Protein Interaction (4) Yeast One-Hybrid Assay

Principle of Y1H Assay

Yeast one-hybrid (Y1H) assay is an in vitro method to analyze the intracellular interaction between DNA and proteins. The general principles of Y1H rely on the yeast two-hybrid (Y2H) assay. The main difference between Y2H and Y1H is that Y2H assay measures the interactions between proteins and proteins, while Y1H assay measures the interactions between DNA and proteins. In brief, target proteins are exogenously expressed in yeast, and in vivo interactions between target proteins and DNA baits are measured by the downstream reporter gene activation.

There are two components in Y1H system: “DNA baits” and “protein preys”. The Matchmaker Gold System is most common used in Y1H assay which employs the strong selective capacity of Aureobasidin A resistance to screening with extreme low background. In Matchmaker Gold System, the bait DNA sequence is cloned into the pAbAi vector as a single copy or multicopies (usually three copies). Then the reconstructed pBait-AbAi plasmid is transformed into Y1HGold yeast strain and efficiently integrated into the genome by homologous recombination to establish a bait-specific reporter strain. The pAbAi plasmid encodes the biosynthesis gene of wild type URA3 which enables plasmid integrant active in the parent strain, and allows the yeast to grow in the without the presence of uracil. Meanwhile, low recognition of target DNA sequence by yeast endogenous transcription factors also promotes the successful use of Y1H system. The Y1HGold strains for Aureobasidin A resistance need to be tested before the transformation of protein preys (potential DNA-binding proteins).

The prey proteins are expressed as fusion proteins with the yeast GAL4 transcription activation domain (GAL4 AD). The coding sequences of potential DNA-binding proteins are cloned into pGADT7 AD vector, and transcription of GAL4 AD fusion protein is driven by constitutively active ADH1 promoter. Then, the strains with interested promoter integrated genomes would express the prey proteins. GAL4 transcription activation domain sequence and nuclear localization sequence (NLS) in vector are arranged before the cloned potential transcription factor, and the constructed plasmid can be expressed into protein in yeast that localized into nucleus and recognize the promoter of interest.

Technical Characteristics of Y1H Assay

The Y1H system has shown great power in the field of biological research since it was established in 1993 by Wang and Reed. Application Y1H system has verified a number of known interactions between DNA and proteins and found a variety of new transcription factors. Recently, research about disease diagnosis by Y1H system has been reported. With the development of Y1H system, it will be more and more widely used in scientific research and medical treatment.

Y1H system can recognize the combination between DNA and proteins in a simple experimental procedure, meanwhile directly find the encoding sequence of prey proteins from the gene bank without the separation and purification of proteins. Due to the detected DNA binding protein is under natural conformation from Y1H system, it overcomes the disadvantage of unnatural conformation protein in traditional in vitro studies, thus has a high sensitivity. At present, a variety of Y1H assay kits and corresponding cDNA libraries have been commercialized to provide favorable conditions for the use of Y1H system.

However, there are also disadvantages of Y1H assay. False positive results due to the interactions between the insert target elements and endogenous yeast transcription activation factors, or the activation of report gene transcription does not require transcription factors. False negative results due to the combining capacity of AD fusion protein and target element are interfered. It happens when yeast expression of AD fusion protein has toxicity to cells, or fusion protein can’t be expressed steady or misfolding in host cells, or protein can’t be located in the yeast cell nucleus, as well as the fusion Gal4 AD blocks the binding site of proteins and DNA.

Reagents and Materials

• For yeast culture

Yeast strain	Yeast Peptone Dextrose complete medium
Minimal medium	10×Dropout (DO) Solution
Aureobasidin A (AbA)	SD/-Leu with/without Agar
SD/-Ura with/without Agar	SD/-Ura/-Leu with/without Agar

 

• For yeast transformation

10× LiAc stock	50% (w/v) polyethyleneglycol
10× TE stock	Restriction enzymes BstBI or BbsI
100% DMSO	PEG/LiAc solution

 

• For plasmid construction

E.coli strain	Matchmaker Insert Check PCR Mix 1
Plasmid extraction Kit	Matchmaker Insert Check PCR Mix 2
GoTaq DNA polymerase	pGADT7 AD Vector as AD vector
Phusion DNA Polymerases	pAbAi Vector as Prey vector
Restriction enzymes	PCR Purification Kit

 

Procedures

1. Clone your DNA bait sequence into pAbAi vector.

• Design and synthesize two antiparallel oligonucleotides for each DNA bait sequence with overhanging sticky ends compatible with the sticky ends of the appropriately digested pAbAi Vector.
• Anneal the two oligonucleotides.
• Using restriction enzymes to generate overhanging sticky ends compatible with your DNA bait sequence oligonucleotides to linearize 1μg of pAbAi. Purify the linear DNA by using a spin column method or on an agarose gel.
• Ligate the annealed oligonucleotides into the linearized pAbAi Vector.

