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Bolliger, Gabriella. "Heterologe Expression einer funktionellen Serin-Palmitoyltransferase." Zürich : ETH, Eidgenössische Technische Hochschule Zürich, Departement Chemie und Angewandte Biowissenschaften, 2005. http://e-collection.ethbib.ethz.ch/show?type=dipl&nr=176.
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Toepfer, Cornelia. "Heterologe Expression einer funktionellen Domäne des nikotinischen Acetylcholinrezeptors." [S.l. : s.n.], 2002. http://www.diss.fu-berlin.de/2002/203/index.html.
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Höffle, Anja. "Ektopische Expression schwer exprimierbarer nikotinischer Acetylcholinrezeptoren in Zellinien." [S.l.] : [s.n.], 2001. http://ArchiMeD.uni-mainz.de/pub/2002/0037/diss.pdf.
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Janzik, Ingar. "Heterologe Expression und Charakterisierung von Subtilasen aus Lycopersicon esculentum /." [S.l.] : [s.n.], 2000. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=13658.
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Kelle, Sebastian [Verfasser]. "Heterologe Expression von Enzymen aus Basidiomyceten in Pichia pastoris / Sebastian Kelle." Hannover : Technische Informationsbibliothek (TIB), 2015. http://d-nb.info/1084239795/34.
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Pechlivanis, Ioannis. "Heterologe Expression des AAA-Proteins Pex6p in der Hefe Saccharomyces cerevisiae." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975790919.
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Scheltz, Tineke [Verfasser]. "Dictyostelium-Amöben : Charakterisierung endogener und heterologe Expression humaner Tetraspanine / Tineke Scheltz." Kiel : Universitätsbibliothek Kiel, 2017. http://d-nb.info/1129685403/34.
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Pfeffer, Jan Christoph. "The lipases from Candida antarctica cloning, expression and their application in the synthesis of structured lipids /." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-34375.
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Büchi, Ruth. "Charakterisierung und heterologe Expression des citratbindenden Proteins aus Lutoiden von Hevea brasiliensis /." [S.l.] : [s.n.], 1997. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=12402.
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Galperin, Ilya [Verfasser]. "Das ligninolytische System von Pleurotus sapidus: Transkriptomanalyse und heterologe Expression einer Arylalkoholoxidase / Ilya Galperin." Gießen : Universitätsbibliothek, 2018. http://d-nb.info/1162053968/34.
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Solem, Even [Verfasser]. "Heterologe Expression und physikochemische Charakterisierung der Polyphenoloxidasen aus dem Cabernet Sauvignon (Vitis vinifera) / Even Solem." Mainz : Universitätsbibliothek Mainz, 2017. http://d-nb.info/114214044X/34.
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Theis, Stephan. "Heterologe Expression und funktionelle Charakterisierung intestinaler und renaler Peptidtransporter in der methylotrophen Hefe Pichia pastoris." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964209551.
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Bonacker, Eckart. "Expression von DNA aus Bodenproben in Streptomyces für den Nachweis neuer Enzymaktivitäten." [S.l. : s.n.], 2002. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10316295.
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Koschorreck, Katja. "Expression, Charakterisierung und Optimierung mikrobieller Laccasen für die Biokatalyse." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-38663.
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Kottwitz, Denise. "Heterologe Expression und strukturelle Charakterisierung der intrazellulären Domäne der d-Untereinheit [Delta-Untereinheit] des nikotinischen Acetylcholinrezeptors." [S.l.] : [s.n.], 2005. http://www.diss.fu-berlin.de/2005/255/index.html.
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Schlecht, Sabine. "Entwicklung eines anwendungssicheren Vakzine-Prototyps auf Basis replikations-defizienter Sendai-Viren zur simultanen Expression von Antigenen heterologer Viren." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=974282286.
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Romanski, Annette. "Das Elicitorprotein NPP1 Isolierung und Charakterisierung der korrespondierenden cDNA, heterologe Expression des Proteins und Studien zur Signalperzeption /." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963984098.
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Mreyen, Silke. "Funktionelle Untersuchungen zur Wechselwirkung des archaebakteriellen Photorezeptors NpSRII mit seinem Transducer NpHtrII durch heterologe Expression in E.coli." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968538266.
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Thalmann, Sophie. "Korrelation zwischen der genetischen und der funktionellen Diversität humaner Bitterrezeptoren." Phd thesis, Universität Potsdam, 2013. http://opus.kobv.de/ubp/volltexte/2013/6684/.
