employment of two hybrid assay to identify interaction-impaired mutants in yeast

Biomolecular Interaction Studies Using Yeast Two-Hybrid Technique 

Interactions between biomolecules are the most basic part of biological phenomena. Protein-protein interactions constitute a significant part of such interactions. Therefore, identification of the interaction partner(s) of a protein of interest is critical to further elucidate the function of the protein/gene of interest. The yeast two-hybrid (Y2H) method is a popular technique to identify protein-protein interactions in vivo1. In this system, two proteins (X and Y) whose interaction is to be tested are fused to the DNA-binding (DB) domain and transcriptional activation domain (AD), respectively. The DB-X fusion protein binds to a recognition sequence of the DB domain; therefore, when proteins X and Y interact, the AD-Y fusion protein comes into the proximity of the recognition sequence. Consequently, transcription of the reporter gene downstream of the recognition sequence is activated. Therefore, the presence or absence of reporter gene activity can be used to determine the presence or absence of the protein-protein interaction1.
Once a specific interaction partner of the protein of interest is identified, further analyses should be performed to elucidate the biological function of the interaction. For this purpose, if mutants of the proteins that impair or remove the specific protein-protein interaction can be isolated, they will serve as powerful tools. The Y2H system can be used directly to isolate such mutants by screening 'interaction-negative' clones, starting with the wild-type 'interaction-positive' clone. To accelerate this process, 'reverse' Y2H (rY2H) systems were developed2,3. In rY2H systems, the host yeast strains harbor counter-selectable marker genes as reporter genes, meaning yeast cells grow only when the AD-Y and DB-X proteins do not interact.
Although both the Y2H and rY2H systems allow the isolation of interaction-negative mutants, the process of isolating the mutants is laborious because not all of the candidates obtained by screening carry the desired type of mutations (usually missense mutations). The most serious issue is that a significant fraction of candidates harbor frameshift or nonsense mutations, and it is necessary to perform western blotting to exclude undesired clones. To overcome this problem, new plasmid vectors have been developed4. In these vectors, KanMX, a drug resistance marker, is positioned out-of-frame downstream of the DB domain or AD. The marker gene becomes in-frame with the DB domain or AD only when the gene of interest is inserted. When a random mutation(s) is introduced in the gene of interest, undesirable mutants, such as those with frameshift or nonsense mutations, can be easily eliminated by performing drug resistance selection, and candidates carrying desirable missense mutations can be easily identified with the Y2H screen4. This article presents a protocol to isolate interaction-null/impaired mutants of a protein of interest using this strategy.

Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

We are interested in understanding the regulatory mechanisms of the cell cycle in Eukaryotes, especially the relationship between DNA replication, stable germ maintenance, and cell proliferation. The progression of the cell cycle is regulated by cyclin-dependent kinases, or CDKs, in Eukaryotes. This is the case for the initiation of DNA replication.We have identified a way, on substrates of CDKs, to initiate DNA replication. To understand the function of the protein of your interest, a comparison of the wild-type and mutant is a direct and effective way. Yeast two-hybrid assay, being simple detection and will enhance the process of isolating interaction-null mutants.This will contribute to advancement of research in the field.

Employment of Two Hybrid Assay to Identify Interaction-Impaired Mutants in Yeast

To begin, obtain the host yeast strain, transformed with the bait plasmid. After introducing the mutant library into it, select a plate with 500 to 2000 colonies. Set two sheets of filter paper on a replica block and make several replicas of the plate, and incubate the plates at 30 degrees Celsius for one to two days.For the yeast two hybrid color assay, place a sheet of filter paper on the replica plate until it is completely moistened. Remove excess moisture using several sheets of paper towel, twice. Then, using tweezers, peel the filter paper off the plate from the edge and quickly immerse it in liquid nitrogen.Thaw the frozen filter paper on a dry paper towel with the colony side up. Once thawed, immediately transfer it onto another filter paper soaked with buffer. Cover the plastic tray containing the filter paper, put it into an airtight container, and incubate it at 37 degrees Celsius.Once blue color appears, place the filter paper on 500 microliters of stop solution taken in a plastic tray and immediately dry it on a fresh paper towel. The color assay produced blue or white dots, differentiating the mutants that did not interact with the bait.

employment-two-hybrid-assay-to-identify-interaction-impaired-mutants

Research

JoVE Journal

Biology

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...