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Chapter 30 : Cool Tools 1: Development and Application of a Two-Hybrid System

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Cool Tools 1: Development and Application of a Two-Hybrid System, Page 1 of 2

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Abstract:

Interactions between proteins lie at the core of many crucial processes in the cell, including signal transduction, translation, transcription, DNA replication, and metabolic pathways. As a genetic approach, the yeast two-hybrid system has been highly successful in confirming and discovering protein-protein interactions. The principle of this method is based on the creation of two hybrid proteins, one fused to a DNA-binding domain (DBD; the "bait") and the other one fused to a transcription activation domain (AD; "prey"). Recently, an alternative two-hybrid system was developed, called the vesicle capture assay. This method is based upon the construction of two hybrid proteins, one protein that is fused to the vesicle targeted protein Vps32 and one protein fused to green fluorescent protein (GFP). This chapter describes the two-hybrid system based upon the classic approach of transcription factor complementation. To develop a two-hybrid system suitable for , all components of a yeast two-hybrid system needed to be included, with the requirement that each component was functional in . In conclusion, this two-hybrid system can be used to enhance one's knowledge of protein-protein interactions in . Although only one-to-one interactions were tested until now, the method should be compatible for screening experiments due to the selective step involved.

Citation: Stynen B, Van Dijck P, Tournu H. 2012. Cool Tools 1: Development and Application of a Two-Hybrid System, p 483-487. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch30

Key Concept Ranking

Simian virus 40
0.4436144
Candida albicans
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0.4436144
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Figures

Image of FIGURE 1
FIGURE 1

Overview of the two-hybrid system. (A) Bait and prey constructs. Bait and prey genes are expressed under the control of an inducible promoter. The bait fusion protein includes LexA as DNA binding domain, and the prey protein comprises a codon-optimized VP16 activation domain. Both constructs contain SV40 NLSs and epitope tags (HA tag for bait and FLAG [FL] tag for prey). (B) Interaction leading to reporter gene expression. The bait protein is located at the promoter region of reporter genes and by the binding of LexA with its target LexA operator (LexAOp) sequences. When proteins X and Y interact, the VP16 activation domain is brought in close proximity to the reporter genes, resulting in their transcription. Analysis of an interaction is performed on selective medium () or by a galactosidase assay (). doi:10.1128/9781555817176.ch30.f1

Citation: Stynen B, Van Dijck P, Tournu H. 2012. Cool Tools 1: Development and Application of a Two-Hybrid System, p 483-487. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch30
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Image of FIGURE 2
FIGURE 2

(A) The two-hybrid strain SC2H3 contains two integrated reporter constructs. Plasmid pC2H-HIS1 includes reporter gene in front of a basal promoter and five LexA operator (LexAOp) sequences for binding of the bait protein. Plasmid pC2H-LACZ contains reporter gene preceded by a basal promoter sequence and five LexAOp sequences for binding of the bait protein. The complete plasmid is flanked by restriction sites used for integration. Linearized pC2H-HIS1 integrates at the HpaI site located between genes and on chromosome 4, while pC2H-LACZ integrates at the locus on chromosome 1, at the NcoI site. ADH1b, basal promoter of ; , ampicillin resistance gene; Ori, origin of replication; t, terminator; p, promoter. (B) Bait and prey plasmids, pC2HB and pC2HP. A bait gene of interest (left panel) is cloned into the multiple cloning site downstream of the DNA-binding protein Sa and under the control of the promoter. The pC2HB plasmid is integrated between loci and on chromosome 1 after linearization at restriction site NotI, and selection is obtained with the auxotrophic marker from For the prey plasmid pC2HP (right panel), a prey gene of interest is cloned into the multiple cloning site, downstream of AD VP16. The promoter controls expression of the prey gene. An integration sequence, situated in the intergenic region between genes and on chromosome 2, makes insertion in the genome possible after linearization of the plasmid at restriction site NotI. is the auxotrophic marker for transformation. Reprinted from ( ) with permission of the publisher. doi:10.1128/9781555817176.ch30.f2

Citation: Stynen B, Van Dijck P, Tournu H. 2012. Cool Tools 1: Development and Application of a Two-Hybrid System, p 483-487. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch30
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Image of FIGURE 3
FIGURE 3

(A) Two-hybrid interaction of Kis1 with Snf4. Kis1 and Snf4 interact with each other as shown by growth of SC2H3 on SC-HIS and a high galactosidase activity. For the reporter assay, cells were incubated for up to 2 days. () Coimmunoprecipitation of Kis1 and Snf4. The interaction between bait LexA-HA-Kis1 (73 kDa) and prey VP16-FLAG-Snf4 (47 kDa) is confirmed in a coimmunoprecipitation experiment. Total protein concentrations were equal for each sample. Lane 1, loading control of Kis1; lane 2, immunoprecipitation of Kis1 with anti-HA antibodies; lane 3, coimmunoprecipitation of Kis1 with anti-FLAG antibodies; lane 4, negative control of Kis1 immunoprecipitation without antibodies; lane 5, loading control of Snf4; lane 6, coimmunoprecipitation of Snf4 with anti-HA antibodies; lane 7, immunoprecipitation of Snf4 with anti-FLAG antibodies; lane 8, negative control of Snf4 immunoprecipitation without antibodies. The antibodies used for Western blotting are indicated on the right side of each blot, and the antibodies for immunoprecipitation are shown below the blot. IP-AB, antibody used for immunoprecipitation; LC, loading control; αFL, anti-FLAG antibody; αHA, anti-HA antibody. Reprinted from ( ) with permission of the publisher. doi:10.1128/9781555817176.ch30.f3

Citation: Stynen B, Van Dijck P, Tournu H. 2012. Cool Tools 1: Development and Application of a Two-Hybrid System, p 483-487. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch30
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Download as Powerpoint

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