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Der Mensch besitzt ~25 funktionelle Bitterrezeptoren (TAS2R), die für die Wahrnehmung potenziell toxischer Substanzen in der Nahrung verantwortlich sind. Aufgrund der großen genetischen Variabilität der TAS2R-Gene könnte es eine Vielzahl funktionell unterschiedlicher TAS2R-Haplotypen geben, die zu Unterschieden der Bitterwahrnehmung führen. Dies konnte bereits in funktionellen Analysen und sensorischen Studien für einzelne Bitterrezeptoren gezeigt werden. In dieser Arbeit wurden die häufigsten Haplotypen aller 25 Bitterrezeptoren verschiedener Ethnien funktionell charakterisiert. Das Ziel war eine umfassende Aussage über die funktionelle Diversität der TAS2Rs, die die molekulare Grundlage für individuelle Bitterwahrnehmung bildet, treffen zu können. Fehlende Varianten wurden aus genomischer DNA kloniert oder durch gezielte Mutagenese bereits vorhandener TAS2R-Konstrukte generiert. Die funktionelle Analyse erfolgte mittels Expression der TAS2R-Haplotypen in HEK293TG16gust44 Zellen und anschließenden Calcium-Imaging-Experimenten mit zwei bekannten Agonisten. Die Haplotypen der fünf orphanen TAS2Rs wurden mit über hundert Bitterstoffen stimuliert. Durch die gelungene Deorphanisierung des TAS2R41 in dieser Arbeit, wurden für die 21 aktivierbaren TAS2Rs 36 funktionell-unterschiedliche Haplotypen identifiziert. Die tatsächliche funktionelle Vielfalt blieb jedoch deutlich hinter der genetischen Variabilität der TAS2Rs zurück. Neun Bitterrezeptoren wiesen funktionell homogene Haplotypen auf oder besaßen nur eine weltweit vorherrschende Variante. Funktionell heterogene Haplotypen wurden für zwölf TAS2Rs identifiziert. Inaktive Varianten der Rezeptoren TAS2R9, TAS2R38 und TAS2R46 sollten die Wahrnehmung von Bitterstoffen wie Ofloxacin, Cnicin, Hydrocortison, Limonin, Parthenolid oder Strychnin beeinflussen. Unterschiedlich sensitive Varianten, besonders der Rezeptoren TAS2R47 und TAS2R49, sollten für Agonisten wie Absinthin, Amarogentin oder Cromolyn ebenfalls zu phänotypischen Unterschieden führen. Wie für den TAS2R16 bereits gezeigt, traten Haplotypen des funktionell heterogenen TAS2R7 und TAS2R41 ethnien-spezifisch auf, was auf lokale Anpassung und verschiedene Phänotypen hinweisen könnte. Weiterführend muss nun eine Analyse der funktionell-variablen TAS2Rs in sensorischen Tests erfolgen, um ihre phänotypische Relevanz zu prüfen. Die Analyse der funktionsmodulierenden Aminosäurepositionen, z.Bsp. des TAS2R44, TAS2R47 oder TAS2R49, könnte weiterführend zum besseren Verständnis der Rezeptor-Ligand- und Rezeptor-G-Protein-Interaktion beitragen.Bitter taste perception varies markedly from person to person, due to a high number of polymorphisms present in the 25 known functional bitter receptors (TAS2Rs). These polymorphisms lead to a number of haplotypes for each receptor, which are common in different populations, but vary in frequency. The individual combination of receptor variants seems to determine the person’s sensitivity of bitter perception, as could already be shown for single TAS2Rs. Bitter is an aversive taste quality, indicating the ingestion of harmful substances. Different sensitivity could have an impact on food choice. In order to characterize functional consequences of the genetic diversity, we performed calcium imaging experiments with all main haplotypes for the 25 bitter receptors. The obtained information about receptor properties enables us on the one hand to analyze structure-function relationships and on the other hand gives us the functional diverse candidates to focus on in psychophysical studies. The overall aim is to show genotype-phenotype correlation for bitter taste perception and their impact on food choice and therefore diet and health. Our first aim was to identify agonists for the 5 receptors, which could not be deorphaned in previous screens. We challenged all main haplotypes of these TAS2Rs with 106 bitter compounds and could identify the antibiotic chloramphenicol as agonist for bitter receptor TAS2R41. In total we identified 36 functionally different receptor variants of the 21 deorphaned TAS2Rs. Main haplotypes of nine TAS2Rs were functionally homogeneous while twelve TAS2Rs possessed between two and three functionally heterogeneous receptor variants. In summary the observed functional diversity is not as big as expected. Based on our in vitro findings the shown functional diversity of these twelve bitter receptors might be the molecular basis for individual differences in bitter taste perception and will be further analyzed in psychophysical studies.
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Blaschke, Lisa [Verfasser], and Steffen [Akademischer Betreuer] Rupp. "Heterologe Expression, Charakterisierung und Anwendung einer Formaldehyd-Dismutase zur Gewinnung von Methanol aus Methan / Lisa Blaschke ; Betreuer: Steffen Rupp." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2017. http://d-nb.info/1163603988/34.
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Appert, Christoph. "Heterologe Expression der Phenylalanin Ammonium-Lyasen von Petroselium crispum : biochemische Charakterisierung der Enzyme und Analyse der Interaktion mit spezifischen Hemmstoffen /." [S.l.] : [s.n.], 1994. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10788.
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Follmann, Kara Karen. "Deletion der Untereinheit VIa der Cytochrom-c-Oxidase in Hefe und heterologe Expression des entsprechenden Rindergens sowie Charakterisierung der Mutanten." [S.l. : s.n.], 1998. http://archiv.ub.uni-marburg.de/diss/z1999/0061.
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Scheich, Christoph. "High-throughput evaluation of protein folding conditions and expression constructs for structural genomics." Phd thesis, [S.l. : s.n.], 2004. http://pub.ub.uni-potsdam.de/2004/0055/scheich.pdf.
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Stockmeyer, Kerstin [Verfasser]. "Heterologe Expression und Funktionsanalyse des CMS-assoziierten offenen Leserahmens orf107 aus Sorghum bicolor in Nicotiana tabacum und Arabidopsis thaliana / Kerstin Stockmeyer." Kiel : Universitätsbibliothek Kiel, 2008. http://d-nb.info/1019543426/34.
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Druzinec, Damir [Verfasser]. "Heterologe Expression eines von Galleria mellonella abgeleiteten antimikrobiellen Peptids (AMP) mittels insektenzellbasierter Expressionssysteme : Auswirkungen hydrodynamischer Beanspruchungen im mikroblasenbegasten Rührreaktor / Damir Druzinec." Aachen : Shaker, 2017. http://d-nb.info/1139583468/34.
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Eidhoff, Ulf Benno. "Heterologe Expression, Kristallisation und Untersuchungen zur Struktur von Bos taurus [beta]-Arrestin-1 [Beta-Arrestin-1] und Rattus norvegicus PAR-4." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=961407883.
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Lang, Manuel. "Herstellung und heterologe Expression neukombinierter Gene nichtribosomaler Peptidsynthetasen (NRPS) aus Streptomyces unter Einbeziehung katalytischer Domänen aus Bacillus und funktionelle Charakterisierung ihrer Genprodukte." [S.l.] : [s.n.], 2007. http://opus.kobv.de/tuberlin/volltexte/2007/1590.
