An important topic in research is the study of interactions between proteins. Therefore, many strategies have been developed to better understand these interactions. One method is the yeast two-hybrid system.
The glo-1(QL) gene encodes a Rab GTPase and is localized in lysosomal organelles. Several effector proteins have been found for the glo-1 gene. One of them is C35B1.2a.
Using the yeast two-hybrid system, the interaction between GLO-1(QL) and C35B1.2a should be shown. In addition, the expression rate of the interaction partners should be documented by promoter exchange. However, this system is not suitable for all proteins. For this reason, the reverse split ubiquitin system was developed. A yeast-two-hybrid system-based method to study protein-protein interactions (PPI) of membrane proteins. In this case, the ubiquitin is cleaved into an N-terminal domain (Nub) and C-terminal (Cub) domain, each domain being fused to a protein. The C-terminal domain additionally consists of an arginine and subsequent reporter gene. If there is an interaction of the two fusion proteins, this is recognized by so-called ubiquitin-specific proteases (USPs) and cut the ubiquitin apart. Subsequently, the reporter gene is degraded due to the arginine.
In this study, the interaction could not be demonstrated because no clones grew on selective plates. Even by the replacement of the promoters no growth could be found. To optimize the interaction, the split ubiquitin system should be further refined.
Table of Contents
1. Introduction
1.1 Autophagy
1.1.1 The lysosomal pathway
1.2 The Rab GTPase family
1.2.1 The glo-1 gene
1.2.2 The Rab cycle
1.3 Protein-protein interactions
1.4 Yeast Two Hybrid System
1.4.1 The Split Ubiquitin System
1.4.2 The rUra3p based split-ubiquitin system
1.4.3 Screening with the reverse split-ubiquitin system
1.5 Aim
2. Material and Methods:
2.1 Yeast strains:
2.2 Yeast transformation
2.3 Polymerase Chain reaction:
2.4 Ligation into the pGEMT vector
2.5 Cloning into target vectors:
2.6 Bacterial Transformation
2.7 DNA Mini-Preparation
2.8 Control digestion
2.9 Sequencing
3. Results
3.1 Creating vectors with TEF1 promoter
3.1.1 GLO-1 and C35B1.2a
3.1.2 C35B1.2a (Aa95-301)
3.2 Creating vectors with SSA1 and SSB1 promoter
3.3 Yeast Two Hybrid Assay
4. Discussion
Research Objectives & Topics
The primary aim of this bachelor's thesis is to establish and optimize a variant of the split-ubiquitin system to characterize protein-protein interactions (PPI) between the Rab GTPase GLO-1 and its effector protein C35B1.2a in yeast, focusing on the use of different promoters to regulate expression levels.
- Functional principles of the yeast two-hybrid and split-ubiquitin systems.
- Molecular cloning of bait and prey constructs using various promoters (TEF1, SSA1, SSB1).
- Analysis of protein interaction via yeast growth assays on selective media.
- Troubleshooting and optimization strategies for split-ubiquitin based protein interaction studies.
Excerpt from the Thesis
1.4.1 The Split Ubiquitin System
With the split-ubiquitin system, designed by Johnsson and Varshavski in 1994, protein-protein interactions between membrane proteins can be characterized (Johnsson and Varshavski 1994). In contrast to the classical yeast two-hybrid system, the interaction does not have to take place in the nucleus but can be studied directly on the cellular membrane.
An important function of ubiquitin is the control of defective or damaged proteins. These proteins are labeled with a poly-ubiquitin tail and transported to the proteasome. There will be recognized by certain ubiquitin-specific proteases (USPs), which ultimately cut and inactivate the protein.
To use the split ubiquitin system in analysing protein-protein interactions ubiquitin is divided into two independent domains, a N-terminal (Nub) and a C-terminal domain (Cub). These two domains have a basic affinity for each other, so they can spontaneously reassociate to native ubiquitin. Mutations were placed into Nub by replacing the aminoacids isoleucine into glycine, alanine or valine. This will reduce the affinity for the C-terminal Ubiquitin and thereby suppress the spontaneous reassembly of Cub and mutated Nub (Johnsson & Varshavsky 1994).
A reassociation is only observed, if the corresponding parts are near each other by the fusion two two interacting proteins P1 and P2 (Fig. 4).
Subsequently, a Cub-attached reporter is cleaved off from split-ubiquitin. There are different ways in which the split ubiquitin system can be applied. An example is the rUra3p based split ubiquitin system.
Summary of Chapters
1. Introduction: Provides an overview of autophagy, the Rab GTPase family, and the theoretical background of the yeast two-hybrid and split-ubiquitin systems.
2. Material and Methods: Details the experimental procedures including yeast transformation, polymerase chain reaction (PCR), cloning strategies, and DNA preparation techniques.
3. Results: Documents the successful construction of vectors with different promoters and the subsequent yeast two-hybrid assays conducted to test protein interactions.
4. Discussion: Evaluates the experimental outcomes, addresses potential reasons for the lack of observed interaction growth, and suggests further optimizations for the system.
Keywords
Split-Ubiquitin System, Yeast Two-Hybrid, Protein-Protein Interaction, GLO-1, C35B1.2a, Rab GTPase, TEF1, SSA1, SSB1, Molecular Cloning, Yeast Transformation, Autophagy, HIS3, Reporter Gene, N-end rule
Frequently Asked Questions
What is the core subject of this bachelor thesis?
The work focuses on developing and optimizing a reverse split-ubiquitin system to identify and characterize protein-protein interactions, specifically between the Rab GTPase GLO-1 and its effector C35B1.2a.
What are the primary research themes?
The main themes include protein interaction screening methods, molecular cloning techniques, the influence of different promoters on protein expression, and the troubleshooting of yeast-based interaction assays.
What is the central research question?
The study aims to confirm the interaction between GLO-1 and C35B1.2a and to determine whether replacing a strong promoter (TEF1) with weaker chaperonin promoters (SSA1, SSB1) can optimize the specificity of the split-ubiquitin system.
Which scientific methods were applied?
The research employed molecular cloning (PCR, ligation, bacterial transformation), yeast transformation, and yeast two-hybrid assays on selective media to detect protein interactions.
What is covered in the main section?
The main section describes the theoretical framework of the split-ubiquitin system, the materials and methods used for vector construction, the results of the test digestions and yeast growth assays, and a critical discussion of the results.
Which keywords characterize this thesis?
Key terms include Split-Ubiquitin System, Yeast Two-Hybrid, GLO-1, Rab GTPase, Protein-Protein Interaction, and promoter optimization.
Why was the TEF1 promoter replaced?
The TEF1 promoter was replaced because it proved to be too strong, leading to overexpression of the C-terminal ubiquitin fusion and preventing the proper degradation of the reporter gene, which caused false-positive growth signals.
What were the results regarding the interaction testing?
The interaction between GLO-1 and C35B1.2a could not be demonstrated in this study, as no growth was observed on selective plates containing 3-AT, suggesting potential issues with the system's sensitivity or the experimental setup.
- Citation du texte
- Fatih Sahin (Auteur), 2018, Development of a reverse split-ubiquitin system to characterize protein-protein interactions, Munich, GRIN Verlag, https://www.grin.com/document/477201
