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Cell-free protein synthesis is gaining increasing importance in basic and applied research as a result of advances that have been made in the past years. This study successfully established a commercially available coupled in vitro transcription/translation system for DNA expression templates based on HeLa cell lysate and suitable detection methods. Possibilities to establish an inexpensive in-house CFPS system based on E. coli lysate were elucidated. Cell-free extract from E. coli strains HB101, JM109 and B (Luria) was generated using sonication and a streamlined procedure for the creation of a reaction mix for convenient and simple application in CFPS systems was established. Of the three strains tested using the in-house protocol, neither exhibited any CFPS activity.
Im Rahmen dieser Arbeit wurde die sekretorische Produktion des DESIGNER-Proteins DP10A durch Pichia pastoris untersucht. Dazu wurde die DNA-Sequenz des Zielproteins in den Vektor pPICZαA kloniert und in das Genom von Pichia pastoris durch homologe Rekombination integriert. Für die Auswahl eines geeigneten Klons wurde ein Klon-Screening durchgeführt. Zusätzlich wurde die Produktion des Zielproteins bei verschiedenen pH-Werten sowie die Löslichkeitsverteilung von intrazellulär vorliegendem DP10A untersucht.
Das DESIGNER-Protein DP10A konnte mit P. pastoris produziert werden. Allerdings konnte das Ziel einer sekretorischen Produktion nicht erreicht werden, da DP10A nur intrazellulär und hauptsächlich unlöslich in der Zelle vorlag.
In der vorliegenden Bachelorarbeit werden zwei gentechnisch veränderte Escherichia coli Stämme unter verschiedenen Wachstumsbedingungen mittels flowzytometrischer und massenspektrometrischer Analysen untersucht. Zum Einen wird die Quantität des rekombinanten Proteins L - Prolin - trans - 4 - Hydroxylase auf Populations -, sowie Subpopulationsebene, analysiert. Zum Anderen werden weitere Proteine des Proteoms einbezogen, um auftretende Subpopulationen genauer zu charakterisieren.
In this work a new method for the prediction of the Xaa-proline (where Xaa is any amino acid) cis/trans isomerization was investigated. By extraction of twelve structural features (real secondary structure, inside/outside classification, properties of the environment around proline and proline itself) a support vector machine (SVM) based prediction approach was evolved. The Java software Xaa-PIPT for structural feature extraction was developed. Based on 4397 (2199 cis and 2198 trans) prolines extracted from non-redundant, globular proteins a classifier was trained using the radial basis function (RBF) kernel. In ten-fold cross-validation it achieved an accuracy of 70.0478 % and a Matthews correlation coefficient (MCC) of 0.4223, a sensitivity of 0.5433 and a specificity of 0.8576. Based on this classifier a lightweight and easy-to-use Java software tool, called m Xaa-PIPT, for the prediction of the Xaa-proline cis/trans isomerization was devel-oped. It was shown that there are correlations between the proline surrounding environment and the isomerization state. m Xaa-PIPT can be used for the evaluation of low-resolution protein structures and theoretical models to improve their quality by the prediction of the Xaa-proline isomerization.
Die vorliegende Arbeit befasst sich mit der Sequenzanalyse der für die Legionellose relevanten Schlüsselproteine der Organismen Legionella anisa, Legionella drancourtii und Legionella shakespearei, sowie mit in vitro-Kultivierungsexperimenten der Spezies Legionella pneumophila ATCC 33152. Zunächst wurden die gesetzlichen Gegebenheiten der Legionellenproblematik und die biologischen Hintergründe betrachtet, darunter die intrazellulären Mechanismen der Legionellose. Die Ergebnisse der Arbeit beinhalten Informationen über die Schlüsselproteine der drei Legionellen Spezies und deren mögliche Pathogenität.
The bachelor thesis is about cis-trans isomerization of Xaa-Pro (Xaa = any amino acid), their quantitative acquisition and the selection of 3D structure information for the prediction with a support vector machine (SVM). The quantitative detection of occurrence of cis-, trans- and cis/trans conformation in membrane proteins will be examined and evaluated. The 3D structure informa-tions include 12 features, the amino acids around proline and are including of proline. These include the inside/outside classification, the real secondary structure, energy consideration, as well as five further amino acid occur properties within a defined radius of the proline. From this information, a data set was created for the SVM. This program is used for the prediction of unknown and known Xaa Pro Isomerisms. The methods for the analysis were implemented with the platform independent programming language Java. Two programs have emerged from the work to a Xaa PIPT for the quantitative detection and extracting structural information and m Xaa-PIPT to the pure prediction of Xaa-Pro isomerism in protein structures. 389 Membrane proteins from the PDB (Protein Data Bank) served as a basis. The data were also statistically analysed and evaluated.
The study “Proteomic and systems biological database analysis of changed proteins from rat brain tissue after diving “ is about system biological testing of proteomic data obtained by rat brain after experimental diving in a pressure chamber. Basically, brain tissue from animal decompression sickness (DCS) was analyzed by mass spectrometry and has given two larger sets of modified proteins. Thereupon, the resulting up- and down-regulated proteins wereidentified and later compared by means of systems of biological databases, in this case GeneGo MetaCoreTM, in order to find similar or various affected cell biological signaling pathways when two different mass-spectrometry methods were compared.
A Protein is a large molecule that consists of a vast number of atoms; one can only imagine the complexity of such a molecule. Protein is a series of amino acids that bind to each other to form specific sequences known as peptide chains. Proteins fold into three-dimensional conformations (or so-called protein’s native structure) to perform their functions. However, not every protein folds into a correct structure as a result of mutations occurring in their amino acid sequences. Consequently, this mutation causes many protein misfolding diseases. Protein folding is a severe problem in the biological field. Predicting changes in protein stability free energy in relation to the amino acid mutation (ΔΔG) aids to better comprehend the driving forces underlying how proteins fold to their native structures. Therefore, measuring the difference in Gibbs free energy provides more insight as to how protein folding occurs. Consequently, this knowledge might prove beneficial in designing new drugs to treat protein misfolding related diseases. The protein-energy profile aids in understanding the sequential, structural, and functional relationship, by assigning an energy profile to a protein structure. Additionally, measuring the changes in the protein-energy profile consequent to the mutation (ΔΔE) by using an approach derived from statistical physics will lead us to comprehend the protein structure thoroughly. In this work, we attempt to prove that ΔΔE values will be approximate to ΔΔG values, which can lead the future studies to consider that the energy profile is a good predictor of protein binding affinity as Gibbs free energy to solve the protein folding problem.