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Lorenz, Julia [Verfasser], and Michael [Akademischer Betreuer] Duszenko. "Klonierung und heterologe Expression zur Analyse der Metalloproteasen TbMSP-B und TbMSP-C aus Trypanosoma brucei brucei / Julia Lorenz ; Betreuer: Michael Duszenko." Tübingen : Universitätsbibliothek Tübingen, 2017. http://d-nb.info/116731123X/34.
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Özgür, Armagan Yakup [Verfasser], Karl-Erich [Akademischer Betreuer] Jaeger, and Lutz [Akademischer Betreuer] Schmitt. "Evaluierung eines Rhodobacter capsulatus nif Promotor-basierten Systems für die heterologe Expression von therapeutisch relevanten Membranproteinen / Armagan Yakup Özgür. Gutachter: Karl-Erich Jaeger ; Lutz Schmitt." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/1079971211/34.
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Schwarz, Heiko [Verfasser]. "Cytochrom P450-Enzyme aus Zellkulturen von Centaurium erythraea und Hypericum androsaemum : Molekulare Klonierung und heterologe Expression von P450-Enzymen und NADPH:Cytochrom P450-Reduktasen / Heiko Schwarz." Aachen : Shaker, 2004. http://d-nb.info/1170529577/34.
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Henze, Mathias [Verfasser], Hauke [Akademischer Betreuer] Lilie, Jochen [Akademischer Betreuer] Balbach, and Daniel [Akademischer Betreuer] Huster. "Die humanen Parathormon-Rezeptoren 1 und 2 : heterologe Expression und Charakterisierung von Peptidliganden und ihrer extrazellulären Bindungsdomänen / Mathias Henze ; Hauke Lilie, Jochen Balbach, Daniel Huster." Halle, 2016. http://d-nb.info/1116952122/34.
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Hauptmann, Valeska [Verfasser], Udo [Akademischer Betreuer] Conrad, Karsten [Akademischer Betreuer] Mäder, and Eva [Akademischer Betreuer] Stöger. "Heterologe Expression hochmolekularer repetitiver Spinnenseiden-basierter Proteine in planta sowie deren mechanische und immunologische Charakterisierung [kumulative Dissertation] / Valeska Hauptmann. Betreuer: Udo Conrad ; Karsten Mäder ; Eva Stöger." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2015. http://d-nb.info/1079524258/34.
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Korsten, Anne [Verfasser]. "Das seltene kompatible Solut N-Acetyl-glutaminylglutamin-1-amid (NAGGN): Heterologe Expression des Genclusters aus Pseudomonas putida und Untersuchungen zur Funktion der putativen Biosyntheseenzyme / Anne Korsten. Mathematisch-Naturwissenschaftliche Fakultät." Bonn : Universitäts- und Landesbibliothek Bonn, 2012. http://d-nb.info/1019451696/34.
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Karstens, Katja. "Untersuchungen zur Funktion sauerstofftoleranter, NAD + -reduzierender Hydrogenasen und zu deren Anwendung in der lichtgetriebenen Wasserstoffproduktion in Cyanobakterien." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17123.
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Die lösliche, NAD+-reduzierende Hydrogenase (SH) aus Ralstonia eutropha H16 ist eine Pyridinnukleotid-abhängige Hydrogenase. Das heißt, der Umsatz von H2 im Hydrogenasemodul des Enzyms ist an die Reduktion von NAD(P)+ im NAD(P)H:Akzeptor-Oxidoreduktasemodul gekoppelt. Die SH ist Vertreter des Subtyps, der auch in Gegenwart von O2 katalytisch aktiv ist. Dies wird ermöglicht durch eine reduktive Entfernung von O2, die nach dem aktuellen Modell abhängig ist vom rückläufigen e--Transport vom NADH:Akzeptor-Oxidoreduktasemodul zum aktiven [NiFe]-Zentrum in der großen Hydrogenaseuntereinheit. Der Einfluss des FeS-Clusters in der kleinen Hydrogenaseuntereinheit HoxY auf diesen Prozess wurde hier untersucht. Dabei konnte gezeigt werden, dass die vier hochkonservierten Cysteine C41, C44, C113 und C179 in HoxY an der Koordination des FeS-Zentrums beteiligt sind. Außerdem wurde das nahegelegene Cystein C39 als relevant für die Sauerstofftoleranz identifiziert. Weiterhin wurde gezeigt, dass das Tryptophan W42 aus HoxY essentiell für die Hydrogenaseaktivität der SH ist. Damit bestätigt sich, dass die Kinetik des rückläufigen e--Transports durch die Aminosäureumgebung des FeS-Clusters in HoxY beeinflusst ist. Ferner wurden in dieser Arbeit Ansätze zum Einsatz der SH aus R. eutropha in einer H2-produzierenden cyanobakteriellen Designzelle weiterverfolgt. Dazu wurden Hybridsysteme aus SH und cyanobakteriellem Photosystem I in vitro hinsichtlich ihrer Fähigkeit zur lichtgetriebenen H2-Produktion untersucht. Außerdem wurde an einem heterologen Expressionssystem der SH für Cyanobakterien gearbeitet. Weiterhin wurde die SH aus Rhodococcus opacus MR11 als komplementäres Modellsystem für O2-tolerante Pyridinnukleotid-abhängige Hydrogenasen etabliert. Dieser zur SH aus R. eutropha homologe Komplex hatte in früheren Arbeiten Vorteile für spektroskopische Studien offenbart und wurde hier erstmals im direkten Vergleich zur SH aus R. eutropha biochemisch und spektroskopisch charakterisiert.The soluble, NAD+-reducing hydrogenase (SH) from Ralstonia eutropha H16 is a pyridine nucleotide-dependent hydrogenase. In these types of enzymes the conversion of H2 in the hydrogenase module of the complex is coupled to the reduction of NAD(P)+ in the NAD(P)H:acceptor oxidoreductase module. The SH belongs to a subtype that is catalytically active also in the presence of O2. This O2 tolerance is enabled by a reductive removal of O2, which according to the current model depends on a reverse e- flow from the NADH:acceptor oxidoreductase module to the active [NiFe] site in the large hydrogenase subunit. The impact of the FeS cluster in the small hydrogenase subunit HoxY on this process was analyzed in this study. Thereby it was shown that the four highly conserved cysteines C41, C44, C113 and C179 in HoxY are involved in the coordination of the FeS center. Further the nearby cysteine C39 was identified to be relevant for the O2 tolerance of the SH. Additionally we found the tryptophan W42 to be essential for the hydrogenase activity of the SH. Thus it was confirmed that the kinetic of the reverse e- transport is affected by the amino acid environment of the FeS cluster in HoxY. In addition, approaches for using the SH from R. eutropha in H2 producing cyanobacterial design cells were pursued. On one hand hybrid systems consisting of the SH and cyanobacterial photosystem I were analyzed in vitro for their capacity to produce H2 in a light dependent manner. On the other hand work on a heterologous expression system of the SH for Cyanobacteria was continued. Furthermore the SH from Rhodococcus opacus MR11 was established as complementary model system for O2-tolerant pyridine nucleotide-dependent hydrogenases. This complex, which is homologous to the SH from R. eutropha, has revealed advantages for spectroscopic analysis in earlier studies. Here it was characterized biochemically and spectroscopically for the first time in direct comparison with the SH from R. eutropha.
