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Autodisplay der humanen Proteinkinase CK2 und Entwicklung eines Verfahrens zur Inhibitionstestung 9783832525736

Autodisplay der humanen Proteinkinase CK2 und Entwicklung eines Verfahrens zur Inhibitionstestung

ISBN-10: 3832525734
ISBN-13: 9783832525736
PUBLISHER: Logos Verlag Berlin
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Note: Not guaranteed to come with supplemental materials (access codes, CDs, DVDs)
Product Description: Die humane Proteinkinase CK2 ist ein Enzym, dessen Aktivität in einer Vielzahl von Tumoren erhöht ist. Es wird als Zielmolekül für neuartige Cytostatika diskutiert. In der vorliegenden Dissertation wurden Strategien erarbeitet, um Inhibitoren der CK2 möglichst einfach zu identifizieren. Um einen schnellen Zugang zu der CK2 zu erhalten, wurde dieses Enzym mittels Autodisplay an der Zelloberfläche von Escherichia coli präsentiert. Mit Hilfe eines radiometrischen Testverfahrens konnte die Aktivität der oberflächenpräsentierten CK2 belegt und Inhibitionstestungen durchgeführt werden.Im zweiten Teil der Arbeit wurden Testverfahren entwickelt, die als mögliche Alternativen zu dem gängigen radiometrischen CK2-Aktivitätstest dienen sollten. Ein Testverfahren nutzt in einer gekoppelten Enzymreaktion die unterschiedliche Proteasesensibilität eines unphosphorylierten CK2-Substratpeptid gegenüber einem phosphorylierten CK2-Produktpeptid aus. Das Peptid wurde N- und C-terminal mit einem FRET-Donor bzw. -Akzeptor markiert. Die nach Proteaseinkubation entstehende Fluoreszenz wurde als Maß für die Proteolyse herangezogen und war umgekehrt proportional zur CK2-Aktivität. Das zweite neu entwickelte Testverfahren für CK2-Aktivität basierte auf dem direkten Nachweis von Substrat und Produkt der CK2-Reaktion. Durch eine kapillarelektrophoretische Trennung und Quantifizierung von unphosphoryliertem und phosphoryliertem CK2-Substratpeptid konnte die Aktivität der CK2 bestimmt werden. Dieses Verfahren eignete sich hervorragend zur Bestimmung von IC50 Werten. Es wurde zur Durchmusterung einer Substanzbibliothek eingesetzt, wodurch ein hochpotenter CK2-Inhibitor mit einem IC50 Wert von 0,03 muidentifiziert werden konnte.

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PAGES: 172

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