Entwicklung einer schaltbaren Protease und eines fluoreszenten Reporters mittels Inteinkassetten

Gespaltene Inteine haben sich in den letzten Jahren als biochemisches und molekularbiologisches Werkzeug zur posttranslationalen Modifikation von Proteinen etabliert. Die Inteinhälften werden dabei separat auf zwei getrennten Fusionsgenen kodiert. Die Proteintrans- Spleißreaktion (PTS) beginnt nach der Assoziation und Faltung der Inteinhälften in den aktiven Komplex und verknüpft die fusionierten Sequenzen des Zielproteins durch eine native Peptidbindung. Ein kritischer Punkt dieser Reaktion ist die Abhängigkeit der Inteine von der Primärstruktur des Zielproteins und insbesondere von den an das Intein angrenzenden Aminosäuren. Diese können die Spleißreaktion beeinträchtigen oder sogar inhibieren. Um diese Effekte möglichst schnell und mit geringem Arbeitsaufwand für die Generierung von Inteinfusionsproteinen zu erkennen und zu umgehen, wurden in dieser Arbeit auf Inteinkassetten basierende Ansätze entwickelt. Der Vorteil dieser Inteinkassetten gründet sich auf der in einem Schritt stattfindenden „spurlosen“ Integration aller Inteinkomponenten in ein Zielgen durch homologe Rekombination oder restriktionsfreie PCR. Dabei ist die freie Wahl der flankierenden Aminosäuren über die zur Amplifikation gewählten Primer möglich. Letztendlich entfallen sowohl aufwendige Klonierungsarbeiten als auch die Addition von Restriktionsschnittstellen an die Inteingene, wobei letztere zu zusätzlichen Aminosäurecodons führen würden. Im ersten Teil der Arbeit wurde eine bereits in der vorangegangenen Diplomarbeit generierte Inteinkassette verwendet, um konditionale Kontrolle über die Tabakätzvirus- (TEV)-Protease auszuüben. Diese Kontrolle ist möglich, weil das verwendete, künstlich gespaltene Saccharomyces cerevisiae VMA-Intein nur in Verbindung mit dem Heterodimerisierungssystem FRB/FKBP und unter Zugabe des Liganden Rapamycin die Spleißreaktion effizient durchführen kann. Die so induzierte PTS-Reaktion wird als konditionales Proteinspleißen (CPS) bezeichnet. Nach der Integration der Inteinkassette an neun unterschiedlichen Positionen des TEV-Proteasegens in Hefe wurden zwei Positionen identifiziert, die in einer schaltbaren TEV-Protease resultierten und zur selektiven Spaltung von Reporterproteinen mit einer TEV-Erkennungssequenz verwendet werden konnten. Dabei konnte die Spleißproduktbildung bereits nach 30 Minuten in Western-Blot-Analysen nachgewiesen und die Spaltungsaktivität der schaltbaren TEV-Proteasen mittels Konfokalmikroskopie in einzelnen Zellen verfolgt werden. Erste Experimente in Säugerzellen wiesen auch in diesem Modellsystem die Aktivität einer der beiden konditionalen Proteasen nach. Innerhalb eines zweiten Projektes wurden drei auf den gespaltenen Ssp DnaB, Npu DnaE und Mxe GyrA Inteinen basierende trans-Inteinkassetten entwickelt, die alle spontane Aktivität in der PTS-Reaktion zeigten. Dabei variierten die einzelnen Kassetten sowohl in ihrer Sequenzpräferenz als auch in den verschiedenen Aminosäuren, die nach der abgelaufenen Spleißreaktion im ligierten Zielprotein als Reste übrig blieben. Die Kassetten wurden so konzipiert, dass die nach der Zielgen-Integration entstandenen Fusionsgene selektiv in Escherichia coli exprimiert werden konnten. Für das Modellprotein gpD-Trx wurde zuerst die Funktionalität des Integrationsprozesses der Ssp DnaB- und Npu DnaE-Inteinkassetten gezeigt und anschließend die selektive Spleißproduktbildung mittels Western-Blot-Analyse verfolgt. Außerdem wurde die Npu DnaE-Inteinkassette in das CobA-Enzym - eine Uroporphyrinogen- III-Methyltransferase - integriert. Infolgedessen konnte rekonstituiertes CobA durch die Produktion von fluoreszierenden Tetrapyrrol-Derivaten als Biosensor für erfolgreiches Proteinspleißen verwendet werden. Die Aktivität des Proteins wurde über selektive Expression der Inteinfusionsgene in E. coli gesteuert. In der Übertragung dieses Systems auf Säugerzellen konnte die PTS-Reaktion sowie die Aktivität des CobA-Proteins mittels Fluoreszenzmikroskopie detektiert werden.In the last couple of years, split inteins were established as a biochemical and molecular biology tool for the posttranslational modification of proteins. To this end, the intein halves are expressed as two separate intein fusion genes. The actual protein trans-splicing (PTS) reaction takes place after the association and folding of the intein halves into an active complex. Eventually the process leads to the formation of a native peptide bond between both target protein sequences which were fused to the intein halves. A critical step in this reaction is the dependence of the intein on the primary structure of the fused target protein and in particular on the amino acids in direct contact with the intein. These flanking amino acids can impair or even inhibit the protein splicing reaction. The chosen way to recognize and to get rid of these intein related effects and additionally to streamline the fusion gene generation was based on intein cassettes. The advantage of the intein cassette is due to the one-step “traceless” integration process which inserts all intein related components into the gene of interest either via homologous recombination or restriction-free cloning. Even the free selection of the flanking amino acids is possible by simply varying the primers used in the amplification step. Moreover, tedious cloning efforts and the addition of restriction sites, which result in additional amino acid codons, are skipped. In the first part of this work, an intein cassette which was generated during my diploma thesis was used to exercise conditional control upon the Tobacco Etch Virus (TEV) protease. This control is possible, because the artificially split Saccharomyces cerevisiae VMA intein can only perform the protein splicing reaction if utilized in combination with the FRB/FKBP heterodimerizer system and by addition of the small molecule rapamycin. The induced PTS reaction is called conditional protein splicing (PTS). After the integration procedure of the intein cassette at nine different positions in the TEV gene in yeast, two switchable proteases were identified, all of which had the ability to cleave a reporter protein. For those conditional proteases the formation of splice product could be identified within 30 minutes after the addition of rapamycin in western blot analysis. Additionally, the cleavage events could be followed on a single cell basis by confocal microscopy. First experiments in cell culture even indicated activity of one of the CPS proteases in this model system. In a second project, three different intein cassettes were developed that were based upon the Ssp DnaB, Npu DnaE, and Mxe GyrA split inteins and all showed spontaneous activity in the PTS reaction. Thus, the intein cassettes differ in their sequence preference as well as in the persisting amino acid, which is the only remnant of the splicing reaction within the ligated target protein. Another feature of the intein cassette integration is that the resulting fusion genes can be selectively expressed in Escherichia coli. By using a model protein (gpDTrx) and the Ssp DnaB and Npu DnaE cassettes, the functionality of the integration procedure was demonstrated and subsequently the selective formation of the splice product was detected in western blot analysis. Furthermore, the Npu DnaE intein cassette was integrated into the CobA enzyme, a uroporphyrinogen III methyltransferase. The reconstituted CobA generates fluorescent tetrapyrrole derivatives which is why it could be used as a biosensor for successful protein splicing. The activity of the protein was controlled via selective expression of the intein fusion genes in E. coli. After the final transfer of this system to cell culture, the PTS reaction, as well as the resulting CobA activity was detected by fluorescence microscopy