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Ryzhkov, Pavel. "Bioengineering of S-layers: molecular characterization of the novel S-layer gene sslA of Sporosarcina ureae ATCC 13881 and nanotechnology application of SslA protein derivatives." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1204126129404-82781.
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S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.
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Ueblacker, Peter. "Gentherapeutischer Ansatz zur Expression heterologer Rezeptoren im Herzen." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=96207179X.
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Ryzhkov, Pavel. "Bioengineering of S-layers: molecular characterization of the novel S-layer gene sslA of Sporosarcina ureae ATCC 13881 and nanotechnology application of SslA protein derivatives." Doctoral thesis, Technische Universität Dresden, 2007. https://tud.qucosa.de/id/qucosa%3A24121.
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S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.
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Petit, Isabelle, and J. M. REICHNART. "Expression de peptides antibacteriens d'insectes dans des systemes heterologues." Université Louis Pasteur (Strasbourg) (1971-2008), 1993. http://www.theses.fr/1993STR13005.
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Les insectes superieurs font face aux agressions bacteriennes grace a un mecanisme de defense tres efficace impliquant la synthese de plusieurs familles de peptides antibacteriens a large spectre d'activite. Au laboratoire, 3 familles de peptides antibacteriens ont ete recemment isolees: les defensines, presentant 3 ponts disulfures; la drosocine et les diptericines, peptides o-glycolyses. La structure de ces peptides ne permet pas une synthese chimique aisee. Vu leur interet potentiel, nous avons choisi d'exprimer les genes de ces molecules dans un systeme heterologue. Nous avons realise l'expression des defensines d'insectes en fusion avec le precurseur du facteur dans la levure. Nous avons ainsi obtenu la production de defensine recombinante identique a la defensine native. Dans certains cas, la coamplification du gene kex2 de levure a permis d'ameliorer la production. La drosocine et la diptericine natives ont une activite antibacterienne liee a leur o-glycosylation. La drosocine recombinante est glycosylee mais la substitution est differente de celle du peptide natif. La dipteridine est produite par la levure en faible quantite. Nous avons choisi un autre systeme d'expression, le baculovirus. La diptericine obtenue est inactive. La levure apparait comme un excellent systeme pour la production de defensines d'insectes mais n'a pas permis d'obtenir la production de peptides avec les memes modifications post-traductionnelles que celles des peptides natifs
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Andrews, Christopher John. "Glutathione transferases in soybean Glycine max (L.) Merr." Thesis, Durham University, 1999. http://etheses.dur.ac.uk/4857/.
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Glutathione transferases, also known as Glutathione S-transferases (GSTs), are a diverse group of enzymes that catalyse the conjugation of the tri-peptide glutathione to a wide range of electrophilic substrates. Their biological function in endogenous metabolism in plants is not well characterised, although their role in herbicide metabolism and herbicide selectivity is well documented. Many herbicides used in soybean. Glycine max (L.) Merr., are selective against weeds due to their rapid detoxification in the crop through conjugation with homoglutathione (γ-glu-cys-β-ala), the predominant free thiol in many legumes. However, an in depth characterisation of the GSTs which can potentially catalyse these reactions in soybean has never been performed. This work describes the biochemical and molecular characterisation of GSTs in soybean with emphasis on the identification of specific isoenzymes involved in herbicide metabolism. GST activity toward the chloroacetanilide herbicides acetochlor and metolachlor, the diphenyl ethers acifluorfen and fomesafen and the sulphonyl urea chlorimuron-ethyl were all detected in crude protein extracts from five-day-old suspension cultured soybean cells. GST activity was also determined in five-day-old soybean seedlings, though this activity was significantly lower than that observed with the cell suspension cultures. Treatment of soybean plants with herbicides and herbicide safeners resulted in increased GST activity toward the model substrate l-chloro-2,4-dinitrobenzene (CDNB), but no change in activity toward herbicide substrates. In both plant and cell cultures GST-catalysed conjugation of the diphenyl ethers acifluorfen and fomesafen was over five-fold greater in the presence of homoglutathione as compared with glutathione. The preferential detoxification of these herbicides in the presence of homoglutathione appeared to be an important determinant of their rapid detoxification in soybean and an important factor in herbicide selectivity. GSTs were purified from five-day-old soybean cell cultures using S-hexylglutathione affinity chromatography and anion-exchange chromatography. A combination of reversed-phase HPLC, SDS-PAGE and MALDI-TOF mass spectrometry of the purified fractions indicated the presence of nine putative GST subunits, each with a molecular mass between 25 and 29 kDa. Soybean GST cDNA clones were obtained using a combination of RT-PCR, utilising degenerate oligonucleotides designed to conserved regions within plant GSTs, and screening of cDNA libraries prepared from soybean plants and cell cultures. This process failed to identify any theta-type GSTs, the class associated with herbicide detoxification in maize. In contrast, seven distinct tau-type GSTs were isolated together with a number of clones showing minor variations in individual sequences. Expression of these cDNAs in Escherichia coli showed the purified recombinant GSTs were active toward a diverse range of substrates, and possessed additional glutathione peroxidase activity. GST activities for each recombinant enzyme varied with substrate and thiol type, with a marked preference for homoglutathione with selected substrates. From the work reported in this study it would appear that the tau-type GSTs of soybean are at least as complex as those previously reported in cereals and have an important role in determining herbicide metabolism and selectivity in this major crop.
