Development of a reverse split-ubiquitin system to characterize protein-protein interactions

Inhaltsverzeichnis (Table of Contents)

• Zusammenfassung
• Abstract
• List of Abbreviations
• 1 Introduction
• 1.1 Protein-protein interactions
• 1.2 Yeast two-hybrid system
• 1.3 Reverse split-ubiquitin system
• 2 Materials and Methods
• 2.1 Plasmids and strains
• 2.2 Cloning strategy
• 2.3 Transformation
• 2.4 Yeast cultivation and selection
• 2.5 Yeast growth assay
• 2.6 Western blotting
• 3 Results
• 3.1 Cloning of GLO-1(QL) and C35B1.2a into the vectors
• 3.2 Yeast transformation and selection
• 3.3 Yeast growth assay
• 3.4 Western blot analysis
• 4 Discussion
• 5 References

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This bachelor thesis focuses on the development and application of a reverse split-ubiquitin system to characterize protein-protein interactions (PPIs) of membrane proteins. The study aims to demonstrate the interaction between GLO-1(QL), a Rab GTPase, and its effector protein C35B1.2a using this system.

• Protein-protein interactions (PPIs) in biological processes
• Yeast two-hybrid system and its limitations
• Reverse split-ubiquitin system for studying PPIs of membrane proteins
• Interaction of GLO-1(QL) and C35B1.2a
• Optimization of the split-ubiquitin system

Zusammenfassung der Kapitel (Chapter Summaries)

• Chapter 1: Introduction Provides an overview of protein-protein interactions, the yeast two-hybrid system, and the reverse split-ubiquitin system as methods for studying these interactions. It introduces the GLO-1(QL) gene, its role in lysosomal organelles, and its effector protein C35B1.2a.
• Chapter 2: Materials and Methods Details the experimental procedures employed in this study, including cloning strategies, transformation methods, yeast cultivation and selection techniques, yeast growth assays, and Western blotting analysis.
• Chapter 3: Results Presents the results of the cloning, transformation, selection, growth assays, and Western blotting experiments, including the challenges encountered in demonstrating the interaction between GLO-1(QL) and C35B1.2a.
• Chapter 4: Discussion Analyzes the findings, discusses the limitations of the split-ubiquitin system used in this study, and suggests potential improvements to the system for future investigations.

Schlüsselwörter (Keywords)

The study focuses on protein-protein interactions, membrane proteins, yeast two-hybrid system, reverse split-ubiquitin system, GLO-1(QL), C35B1.2a, Rab GTPase, lysosomal organelles, PPI, Nub, Cub, USPS, reporter gene, optimization.