore the potential of SELPs for applications in the engineering of load-bearing tissues such as arteries.
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Mikhaylova, E. E., I. K. Baykov i N. V. Tikunova. "MODIFICATION OF AN ANTIBODY AGAINST TICK-BORNE ENCEPHALITIS VIRUS USING DIRECTED PROTEIN EVOLUTION". W X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-348.
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It is possible to obtain modified protein molecules with the properties necessary for the researcher by various methods. One of them is the directed evolution of proteins, which mimics the process of natural selection. The use of this method allowed to increase the affinity of the single-stranded antibody sc14D5 to the recombinant domain III of glycoprotein E of the European and Siberian subtypes of tick-borne encephalitis virus.
Raporty organizacyjne na temat "Recombinase protein"
1
Hodges, Thomas K., i David Gidoni. Regulated Expression of Yeast FLP Recombinase in Plant Cells. United States Department of Agriculture, wrzesień 2000. http://dx.doi.org/10.32747/2000.7574341.bard.
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Research activities in both our laboratories were directed toward development of control of the FLP/frt recombination system for plants. As described in the text of the research proposal, the US lab has been engaged in developing regulatory strategies such as tissue-specific promoters and the steroid-inducible activation of the FLP enzyme while the main research activities in Israel have been directed toward the development and testing of a copper-regulated expression of flp recombinase in tobacco (this is an example of a promoter activation by metal ions). The Israeli lab hat additionally completed experiments of previous studies regarding factors affecting the efficiency of recombinase activity using both a gain-of-function assay (excisional-activation of a gusA marker) and loss of function assay (excision of a rolC marker) in tobacco. Site-specific recombinase systems, in particular the FLP/frt and R/RS systems of yeast and the Cre/lox system of bacteriophage P1, have become an essential component of targeted genetic transformation procedures both in animal and plant organisms. To provide more flexibility in transgene excisions by the recombinase systems as well as gene targeting, and to widen possible applications, the development of controlled or regulated recombination systems is highly desirable and was therefore the subject of this research proposal. There are a few possible mechanisms to regulate expression of a recombinase system. They include: 1) control of the recombination system by having the target sites (e.g. frt) in one plant and the flp recombinase gene in another, and bringing the two together by cross fertilization. 2) regulation of promoter activities by external stimuli such as temperature, chemicals, metal ions, etc. 3) regulation of promoter activities by internal signals, i.e. cell- or tissue-specific, or developmental regulation. 4) regulation of enzyme activity by providing cofactors essential for biochemical reactions to take place such as steroid molecules in conjunction with a steroid ligand-binding protein (domains). During the course of this research our major emphasis have been focused toward studying the feasibility of hybrid seed production in Arabidopsis, using FLP/frt. Male-sterility was induced using the antisence of a pollen- and tapetum-specific gene, bcp1, isolated from Arabidopsis. The sterility inducing gene was flanked by frt sites. Upon cross pollination of flowers of male-sterile plants with pollen from FLP-containing plants, viable seeds were produced, and the progeny hybrid plants developed normally. The major achievement from this work is the first demonstration of using a site-specific recombinase to restore fertility in male-sterile plants (see attached paper, Luo et al., Plant J 2000; 23:423-430). The implication from this finding is that site-specific recombination systems can be applied in crop plants as a useful alternative method for hybrid seed production.
2
Banai, Menachem, i Gary Splitter. Molecular Characterization and Function of Brucella Immunodominant Proteins. United States Department of Agriculture, lipiec 1993. http://dx.doi.org/10.32747/1993.7568100.bard.
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The BARD project was a continuation of a previous BARD funded research project. It was aimed at characterization of the 12kDa immunodominant protein and subsequently the cloning and expression of the gene in E. coli. Additional immunodominant proteins were sought among genomic B. abortus expression library clones using T-lymphocyte proliferation assay as a screening method. The 12kDa protein was identified as the L7/L12 ribosomal protein demonstrating in the first time the role a structural protein may play in the development of the host's immunity against the organism. The gene was cloned from B. abortus (USA) and B. melitensis (Israel) showing identity of the oligonucleotide sequence between the two species. Further subcloning allowed expression of the protein in E. coli. While the native protein was shown to have DTH antigenicity its recombinant analog lacked this activity. In contrast the two proteins elicited lymphocyte proliferation in experimental murine brucellosis. CD4+ cells of the Th1 subset predominantly responded to this protein demonstrating the development of protective immunity (g-IFN, and IL-2) in the host. Similar results were obtained with bovine Brucella primed lymphocytes. UvrA, GroE1 and GroEs were additional Brucella immunodominant proteins that demonstrated MHC class II antigenicity. The role cytotoxic cells are playing in the clearance of brucella cells was shown using knock out mice defective either in their CD4+ or CD8+ cells. CD4+ defective mice were able to clear brucella as fast as did normal mice. In contrast mice which were defective in their CD8+ cells could not clear the organisms effectively proving the importance of this subtype cell line in development of protective immunity. The understanding of the host's immune response and the expansion of the panel of Brucella immunodominant proteins opened new avenues in vaccine design. It is now feasible to selectively use immunodominant proteins either as subunit vaccine to fortify immunity of older animals or as diagnostic reagents for the serological survaillance.