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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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Jacob, Daniela. "Erkennung von Regulationssequenzen für die Transkription in heterologen Systemen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2003. http://dx.doi.org/10.18452/14985.
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Während der Evolution entwickelten sich Promotorelemente von Prokaryonten, Eukaryonten und Plastiden in Sequenz und Struktur unterschiedlich. Ein Transfer von Promotorsequenzen zwischen Prokaryonten, Eukaryonten und Plastiden sollte daher zu keiner effizienten Genexpression führen. Mit dieser Arbeit sollte die Spezifität von Promotoren analysiert und ihre Funktionalität über `kingdom´-Grenzen hinaus untersucht werden. Hierfür wurden zwölf pflanzenspezifische Promotoren, welche in der Gentechnik zur Herstellung von transgenen Pflanzen eingesetzt werden, auf Genexpression in fünf Bakterienarten untersucht. Weiterhin wurden drei bakterielle und sechs Plastiden Promotoren auf Genexpression in Nicotiana tabacum untersucht. Die Frequenz, mit der die pflanzenspezifischen Promotoren (P) in den untersuchten Bakterienarten zu einer Expression führten, lag bei 50 % der getesteten Kombinationen. Für den P ST-LS1 aus Solanum tuberosum wurde eine Expression in den fünf untersuchten Bakterienarten nachgewiesen. Zwei der getesteten pflanzenspezifischen Promotoren, P RolC aus Agrobacterium rhizogenes und P 247 aus N. tabacum zeigten keine Expression in den untersuchten Bakterienarten. Die Charakterisierung der mRNA-Transkripte ausgewählter Fusionen der pflanzenspezifischen Promotoren mit den Reportergenen zeigten eindeutig, dass für die Transkriptionsinitiation durch die bakterielle RNA-Polymerase Sequenzelemente der pflanzenspezifischen Promotoren genutzt wurden. Mit einer ortsspezifischen Mutagenese des P ST-LS1 konnte die von der bakteriellen RNA-Polymerase genutzte -10-Region in Escherichia coli und Acinetobacter sp. ermittelt werden. Die `down-Mutanten´ führten in E. coli und Acinetobacter sp. zu einer Reduktion der Expression, wohingegen sie nach stabiler Integration in das Pflanzengenom von N. tabacum eine unverminderte Expression in bezug auf den Wildtyp-Promotor zeigten. Die Untersuchung der bakteriellen und Plastiden Promotoren ergab nur in einem einzigen Fall von neun getesteten Promotoren eine sehr geringe transiente Expression.During evolution the promoter elements from prokaryotes, eukaryotes and plastids have developed differently with regard to their sequence and structure, implying that in general a transfer of promoter sequences between prokaryotes, eukaryotes and plastids will not cause an efficient gene expression. The aim of this study was to investigate the specificity of promoter sequences and their functionality in different kingdoms. Therefore 12 different plant-specific promoters, all used for the construction of genetically modified plants, were tested for their capability to direct a gene expression in various bacteria. Furthermore three bacterial and six plastid promoters were tested with respect to their ability to direct a gene expression in Nicotiana tabacum. The frequency of plant-specific promoters (P) directing gene expression in bacteria was 50 % of the combinations analysed. The promoter P ST-LS1 of Solanum tuberosum was functional in all bacteria tested. Two of the plant-specific promoters, P RolC of Agrobacterium rhizogenes and P 247 of N. tabacum, caused no gene expression in the bacteria tested. The characterisation of mRNA-transcripts of fusions between the plant-specific promoter sequences and the reporter genes proved that sequence elements of the plant-specific promoters themselves were used for transcription initiation by the bacterial RNA polymerase. By site-directed mutagenesis of the P ST-LS1 the -10 region used by the bacterial RNA polymerase of E. coli and Acinetobacter sp. was identified. The generated `down-mutants´ of the P ST-LS1 promoter showed a reduction of expression in E. coli and Acinetobacter sp. while these mutants were still fully active after stable integration in the genome of N. tabacum compared to the wildtype promoter. The investigation of the bacterial and the plastid promoters showed a very low transient expression of one out of nine promoters tested.
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Hayashi, Koji. "Functional Analysis of Mammalian Cytochromes P450 by Using Heterologus Expression Systems." Kyoto University, 2001. http://hdl.handle.net/2433/150789.
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Mathes, Tilo. "Photochemie und Signaltransduktion von Blaulichtrezeptorproteinen aus photosynthetisierenden Mikroorganismen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2008. http://dx.doi.org/10.18452/15692.
