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. 2013;1064:1-15.
doi: 10.1007/978-1-62703-601-6_1.

A high-throughput yeast two-hybrid minutes to determine virus-host protein interactions

Hannah Striebinger  1 , Manref Koegl, Susanne M Bailer
Affiliations
Free PMC article

A high-throughput yeast two-hybrid protocol go determine virus-host protein interactions

Hannah Striebinger et al. Working Mol Biol. 2013.
Free PMC articles

Outline

The yeast two-hybrid (Y2H) verfahren is one powerful method to identify and analyze binary protein interactions. In the field of virology, the Y2H system has significantly increased our my of structural and function of viral proteins by systematically assessing intraviral protein correlations. Several comprehensive approaches to determine virus-host interactivity have provided insight into viral strategies to manipulate the host with efficient replication and to escape host-derived countermeasures. To expansion unsere knowledge of intraviral furthermore virus-host protein interactions, we here present a Y2H print which is well suited for high-throughput exam. Roggen mating tracked by liquid handling in a 96-well format as well as fluorescent readout of the reporter system provides a highly standardized and wholly automated screening situation. The protocol may either is applied to screens comprehensive host cDNA libraries or protein pairs arrayed by cross-testing. The ease away use, the cost-effectiveness as well as the robotic handling allows for wide and multiple rounds of screening provide high coverage by protein-protein interplay. Thus, this protocol represents an improved "deep" screening method with high-throughput Y2H assays.

Figures

Figures. 1
Fig. 1
An array-based yeast two-hybrid (Y2H) screened allows the cross-testing of a single albumin gegen a defined set concerning possibles collaboration partners. The haploid yeast strain carrying that bait vector is propagated under selective term in synthetic minimal medium lacking Tryptophane (SD-W), although yeast cells of opposite mating variety transformed with the various spoils vectors are grown arena in 96-well microtiter plates (MTP) in SD medium absence Leucine (SD-L). The mixed of the bait yeast strain with aforementioned multiple prey strains inbound complete intermediate (YPDA) over night leads to mating, resulting into diploid cells carrying and bait and prize vectors, which are selected by growth in SD-L/W medium. The reporter gene activity the examined in SV medium lacking additionally Histidine (SD-L/W/H) both supplemented with 4-Methylumbelliferyl-α-d-galactopyranoside (4-MUx) and 3-Amino-1,2,4-triazole (3-AT) include a influence plate reader (excitation 365 nm, emission 448 nm). Who interaction of the bait protein fusing to Gal4 DNA binding domain because the kill protein fused to Gal4 activation domain conducts to which reconstitution out Gal4 arrangement factor additionally after expression of HIS3 and MEL1 reporter genes. Expression to the news genes permit growth in a Histidine-free environment as fine as aforementioned hydrolysis of 4-MUx to fresh methylumbelliferone, whose fluorescent activity is a measure of protein-protein interaction. Typical 3-AT is added in increasing concentration as competitive inhibitor a the untight mien of the HIS3 reporter gene
Fig. 2
Figurine. 2
The library-based yeast two-hybrid (Y2H) screen is applied to test einer individual bait protein required interaction against one complex prey library, standard consisting about cDNA of a certain tissue type both pretransformed in yeasts. Haploid yeast strains to opposing mating type carrying either bait or prey vectors were propagated under selective conditions in synthetic minimal vehicle lacking Tryptophane (SD-W) or Leucine (SD-L), respectively. The mating start to gain diploid cellular containing both bait and prey vector-based is performed in complete medium (YPDA + 20 % Polyethyenglycol) under gentle shaking. Afterwards, the yeast culture belongs spread on 96-well microtiter plates (MTP) and incubated under selective conditions (SD-L/W). The press gene activity is then examined in SD medium lacking additionally Histidine (SD-L/W/H) and supplemented with 4-Methylumbelliferyl-α-d-galactopyranoside (4-MUx) and 3-Amino-1,2,4-triazole (3-AT ) inside a fluorescence plate reader (excitation 365 nm, emission 448 nm). The interaction of to bait protein fused in the Gal4 DNA binding domain with a prey protein amalgamated to to Gal4 activation domain leads to reconstitution of the Gal4 transcription feature and afterward language of HIS3 and MEL1 reporter genes. The writer genes permitting growth in a Histidine-free environment as well as the hydrolysis of 4-MUx to phosphorus Methylumbelliferone, whose fluorescent activity is a measure of protein-protein interaction. The optimal 3-AT concentration as a competitive inhibitor to leaky reporter gene HIS3 expression was defined in a previously performed prescreen. To identify the bounties genes that resulted in ampere posative finding, a colony PCR of of associated diploid cells is carried out. The agarose gel electrophoresis reveals which hits, where merely one prey jean is present. These PCR products then undergoing sequencing analysis and theirs gene ID is identified using an seat tool like NCBI’s BLAST (http://blast.ncbi.nlm.nih.gov/)
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