Refine
Document Type
- Bachelor Thesis (9)
- Master's Thesis (5)
- Diploma Thesis (1)
Year of publication
Keywords
- Proteine (15) (remove)
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.
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.
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.
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.