3
Galili, Gad, i Alan Bennett. Role of Molecular Chaperone in Wheat Storage Protein Assembly. United States Department of Agriculture, kwiecień 1995. http://dx.doi.org/10.32747/1995.7604926.bard.
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Following sequestration into the ER, wheat gliadins assemble into complexes that initiate the formation of protein bodies. In the present work we have characterized the DNA sequence and regulation of expression of a plant BiP and also studied its interaction with wheat storage proteins as well as its role in the maturation of these storage proteins. In the Israeli lab, immunoprecipitation studies were made using anti BiP and anti storage proteins sera, both in wheat and in transgenic tobacco plants expressing a wheat gliadin storage proteins. In both cases, we could show that BiP interacts with the gliadin storage proteins. In addition, we could show that BiP also played an important role in the initial assembly of the gliadins. In the American lab, the complexity, structure and properties of tomato BiP was characterized at the molecular and biochemical levels. In addition, tomato BiP was also overexpressed in bacteria and the overexpressed protein was found to be active. The cooperative findings of the Israeli and American labs clearly improves our understanding of the structure and expression of a plant BiP as well as its role in the maturation of storage proteins in plants seeds. In addition, it will serve as a foundation for future studies of the mechanisms of BiP function in in vitro studies using purified storage proteins and purified recombinant active BiP.
4
Bercovier, Herve, Raul Barletta i Shlomo Sela. Characterization and Immunogenicity of Mycobacterium paratuberculosis Secreted and Cellular Proteins. United States Department of Agriculture, styczeń 1996. http://dx.doi.org/10.32747/1996.7573078.bard.
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Our long-term goal is to develop an efficient acellular vaccine against paratuberculosis based on protein antigen(s). A prerequisite to achieve this goal is to analyze and characterize Mycobacterium paratuberculosis (Mpt) secreted and cellular proteins eliciting a protective immune response. In the context of this general objective, we proposed to identify, clone, produce, and characterize: the Mpt 85B antigen and other Mpt immunoreactive secreted proteins, the Mpt L7/L12 ribosomal protein and other immunoreactive cellular proteins, Mpt protein determinants involved in invasion of epithelial cells, and Mpt protein antigens specifically expressed in macrophages. Paratuberculosis is still a very serious problem in Israel and in the USA. In the USA, a recent survey evaluated that 21.6% of the dairy herd were infected with Mpt resulting in 200-250 million dollars in annual losses. Very little is known on the virulence factors and on protective antigens of Mpt. At present, the only means of controlling this disease are culling or vaccination. The current vaccines do not allow a clear differentiation between infected and vaccinated animals. Our long-term goal is to develop an efficient acellular paratuberculosis vaccine based on Mpt protein antigen(s) compatible with diagnostic tests. To achieve this goal it is necessary to analyze and characterize secreted and cellular proteins candidate for such a vaccine. Representative Mpt libraries (shuttle plasmid and phage) were constructed and used to study Mpt genes and gene products described below and will be made available to other research groups. In addition, two approaches were performed which did not yield the expected results. Mav or Mpt DNA genes that confer upon Msg or E. coli the ability to invade and/or survive within HEp-2 cells were not identified. Likewise, we were unable to characterize the 34-39 kDa induced secreted proteins induced by stress factors due to technical difficulties inherent to the complexity of the media needed to support substantial M. pt growth. We identified, isolated, sequenced five Mpt proteins and expressed four of them as recombinant proteins that allowed the study of their immunological properties in sensitized mice. The AphC protein, found to be up regulated by low iron environment, and the SOD protein are both involved in protecting mycobacteria against damage and killing by reactive oxygen (Sod) and nitrogen (AhpC) intermediates, the main bactericidal mechanisms of phagocytic cells. SOD and L7/L12 ribosomal proteins are structural proteins constitutively expressed. 85B and CFP20 are both secreted proteins. SOD, L7/L12, 85B and CFP20 were shown to induce a Th1 response in immunized mice whereas AphC was shown by others to have a similar activity. These proteins did not interfere with the DTH reaction of naturally infected cows. Cellular immunity provides protection in mycobacterial infections, therefore molecules inducing cellular immunity and preferentially a Th1 pathway will be the best candidate for the development of an acellular vaccine. The proteins characterized in this grant that induce a cell-mediated immunity and seem compatible with diagnostic tests, are good candidates for the construction of a future acellular vaccine.
