The
Yeast Two Hybrid (Y2H) screening is a molecular biology technique used to discover
protein–protein interactions (PPIs). By leveraging the simple eukaryotic system of yeast, scientists can identify and map interactions between various proteins. This technique employs a bait and prey system, where the protein of interest (bait) is fused to a DNA-binding domain, and a potential interacting protein (prey) is fused to a transcriptional activation domain. If the bait and prey proteins interact, a reporter gene is activated, signaling a successful interaction.
Cancer is characterized by complex alterations in cellular signaling pathways, often driven by
mutations and aberrant
protein interactions. Understanding these interactions can provide critical insights into the mechanisms driving cancer progression. Y2H screening allows researchers to:
The Y2H screening process involves several key steps:
Construct the bait and prey vectors by cloning the genes of interest into appropriate yeast expression plasmids.
Transform the yeast cells with the bait and prey plasmids.
Co-culture the transformed yeast on selective media to screen for interactions.
Analyze the positive interactions using reporter gene activity, often visualized as color changes or growth on selective media.
Y2H screening offers several advantages in cancer research:
High throughput: Large libraries of potential interacting proteins can be screened simultaneously.
Versatility: It can be adapted to study various types of proteins, including membrane-bound and soluble proteins.
Cost-effective: Compared to other interaction mapping techniques, Y2H is relatively inexpensive.
Functional interactions: It screens for interactions in a eukaryotic context, increasing the relevance of the findings.
Despite its advantages, Y2H screening has certain limitations:
False positives: Non-specific interactions can sometimes be detected, leading to misleading results.
False negatives: Some genuine interactions may not be detected if the fusion proteins are not properly expressed or localized.
Post-translational modifications: Yeast cells may not perform certain post-translational modifications present in human cells, potentially affecting interaction outcomes.
Examples of Y2H Screening Applications in Cancer Research
Y2H screening has been instrumental in several cancer research studies:
Future Directions and Innovations
Advances in Y2H technology and complementary techniques are poised to further enhance cancer research:
