CytoTrap双杂交筛选系统

Lei Zhang; Liqun Du; B. W. Poovaiah

doi:10.21769/BioProtoc.1310

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Peer-reviewed

CytoTrap Two-Hybrid Screening Assay

CytoTrap双杂交筛选系统

LZ Lei Zhang

LD Liqun Du

B. W. Poovaiah email

发布: 2014年12月05日第4卷第23期 DOI: 10.21769/BioProtoc.1310 浏览次数: 12846

评审: Tie LiuSaminathan Thangasamy

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The authors used this protocol in:

Cover of The Plant Journal, featuring study using the protocol.

Apr 2014

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Abstract

CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein (Li et al., 1993), and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. cerevisiae) strain cdc25H at 36 °C. In the CytoTrap two-hybrid system, detection of protein interaction occurs in the cytoplasm near cell membrane and is not dependent on transcription activation of reporter genes. Hence, the system is particularly useful for identifying interaction partners of transcription factors and proteins that need post-translational modification in the cytoplasm, which could not be used as bait proteins in conventional transactivation-based yeast two-hybrid systems. Here we describe the construction of a cDNA library from the model plant Arabidopsis and a procedure for screening interaction proteins of AtSR1/CAMTA3, a Ca²⁺/CaM-regulated transcription factor from this library. This procedure could be adapted to identify interacting partners of interested proteins from other organisms.

Keywords: Cytotrap system (CytoTrap系统)

Yeast two-hybrid screen (酵母双杂交筛选)

Protein interaction (蛋白质相互作用)

Materials and Reagents

XL10-Gold Kanr Ultracompetent cells (Agilent, catalog number: 200317 )
RNeasy plant mini kit (QIAGEN, catalog number: 74904 )
Oligotex mRNA mini kit (QIAGEN, catalog number: 70022 )
CytoTrap two-hybrid system (Agilent, catalog number: 200444 )
Yeastmaker^TM yeast transformation system 2 (Takara Bio Company, Clontech, catalog number: 630439 )
AccuScript (Agilent, catalog number: 200820 )
RNase H (New England Biolabs, catalog number: M0297S )
DNA polymerase I (New England Biolabs, catalog number: M0209S )
UltraPure^TM phenol: chloroform: isoamyl alcohol (25:24:1, v/v) (Life Technologies, catalog number: 15593-031 )
T4 ligase (New England Biolabs, catalog number: M0202S )
T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
Xho I (New England Biolabs, catalog number: R0146M )
Acid-washed glass beads (Sigma-Aldrich, catalog number: G8772 )
Triton^TM X-100 (Thermo Fisher Scientific, catalog number: BP151-100 )
Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: BP166-100 )
Sodium chloride (NaCl) (J.T.Baker^®, catalog nuber: JT3628-1 )
Tris (J.T.Baker^®, catalog number: JT4109-1 )
EDTA (J.T.Baker^®, catalog number: JT8993-1 )
Yeast extract (Thermo Fisher Scientific, catalog number: BP9727-500 )
Peptone (Thermo Fisher Scientific, catalog number: BP9725-500 )
Dextrose (J.T.Baker^®, catalog number: JT1919-1 )
Adenine sulfate (Sigma-Aldrich, catalog number: A2545 )
Yeast nitrogen base without amino acids (BD, catalog number: 291940 )
DO supplement -His/-Leu/-Trp/-Ura (Takara Bio Company, Clontech, catalog number: 630425 )
Histidine (Sigma-Aldrich, catalog number: H6034 )
Tryptophan (Sigma-Aldrich, catalog number: T0254 )
Galactose (Sigma-Aldrich, catalog number: G0750 )
Raffinose (Sigma-Aldrich, catalog number: R0250 )
Yeast lysis solution (see Recipes)
YPDA/YPAD (see Recipes)
SC/-LU (glucose) (see Recipes)
SC/-LU (galactose) (see Recipes)
10x STE buffer (see Recipes)