Isolating Interaction-Null/Impaired Mutants Using the Yeast Two-Hybrid Assay

Summary

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.

Abstract

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.

Introduction

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 reco....

Protocol

1. Construction of the mutant library

1. Set up the polymerase chain reaction (PCR) (an example is shown below). Generally, prepare 50 µL of the reaction, which is divided into ten aliquots of 5 µL, and amplify the target gene fragment in a PCR machine⁴.
   NOTE: Users should select an appropriate polymerase to efficiently introduce mutations. If the DNA fragment to be amplified is short, then a polymerase with a higher error rate, such as Taq polymerase, wh.......

Discussion

This article describes how to isolate interaction-null/impaired mutants using the Y2H assay. Such mutants are powerful tools to analyze the function of a protein of interest. To isolate such mutants, rY2H assays were developed previously by modifying the Y2H host strain^2,3. However, they have not greatly reduced the amount of labor. By contrast, mutants can be isolated with this method without a significant amount of labor. In this method, modification of the Y2H.......

Materials

Name	Company	Catalog Number	Comments
0.5 M EDTA (8.0)	Nacalai Tesque Inc.	14347-21
10% SDS Solution	Fujifilm Wako Pure Chemical Corp.	313-90275
2-mercaptoethanol	Fujifilm Wako Pure Chemical Corp.	135-07522
2-propanol	Kishida Chemical Co. Ltd.	110-64785
5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal)	Fujifilm Wako Pure Chemical Corp.	021-07852
Agar	Formedium	AGR60
Ampicillin Sodium	Fujifilm Wako Pure Chemical Corp.	68-52-3
Anti-HA-tag mAb-HRP-DirecT	Medical & Biological Laboratories Co. Ltd.	M180-7
DNA from salmon testes	Merck KGaA.	D1626
Ethanol	Merck KGaA.	9-0770-4-4L-J
Filter paper for colony lift (Grade 50)	Whatman, Cytiva	1450-090
Filter paper for colony lift (No.4A)	Advantec Toyo Kaisha, Ltd.	01411090
Filter paper for replicaplating (No.1)	Advantec Toyo Kaisha, Ltd.	00011150
G-418 Sulfate	Fujifilm Wako Pure Chemical Corp.	075-05962
Hydrochloric acid	Kishida Chemical Co. Ltd.	230-37585
KCl	Fujifilm Wako Pure Chemical Corp.	163-03545
Lithium Acetate Dihydrate	Nacalai Tesque Inc.	20604-22
MgSO4•7H2O	Fujifilm Wako Pure Chemical Corp.	131-00405
Na2HPO4•12H2O	Nacalai Tesque Inc.	10039-32-4
NaCl	Nacalai Tesque Inc.	31319-45
NaH2PO4•2H2O	Nacalai Tesque Inc.	31717-25
Paper towel	AS ONE Corp.	7-6200-02
Phenol:Chloroform:Isoamyl Alcohol 25:24:1	Nacalai Tesque Inc.	25970-56
Plasmid DNAs	the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)
Plasmid isolation Kit	Nippon Genetics Co. Ltd.	FG-90502
Polyethylene Glycol #4,000	Nacalai Tesque Inc.	11574-15
SC double drop-out mix -Leu -Trp	Formedium	DSCK172
Seamless cloning kit (In-Fusion assembly )	Takara Bio Inc.	#639648
Skim milk powder	Fujifilm Wako Pure Chemical Corp.	190-12865
Streptomycin Sulfate	Fujifilm Wako Pure Chemical Corp.	3810-74-0
Taq polymerase (GoTaq Green Master Mixes)	Promega Corp.	M7122
TRIS (hydroxymethyl) aminomethane	Formedium	TRIS01
Triton X-100	Nacalai Tesque Inc.	12967-45
Tryptone	ThermoFisher scientific Inc.	211705
Tween 20	Nacalai Tesque Inc.	35624-15
Yeast Extract	ThermoFisher scientific Inc.	212750
Yeast Nitrogen Base (YNB)	Formedium	CYN0210
Zymolyase 100T	Nacalai Tesque Inc.	07665-55