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Die lichtaktivierte Kinase Phototropin aus Chlamydomonas reinhardtii, die photoaktivierte Adenylatcyclase (PAC) aus Euglena gracilis und das BLUF-Protein Slr1694 aus Synechocystis sp. PCC 6803 wurden in Hinblick auf die molekularen Details der primären photochemischen Prozesse sowie der Signalweiterleitung untersucht. Phototropin wurde mit Hilfe von Arginin aus Escherichia coli in Milligramm Mengen isoliert. Ohne Arginin wurde E. coli cAMP Rezeptorprotein assoziiert aufgefunden, welches eine hohe Homologie zu einer cAMP aktivierten Kinase aus C. reinhardtii besitzt. Volllängen Phototropin bildet wie einzelne LOV-Domänenkonstrukte ohne Kinasedomäne den Flavin-Triplettzustand und das kovalente Cysteinyl-Addukt. Der Zerfall des Signalzustandes ist in Anwesenheit von ATP beschleunigt und deutet auf Photorezeptor-Kinase Interaktion hin. Strukturelle Änderungen in der Kinasedomäne wurden durch FTIR-Differenzspektroskopie gezeigt. Über ELDOR-Spektroskopie wurde der Abstand der Photorezeptordomänen auf etwa 25 Angstrom bestimmt. Mutationen in Slr1694 an S28, N31 und W91 zeigten keine konservierten Einfluss auf die Dynamik des Signalzustands. Die Entfernung der Seitenkette von S28 führte zu einer 15 nm Rotverschiebung des Absorptionsspektrums aufgrund veränderter Wasserstoffbrückenkoordination des Kofaktors. Die Einführung von positiv geladenen Seitenketten an Stelle von N31 erhöhte die Kofaktorbindung von phosphorylierten Flavinen. Künstliche Kofaktoren wie Roseoflavin konnten in Slr1694 durch Koexpression eines prokaryotischen Flavintransporters erreicht werden. Die Rolle von M152 in PAC für die Signalweiterleitung wurde anhand der lichtaktivierten cAMP Synthese-Aktivität gezeigt. Durch ultraschnelle IR-Spektroskopie wurde die Beteiligung der Seitenketten von Y8 sowie Q50 bestätigt und eine genauere Beschreibung der Wasserstoffbrücken im langlebigen Signalzustand ermöglicht.The light activated kinase Phototropin from Chlamydomonas reinhardtii, the photoactivated adenylylcyclase (PAC) from Euglena gracilis and the BLUF protein Slr1694 from Synechocystis sp. PCC 6803 were investigated concerning the molecular details of the primary photochemistry as well as signal transduction. Phototropin was isolated from Escherichia coli in mg amounts after solubilization with arginine. Without arginine E. coli cAMP receptor protein, which shows high homology to a cAMP activated kinase from C. reinhardtii, was copurified. Full length Phototropin shows similar photochemistry to LOV-domain containing proteins without the kinase including triplet and covalent cysteinyl adduct formation. Signaling state decay is accelerated in the presence of ATP and suggests photoreceptor-kinase interaction. FTIR spectroscopy showed light induced structural changes in the kinase domain. The distance of the photoreceptor domains of 25 Angstrom was determined by ELDOR spectroscopy. Mutation of the side chains of S28, N31 and W91 in Slr1694 showed no conserved influence on the dynamic of the signaling state. Removal of the hydroxyl group of S28 lead to a 15 nm red shift of the absorption spectrum as a result of altered hydrogen bond coordination of the cofactor. Introduction of positively charged side chains at the position of N31 strengthened the binding of phosphorylated flavins. An artificial flavin like roseoflavin was introduced in Slr1694 by coexpression of a bacterial flavin transporter. The essential role of M152 in PAC for signal transduction was shown by determination of light activated cAMP synthesis activity. Ultrafast IR spectroscopy confirmed the contribution of Y8 and Q50 in the photocycle and gave a more detailed description of the hydrogen bonding situation in the signaling state.
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Reyes, Luis Fernando. "Proteínas inativadoras de ribossomos: identificação de novas proteínas e estudos de interação da cadeia-A da pulchellina (PAC) com monocamada de Langmuir." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-27052011-114447/.
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Proteínas Inativadoras de Ribossomos (RIPs) são rRNA N-glicosilases capazes de inibir a síntese protéica pela remoção de uma adenina específica do RNA ribossomal. São geralmente classificadas em tipo 1 e tipo 2, sendo as últimas divididas em altamente tóxicas e não tóxicas. A maior parte das RIPs tipo 2 identificadas pertence a espécies de dicotiledôneas, como é o caso da pulchellina. As cadeias tóxicas das RIPs possuem uma região C-terminal hidrofóbica conservada, a qual se atribui a capacidade de interação com a membrana do retículo endoplasmático (RE), durante o transporte retrógrado da toxina para o citosol. Neste trabalho duas abordagens diferentes foram aplicadas para o estudo das RIPs tipo 2: identificação e caracterização de novos integrantes desta família de proteínas, e investigação da interação da cadeia-A da pulchellina (PAC) com sistemas miméticos da membrana celular. Na primeira abordagem, uma busca in silico em bancos de dados genéticos públicos permitiu identificar quatro novas RIPs do tipo 2 de monocotiledôneas. A análise da estrutura primária das proteínas identificadas mostrou a ocorrência de mutações em alguns dos principais aminoácidos que formam o sítio ativo nas RIPs, indicando uma possível perda de função. O representante de Saccharum officinarum (cana-de-açúcar) foi então analisado em maior detalhe, sendo sua cadeia-A clonada (soRIPA), expressa em sistema heterólogo e caracterizada em termos de atividade e estrutura secundária. Os ensaios in vitro mostraram que a soRIPA não foi capaz de depurinar ribossomos eucariotos. Porém, os ensaios de inibição da síntese proteica mostraram uma possível atividade inibitória da soRIP, que precisa ainda ser confirmada. A presença dos transcritos no banco do SUCEST sugere que estes genes não sejam pseudogenes, embora não tenha sido possível purificar a proteína a partir de extratos de folhas. Isto indica que se a soRIP está sendo traduzida, deve sofrer um rápido turnover, tornando difícil a sua detecção e purificação ou, ainda, que a ausência de sítios de ligação à galactose funcionais na cadeia-B impediu sua purificação por cromatografia de afinidade à galactose. A outra abordagem no estudo das RIPs tipo 2 foi centrada na cadeia-A recombinante da pulchellina (rPAC), estudando sua interação com monocamadas de Langmuir. Foram construídos 3 mutantes da rPAC, cada um com diferentes deleções na região C-terminal visando determinar a região responsável pela interação com a membrana