Introduction to the principle
Fields proposed yeast double hybridization in 1989, opening up a fast track for protein interaction screening. The production of this technology is based on the study of the properties of eukaryotic cell transcription factors, especially yeast transcription factor GAL4. GAL4 includes two domains, the DNA binding domain (BD) at the N-terminus and the Activation domain (AD) at the C-end. BD is able to identify upstream activating sequences (UAS) located in the GAL4-responsive gene and bind to them, and AD can initiate transcription of genes downstream of UAS. BD and AD alone do not activate transcription, but when they are spatially close enough, they present full GAL4 transcription factor activity and activate the UAS downstream promoter, transcription promoter downstream genes and allowing them to be expressed.
In a yeast double hybrid system, BD fuses with X protein, AD fuses with Y protein, and if a protein-protein complex is formed between X and Y, AD and BD are spatially close enough to reconstitute the domain and initiate transcription of the reporter gene sequence. Yeasts with bD plasmids can grow on one defective medium, and yeasts with AD plasmids can grow on another defective medium. By mating or co-transformation, the yeast has both AD and BD plasmids, and if the bait protein on the BD interacts with the protein of interest on the AD, such yeast strains can grow on the medium with three and four deficiencies and initiate the transcription of genes β-galactosidase.
Figure Yeast double heterozygotic principle
01 Why is there a false positive for yeast double hybridization? How to solve it?
Possible causes:
According to the principle of yeast double hybridization, if the BD fusion bait protein has a separate activation effect, or its activation effect can be activated by the foreign protein, thereby activating the expression of the downstream reporter gene, which will eventually lead to false positives. Therefore, strict control experiments are required when conducting experiments, and the identification of separate activation reporter genes for bait and target proteins is required to exclude false positives.
Workaround:
(1) Multiple reporter genes (HIS, ADE, MEL1) can be selected for more stringent screening, and the upstream regulatory region of each reporter gene is different, thereby greatly reducing false positives;
(2) by appropriately increasing the concentration of AbA or 3-AT;
(3) The integration of the reporter gene into the chromosome can make the gene expression level stable and eliminate the false positive caused by the fluctuation of gene expression level due to the change of plasmid copy number.
02 Positive clone obtained, PCR detected multiple fragments.
Possible cause: One yeast cell can contain multiple capture proteins.
Solution: Select the monoclonal scribing plate 2-3 times, perform blue-and-white spot screening until there is no separation, and find a positive clone for subsequent experiments.
03 What if the bait protein has self-activating activity and can activate the expression of the reporter gene alone?
(1) Some weaker self-activating effects can be inhibited by adding an appropriate amount of 3-AT, and if higher 3-AT cannot inhibit self-activation, the bait protein needs to be truncated.
(2) If the protein is a transcription factor with a transcriptional activation domain, the transcriptional activation domain needs to be removed; if it is not a transcription factor but still has strong transcriptional activation activity, it is necessary to remove the area where the bait protein has activation activity and then carry out subsequent operations, but this operation may affect the interaction between proteins.
(3) Consideration may be given to switching to Co-IP technology for identification of interoprotective proteins, which is not limited by the self-activation of bait proteins.
The main role of self-activation detection is twofold: first, the concentration of AbA or 3-AT needs to be adjusted according to the degree of self-activation of the bait protein; second, to detect whether the bait protein is toxic, if the colony grows too slowly, the expressed protein may be toxic, and a low-copy plasmid needs to be used to express the bait protein.
04 How to choose nuclear system library and membrane system library?
The membrane library is selected if the bait protein is localized on the membrane or contains a transmembrane domain, and the nuclear system library is selected if the bait protein is localized in the nucleus. Decoy proteins with transmembrane regions may also consider removing transmembrane regions or using nuclear system libraries for sieving libraries in intracellular regions, but this may affect the structure and function of the protein of interest, and there are certain risks.