Особенности обогащения углей с большим содержанием легкоразмокаемой породы

Наведені результати дослідження технологічних процесів при збагаченні вугілля з великим вмістом породи, яка легко розмокає, умовах ЦЗФ "Павлоградська". Визначенні особливості ведення технологічних процесів підготовки машинних класів важкосередовищної сепарації. відсадки, флотації. обробки шламових продуктів та сушіння дрібного концентрату, які відрізняються від загальноприйнятих.Приведены результаты исследований технологический процессов при обогащении углей с большим содержанием легкоразмокаемой породы в условиях ЦОФ "Павлоградская". Определены особенности ведения технологических процессов подготовки машинных классов, тяжелосредной сепарации, отсадки, флотации, обработки шламовых продуктов и сушки мелкого концентрата, которые отличаются от общепринятых

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file

5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed

Pressure to provide milk among mothers of very low birth weight infants: an explorative study

Abstract Background Pump-dependent mothers of very low birth weight (VLBW, < 1500g) infants experience specific challenges achieving sufficient milk supply in the neonatal intensive care unit (NICU) and are therefore less frequently able to achieve (exclusive) breast milk feeding. Stress due to the limitations on participating in the infant’s care may contribute to this problem. Some explorative studies suggest that pressure to provide milk may be an additional stressor in mothers. However, the type of pressure to provide milk perceived by mothers of VLBW infants has rarely been examined. Methods A retrospective and anonymous questionnaire was conducted with mothers of VLBW infants aged 6 to 24 months at the time of data collection. Quantitative data and written comments were used to examine the mothers’ perceptions. Descriptive and bivariate tests (Spearman´s rho, Pearson’s chi2) were performed to show correlations between pressure to provide breast milk, parental stress (PSS:NICU: role alteration subscale), milk volume, and maternal factors. Pressure to provide milk was measured through two self-developed single items to differentiate between internal and external pressures. Results Data of n = 533 mothers of VLBW infants was analysed. More than 70% of the mothers agreed that they pressured themselves to provide milk for their infant. In contrast, 34% of the mothers agreed that they felt pressure from outside to provide milk. Higher milk volume 14 days post-partum was significantly correlated with less internal (Spearman´s rho = 0.2017, p = 0.000) and less external pressure to provide milk (Spearman´s rho = 0.2991; p = 0.000). Higher PSS:NICU parental role alteration scores were significantly correlated with more internal (Spearman´s rho = -0.2865, p = 0.000) and more external pressure to provide milk (Spearman´s rho = -0.1478; p = 0.002). Milk volume 14 days post-partum and the PSS:NICU were not significantly correlated (Spearman´s rho = -0.0190; p = 0.701). Qualitative analyses highlighted these results and enhanced the bidirectional relationships between maternal pressure to provide milk and milk volume. Conclusions Especially internal pressure to provide milk is perceived by many mothers, being mutually dependent on milk supply and parental stress. Pressure to provide milk may be an important factor to decrease maternal stress in the NICU and, therefore, lead to more positive pumping and breastfeeding experiences. More research and validated instruments are needed to adequately measure pressure to provide milk with its different psychological, social, and environmental dimensions