do RE. A cinética de adsorção e pressão superficial exercida pela rPAC sobre a monocamada, assim como o estudo com os mutantes demontraram que a proteína interage fortemente com a monocamada fosfolipídica e que esta interação in vitro é dependente da presença da região C-terminal. De forma geral, os resultados obtidos neste trabalho contribuíram com novas informações sobre esta família de proteínas, identificando e analisando novos integrantes e, ainda, adicionando detalhes do mecanismo funcional do tráfego das toxinas RIPs.Ribosome Inactivating Proteins (RIPs) are rRNA N-glycosilases which are able to inhibit the protein synthesis by removing a specific adenine from the ribosomal RNA. They are usually classified as type 1 and type 2, being the latter divided into highly toxic and nontoxic. The majority of type 2 RIPs currently identified are found in species of dicotyledons, as the pulchellin. The toxic chain of RIPs has a conserved hydrophobic C-terminal region, which is believed to be responsible for the interaction with the lipid membrane of the endoplasmic reticulum ER during the retrograde transport of the toxin to the cytosol. In this work, two different approaches were applied in the study of type 2 RIPs: identification and characterization of new members of this protein family, and investigation of the interaction of the pulchellin\'s A-chain (PAC) with systems that mimic the cellular membrane. In the first approach, an in silico search in public genetic databases was performed and allowed us to identify four new type 2 RIPs in monocots. The primary structure analysis of the identified proteins showed the presence of mutations in key amino acids that form the active site of RIPs, indicating a possible interference on its catalytic activity. The representative of Saccharum officinarum (sugar cane) was then analyzed in greater detail. Its A-chain clone (soRIPA) was expressed in a heterologous system and characterized in terms of activity and secondary structure. In vitro experiments showed that soRIPA was not able to perform the depurination of eukaryotic ribosomes. However, the inhibition of protein synthesis assays presented a possible low inhibitory activity of the soRIP, which still needs to be further investigated. The presence of transcripts on the bank of SUCEST indicates that these genes are not pseudogenes, although it was not possible to purify the protein from leaf extracts. If the soRIP is being translated, this may indicate that it undergoes a quick turnover, preventing its detection and purification. It is also possible that the absence of functional galactose binding sites in the B-chain has prevented its purification by galactose affinity chromatography. Our second approach to the study of type 2 RIPs was focused on the recombinant pulchellin A-chain (rPAC), by investigating its interaction with Langmuir monolayers. We have constructed three mutants of rPAC, each one with different deletions at the C-terminal to determine the region responsible for interaction with the membrane of the ER. The adsorption kinetic and surface pressure applied by rPAC on the monolayer, as well as the study of the mutants, have demonstrated that the protein has a strong interaction with the phospholipid monolayer and that this interaction in vitro is dependent on the presence of the C-terminal. The results of this work have provided new information about the type 2 RIP protein family, identifying and analyzing new members, and also bringing new details about the functional mechanism of the RIP\'s toxin traffic.
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FLEURY, CHRISTOPHE. "Etude fonctionnelle des proteines decouplantes mitochondriales (ucps) par expression heterologue dans la levure saccharomyces cerevisiae." Paris 11, 1998. http://www.theses.fr/1998PA112237.
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La proteine ucp1 (uncoupling protein-1) est un transporteur dans la membrane interne mitochondriale, specifique des adipocytes bruns thermogeniques. Ucp1 permet la rentree des protons dans la matrice mitochondriale et dissipe le gradient de protons genere par la chaine respiratoire. L'energie liberee par ce decouplage entre la respiration cellulaire et la phosphorylation de l'atp est dissipee sous forme de chaleur. Cette activite decouplante est inhibee par les nucleotides et activee par les acides gras libres. L'etude fonctionnelle d'ucp1 par expression heterologue dans la levure saccharomyces cerevisi a necessite la mise au point de techniques et protocoles, notamment de nouveaux outils (mutagenese, banque d'expression, cytometrie de flux). L'analyse de nombreux mutants a permis de mieux comprendre les relations entre la structure, la fonction d'ucp1 et les regulations de son activite. Nous avons ete conduits a postuler l'existence de nouveaux genes codant des proteines tres semblables a l'ucp1 de tissu adipeux brun. De nouveaux genes ont ete mis en evidence chez les mammiferes : - le gene ucp2, exprime dans de nombreux types cellulaires. - le gene ucp3, exprime principalement dans les muscles. - le gene ucp4, exprime preferentiellement dans le cerveau. Un gene homologue aux ucps de mammiferes a egalement ete identifie chez la pomme de terre (stucp) et arabidopsis thaliana (atucp). La constitution d'une famille de proteines decouplantes au sein de la superfamille des transporteurs mitochondriaux suggere que le decouplage des mitochondries doit revetir une importance physiologique et incite a reexaminer les mecanismes moleculaires de la thermogenese. La production de chaleur par tous les types de cellules est liee a un couplage imparfait de la respiration a la synthese d'atp. Les ucps sont donc de bon candidats pour expliquer les fuites de protons dans la membrane interne des mitochondries.
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Hautefort, Isabelle. "Facteurs de colonisation et expression de proteines heterologues chez lactobacillus fermentum (doctorat : microbiologie)." Paris 11, 1998. http://www.theses.fr/1998PA114823.
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Hellmer, Jens. "Untersuchung putativer Kaliumkanäle und Na+/H+-Antiporter von Methanococcus jannaschii anhand heterologer Expression in Escherichia coli." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969248857.
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Paganelli, Fernanda Laroza. "Clonagem e expressão heterologa dos genes responsaveis pela sintese de polihidroxibutirato em Bradyrhizobium elkanii." [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317363.