5
Splitter, Gary, Zeev Trainin i Ruth Meirom. Effector Cell Recognition of Recombinant BHV-1 Proteins. United States Department of Agriculture, sierpień 1994. http://dx.doi.org/10.32747/1994.7604294.bard.
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Veen, Ryan Vander, Mark Mogler, Matthew M. Erdman i D. L. Hank Harris. Preparation of GP5-M Heterodimer Glycantype Specific Recombinant Protein and Replicon Particles. Ames (Iowa): Iowa State University, styczeń 2009. http://dx.doi.org/10.31274/ans_air-180814-698.
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Gafny, Ron, A. L. N. Rao i Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, wrzesień 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
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Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
8
Palmer, Guy H., Eugene Pipano, Terry F. McElwain, Varda Shkap i Donald P. Knowles, Jr. Development of a Multivalent ISCOM Vaccine against Anaplasmosis. United States Department of Agriculture, lipiec 1993. http://dx.doi.org/10.32747/1993.7568763.bard.
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Anaplasmosis is an arthropod+borne disease of cattle caused by the rickettsia Anaplasma marginale and an impediment to efficient production of healthy livestock in both Israel and the United States. Our research focuses on development of a recombinant membrane surface protein (MSP) immunogen to replace current vaccines derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Briefly, we accomplished the following in our BARD supported research: i) characterization of the intramolecular and intermolecular relationships of the native Major Surface Proteins (MSP) in the outer membrane; ii) expression, purification, and epitope characterization of the recombinant MSP-2, MSP-3, MSP-4, and MSP-5 proteins required to construct the recombinant ISCOM; iii) demonstration that the outer membrane-Quil A induces CD4+ T lymphocytes specific for the outer membrane polypeptides; iv) identification of CD4+ T lymphocytes that recognize outer membrane polypeptide epitopes conserved among other wise antigenically distinct strains; v) determination that immunization with the outer membrane-Quil A construct does not affect the ability of ticks to acquire or transmit A. marginale; and vi) demonstration that the outer membrane-Quil A construct induces complete protection against rickettsemia upon homologous challenge and significant protection against challenge with antigenically distinct strains, including tick transmission. Importantly, the level of protection against homologous challenge in the MSP vaccinates was comparable to that induced by live blood-based vaccines and demonstrates that development of a new generation of vaccines is feasible.
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Walls, Lichun H. Isolation and Preliminary Characterization of a Recombinant TAT Protein From Human Immunodeficiency Virus. Fort Belvoir, VA: Defense Technical Information Center, maj 1995. http://dx.doi.org/10.21236/ada298304.
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Brayton, Kelly A., Varda Shkap, Guy H. Palmer, Wendy C. Brown i Thea Molad. Control of Bovine Anaplasmosis: Protective Capacity of the MSP2 Allelic Repertoire. United States Department of Agriculture, styczeń 2014. http://dx.doi.org/10.32747/2014.7699838.bard.
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Anaplasmosis is an arthropod-borne disease of cattle caused by the rickettsia Anaplasmamarginale and is an impediment to efficient production of healthy livestock in both Israel and the United States. Currently, the only effective vaccines are derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Consequently, development of a safe, effective vaccine is a high priority. Despite its drawbacks as a live, blood-based vaccine, the Israel vaccine strain protects against disease upon challenge with wild-type A. marginale in extensive experimental trials and during 50 years of deployment in Israel. Field studies in Australia and Argentina indicate that this protection is broadly effective. Thus, to identify antigens for development of a safe and effective recombinant vaccine, we have used a comparative genomics approach by sequencing the Israel vaccine strain and searching for shared surface antigens with sequenced wild-type U.S. strains. We have focused on Msp2, the immune-dominant but antigenically variable surface protein, based on shared structure among strains and demonstration that antibody from cattle immunized with the Israel vaccine strain binds Msp2 from the genetically and geographically distinct U.S. St. Maries strain, consistent with the ability to protect against St. Maries challenge. Importantly, we have defined the full repertoire of Msp2 simple variants encoded by the vaccine strain and hypothesize that a recombinant vaccine encoding this full repertoire will induce protection equivalent to that induced by the live vaccine strain. Any escape from immunity by generation of complex Msp2 variants is predicted to carry a severe fitness cost that prevents high-level bacteremia and disease— consistent with the type of protection induced by the live vaccine strain. We tested the hypothesis that the Msp2 simple variant repertoires in wild-type A. marginale strains are recognized by antibody from cattle immunized with the Israel vaccine strain and that immunization with the vaccine strain Msp2 repertoire can recapitulate the protection provided by the vaccine strain upon challenge with Israel and U.S. strains of A. marginale. Our findings demonstrate that a set of conserved outer membrane proteins are recognized by immune serum from A. centrale vaccinated animals but that this set of proteins does not include Msp2. These findings suggest that “subdominant” immunogens are required for vaccine induced protection.