Protein transmembrane structure can be predicted using the following website: http://www.cbs.dtu.dk/services/TMHMM/
05 Membrane system yeast double heterozygous screeninghow to choose bait carrier?
Membrane system yeast double heterozygous can choose different vectors according to the protein transmembrane mode of the bait gene, and after determining the protein structure of the bait gene, the appropriate vector can be selected according to the table below:
Here are a few websites for analyzing protein structure:
(1) Sites for analysis of intracellular and extracellular regions of membrane proteins:
http://www.cbs.dtu.dk/services/TMHMM/
http://www.ebi.ac.uk/Tools/pfa/phobius/
(2) Specifically analyze the signal peptide sequence of the protein, and whether there is a cleavge site at the N-terminus:
http://www.cbs.dtu.dk/services/SignalP/
06 What is the screening process for yeast double hybridization?
(1) First analyze the structure of the protein of interest, determine whether it is a membrane protein or whether there is a transmembrane structure, if there is a transmembrane structure, you need to select a suitable bait carrier according to the specific protein structure, if there is no transmembrane structure, generally use pGBKT7 bait carrier;
(2) Convert the yeast into a bait vector after constructing the gene of interest into a bait vector for self-activation detection and toxicity detection, and determine the appropriate filter conditions according to the self-activation test results;
(3) Convert the cDNA library plasmid into the strain, coat it incubate in a suitable screening plate for 4-7 d, and pick a positive cloning point plate from the screening plate to reconfirm the protein interacting with the bait protein in the four deficient plates;
(4) The positive yeast strains obtained by screening were amplified and sequenced with universal primers, and the coding sequences of the fragments were compared in the genebank to study their connection with the biological function of known genes.
07 What should I do if the hybridization efficiency is not high?
Yeast library screening can be performed using mating as well as plasmid transformation.
Mating refers to the incubation of bait bacterial solution (pGBKT7-Bait/AH109) with yeast library Y187 bacterial solution. Mating efficiency is not high, may be the number of hybrid cells is not enough, the bait strain liquid culture overnight, should be selected large, fresh clones for culture, appropriately increase the amount of bait protein liquid culture, and ensure that the yeast library working bacteria droplet titer is more than 10 ^ 8 CFU / mL, at the same time, you can also extend the hybridization time, until the presence of clover shape zygotes is observed by microscopic microscopy, and then follow-up operations.
The low efficiency of plasmid conversion can be solved by the following methods:
(1) Detect the purity of the library plasmid DNA, and if possible, consider re-purification with ethanol;
(2) DNA-BD/bait proteins are likely to be toxic;
(3) Prepare fresh nutrient bases and do control transformation;
(4) Operate strictly in accordance with the operating instructions.
08 How to choose the screening concentration of 3-AT, and is there a recommended concentration range to use?
Normally, the HIS gene will leak slightly, resulting in clones without interactions also growing weakly on trifocal plates, which can be suppressed by adding an appropriate amount of 3-AT. Ideally, a 3-AT plate with a concentration of 10 mM will have a small amount of yeast growth, and a 3-AT concentration of 20 mM will not have or very little yeast growth. If yeast can still be grown on 3-AT plates at 80 mM concentrations, the self-activating activity of the bait protein is too high to be used for double hybrid screening.
09 Does the size of the bait protein have an effect on the results of yeast double hybridization?
Although the literature has reported successful cases of yeast double hybridization from 8-750 amino acids, proteins with large molecular weights may have folding errors in yeast, so in actual work, the success rate of bait proteins with relatively large molecular weights in finding intercropping proteins in yeast will be lower than that of bait proteins of medium molecular weight.
10 Cloning on screening medium is too much.
Possible causes: The bait protein may be self-activating, which can itself activate the expression of downstream screening markers, or the filtering conditions are too loose.
Solution: A more stringent filter was used to inhibit the self-activating activity of the bait protein, and if the effect was not satisfactory, the domain of the bait protein that could cause self-activation activity could be deleted and used for yeast double hybridization for experiments.
11 Too few clones on screening medium.
Possible causes: yeast double hybridization efficiency is inefficient, the filtering is too strict, or there are too few proteins that interact with the protein of interest.
Solution: prolong the hybridization time and improve the efficiency of hybridization; relax the screening conditions; too few proteins interact with the protein of interest are caused by the structure of the bait protein itself and cannot be compensated by experiments.
12 Can the libraries of yeast double heterosis and yeast monozag be shared?
For nuclear libraries, yeast monozyths and yeast bizag libraries can be shared, their construction method is the same, but the yeast monozygritical bait gene is a nucleic acid, generally a promoter sequence, while the yeast heterozygote bait gene is a protein.
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