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Orientadores: Wanderley Dias da Silveira, Eliana Gertrudes de Macedo LemosDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Polihidroxialcanoatos (PHAs) são polímeros de hidroxialcarioatos produzidos, e acumulados, intracelularmente, como fonte de carbono e/ou outros materiais energéticos, em vários microorganismos. Freqüentemente, o acúmulo dos PHAs ocorrem em condições abaixo do limite nutricional de elementos como N, P, S, O ou Mg e excesso de carbono, podendo representar até 80% da massa seca total da célula. Mais de 300 diferentes microorganismos podem sintetizar e acumular PHAs. O polihidroxibutirato (PHB) é o mais conhecido dentre os polímeros bacterianos biodegradáveis denominados polihidroxialcanoatos. Por ter propriedades semelhantes ao polipropileno, o PHB pode ser usado na fabricação do plástico biodegradável. Além da busca por maior produção de tal polímero, pouco é conhecido sobre seu papel biológico, em especial nos rizóbios. Estudos revelaram que há variação na capacidade da produção e acúmulo de PHB nessas bactérias quando em simbiose, dependendo da espécie em questão e das condições de cultivo das mesmas, observando-se bactérias incapazes de acumular PHB quando bacterióides, como é o caso do Rhizobium meliloti, ou capazes de produzir e acumular PHB, nessas condições, como é o caso de Rhizobium etli, Bradyrhizobium japonicum e Bradyrhizobium elkanii. Desta forma, este trabalho teve por objetivo a identificação dos genes responsáveis pela síntese de PHB em Bradyrhizobium elkanii, clonagem e expressão dos mesmos em Escherichia coli, uma vez que esta bactéria é bem conhecida como ferramenta molecular e se multiplica rapidamente, podendo atingir uma alta produção do polímero esperado, em um curto período de tempo. Além disso, objetivou-se, também, aumentar a produção de PHB em B. elkanii através de mutações aleatórias (através da inserção do transposon TnphoA), já que esta é uma bactéria naturalmente produtora de PHB. Para isso, os genes phbA, phbB e phbC foram isolados através da técnica de PCR, amplificando-se os genes inteiros. Estes foram clonados em vetores de expressão tipo pET (NOVAGEN), sendo os genes phbA e phbB clonados em "operon " em um mesmo vetor e o gene phbC clonado separadamente. A expressão dos genes foi analisada, bem como sua capacidade de produzir PHB. Os mutantes de B. eíkanii obtidos através da inserção do transposon TnphoA foram analisados com o uso do corante Sudan Black, procurando-se selecionar linhagens maiores produtoras de PHB. A produção dos mutantes selecionados foi, posteriormente, analisada por cromatografia gasosa. Observou-se que a linhagem de E.coli com os três genes clonados teve a capacidade de produzir PHB, porém com baixa eficiência. Já os mutantes aleatórios de B. elkanii apresentação diferentes acúmulos em relação ao selvagem, com destaque para o MUT33 que teve 72% da sua massa seca acumulada na forma de PHB, enquanto o selvagem acumulou 51 % de PHB, nas mesmas condições.
Abstract: Polyhydroxyalkanoates (PHAs) are polymers of hydroxyalkanoate acids, produced and accumulated intracellularly as a source of carbon and energy storage material, in prokaryotic cells. Often, the PHAs accumulation occurs in conditions when the carbon source is in excess but one or several other nutrients are limited, and may represent up to 80% of the cell dry weight. More than 300 different microorganisms can synthesize and accumulate PHAs. The polyhydroxybutyrate (PHB) is the most studied polymer among the bacterial biodegradable polymers (PHAs). By having properties similar to polypropylene, PHB can be used in the manufacture of biodegradable plastic. On the search for greater production of such polymer, little is known about its biological role, especially in the genus Rhizobium. Studies have shown that there is a variation in the PHB capacity production and accumulation when these bacteria are in symbiosis. Depending on the species and cultivation conditions it has been observed either incapacity of PHB production and accumulation, when Bacteroides, as Rhizobium meliloti; or capacity and accumulation of PHB under bacteroides fase, as Rhizobium etli, Bradyrhizobium japonicum and Bradyrhizobium elkanii. Therefore, the present work aimed the identification, cloning and expression, in Escherichia coli, of responsible genes for PHB synthesis in Bradyrhizobium elkanii. Moreover, it was also expected to increase the production of PHB in B. elkanii, through random mutations (insertion of the TnphoA transposon), since B. elkanii is a good natural producer of PHB. For this, the entier phbA, phbB and phbC genes were isolated by PCR. Those genes were cloned in expression vectors such pET (NOVAGEN), where phbA and phbB genes were cloned in operon, in a single vector, whereas phbC gene was cloned separately, in another vector. The expression of those genes was analyzed, as well as its ability to produce PHB. Mutants B. elkanii, obtained by insertion of the TnphoA transposon, were analyzed using the dye Sudan Black, in order to select different strains that might produce higher quantities of PHB. The production of PHB by mutants was then analyzed by gas chromatography. It was observed that the E. coli with the three cloned genes had the ability to produce PHB, but with low efficiency. The B. elkanii random mutants show different accumulation compared to the wild, especially MUT33 that had 72% of its dry mass accumulated in the form of PHB, while the wild 51% of accumulated PHB under the same conditions.
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular
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Schramm, Christoph [Verfasser]. "Einfluss des selektiven I f -Ionenkanalblockers Ivabradin auf die Schrittmacherionenkanal (HCN)-Expression im heterologen Expressionssystem / Christoph Schramm." Köln : Deutsche Zentralbibliothek für Medizin, 2011. http://d-nb.info/1013613546/34.
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Rückborn, Meike Ute [Verfasser], and Mirko [Akademischer Betreuer] Trilling. "Generierung und Charakterisierung von Cytomegalovirus-Mutanten als Expressions- und Impfstoffplattformen für heterologe Virusproteine / Meike Ute Rückborn ; Betreuer: Mirko Trilling." Duisburg, 2021. http://d-nb.info/123650187X/34.
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