2. Integrate the pBait-AbAi plasmid into the Y1HGold yeast genome to create a bait/reporter strain.

• Linearize 2μg of pBait-AbAi and p53-AbAi.
• Transform 1μg each of the linear plasmids into Y1HGold.
• Plate 100μl from each transformation reaction 1/10, 1/100, and 1/1000 dilutions on SD/-Ura agar medium.
• Pick 5 colonies after 3 days and analyze by PCR to identify correctly integrated clones. Use untransformed Y1HGold colonies as negative controls.
• Pick one colony for each confirmed bait and one for the p53-AbAi control. Spread them onto SD/-Ura agar medium for 3 days at 30°C. They are your newly constructed Y1HGold [Bait/AbAi] strains and [p53/AbAi] control strain.

3. Test the background AbA’ expression of the Y1HGold bait strain.

• Pick a large healthy colony from your bait and control strains. Resuspend each colony in 0.9% NaCl and adjust the OD600 to around 0.002.
• Plate 100μl on SD/-Ura and SD/-Ura with AbA (100/150/200ng/ml) media. Allow colonies to grow 2–3 days at 30°C.
• Use the minimal concentration of AbA, or a concentration that is slightly higher that completely suppress the growth of your bait strain for library screening.

4. Construct and screen a cDNA library by homologous recombination.

A. First-strand SMART cDNA synthesis.

• Prepare or obtain high quality total RNA.
• Combine and mix the RNA sample, CDS III (oligo-dT) and deionized H₂O of 4μl system in a sterile microcentrifuge tube. Meanwhile use a new tube for the control cDNA reaction.
• Incubate at 72°C for 2 min.
• Cool on ice for 2 min, spin briefly.
• To each reaction, add the 5×first-strand buffer, DDT, dNTP, and SMART-RT of 9μl system and mix by tapping or by gentle pipetting. Spin briefly.
• Incubate either primer at 42°C for 10 min.
• Add 1μl SMART III oligo, mix and incubate at 42°C for 1hr.
• Terminate first strand synthesis by placing the tube at 75°C for 10 min.
• Cool to room temperature, add 1μl RNaseH.
• Incubate at 37°C for 20 min.

B. Amplifying SMART cDNA by long distance PCR (LD-PCR).

C. Purifying the dsDNA with CHROMA SPIN+TE-400 columns.

• Prepare one CHROMA SPIN+TE-400 column for each cDNA sample need to be purified.
• Purge the equilibration buffer by centrifuge the columns at 700×g for 5 min. Discard collection tube and buffer. The matrix will appear semi-dry.
• Place each spin column in a second collection tube and apply a cDNA sample to each column at the center of the flat surface of the gel matrix.
• Centrifuge the columns at 700×g for 5 min.
• Mix the two purified cDNA samples into a single tube, measure the mixture volume with a micropipette, and precipitate the cDNA with ethanol.
• Resuspend the cDNA in 20μl deionized H₂O.

D. Creating and screening a one-hybrid cDNA library.

• Create and test your Y1HGold strain on SD/-Leu/AbA.
• Using SMART technology to synthesize 2-5μg of cDNA in a volume of 20μl.
• Using the Yeastmaker Transformation System and setting up library scale and small scale transformation reactions.
• From each of the transformation reactions, spread 100μl of 1/10, 1/100, 1/1000 and 1/10000 dilutions on each of the agar plates (SD/-Leu).
• Plate the remainder of the library transformation reaction on the SD/-Leu/AbA plates using 150μl per plate.
• Incubate the plates for 3-5 days.
• Calculate the number of screened clones by counting the number of colonies on the SD/-Leu 100 mm plates after 3-5 days.

5. Confirm and analysis of results.

A. Too few positives

• Optimize the transformation procedure and repeat the screening procedure.
• Check that your growth media performs as expected with the controls.
• Retest the minimal inhibitory concentration of AbA for your bait strain.
• Try increasing the repeats number of your DNA bait sequence.

B. Too many positives

• Check that your growth media performs as expected with the controls.
• Try increasing the concentration of AbA for screening.
• Pick only large healthy colonies after 3-5 days for further analysis.
• Your bait may interact with a partner which is abundant in the library.

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