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The yeast "two-hybrid" or "interaction trap" assay is a technique used to study protein-protein interactions, which are critical in virtually all cellular processes. The study of protein-protein interactions can be divided into three major parts:
• Identification of binding proteins
• Characterization of known interactions
• Potential to manipulate such interactions
Widely used in the production and preparation of recombinant proteins, especially eukaryotic proteins, the yeast expression system has the advantage of both prokaryotic and advanced eukaryotic systems. It is characterized by common culture conditions, fast growth, low cost, capability of post-translational processing of protein, and easy access to soluble active recombinant protein. Pichia pastoris has become the preferred yeast of choice ffor expressing recombinant proteins due to its rapid growth, abundant commercial expression vectors, and high-efficiency secretory expression.
Application of Yeast Expression System
Yeast Expression System and Method
Yeast two-hybrid technology can be used to screen LKB1 interacting proteins
1. Generate a GAL4 DNA-BD fusion by cloning the gene of interest in the frame with the GAL4 DNA binding domain of pGBKT7.
2. Transform AH109 with bait plasmid, detect bait expression, and test for autoactivation and cell toxicity.
3. Mate the transformed library strain with the bait strain.
4. Restreak colonies that can grow on TDO plates to QDO plates.
5. Restreak positive clones to single colonies on QDO/+X-a-Gal plates.
6. Rescue plasmid DNA from positive yeast clones.
7. Identify and isolate AD/library plasmids from E. coli transformants.
8. Cotransform DNA-BD/bait and AD/library plasmids into AH109.
9. Repeat Step 5.
10. Select actual positive clones and sequence cDNA inserts.
❖Highly reduced rate of false positives
❖Short turn-around time
❖Best price in the market
❖Highly customized service
Medicilon offers a wide range of ready-to-use prey protein cDNA libraries. We can also custom-develop homogenized cDNA libraries according to your specific requirements. The screening and validation process can be done in as short as two months! We also provide a co-immunoprecipitation service to validate your result from the Y2H screening.
Minimizing False Positives:
Run replicates of the experiment to reduce the likelihood of indiscriminate reporter gene activation. Including a prey-only control also provides a baseline level of reporter activation.
Vary the expression levels of bait and prey proteins – overexpression can lead to false interactions. Lowered expression levels will increase the stringency of your binding.
And most importantly: Independently validate identified binding partners with other techniques!
Minimizing False Negatives:
Interactions leading to detectable reporter signals depend on several factors, including protein expression and correct folding, post-translational modification, protein degradation, access to the nucleus in eukaryotic screens, and fusion configuration. The possibility of problems with one or more of these parameters can lead to a high number of false negatives in Y2H screens.
You are testing your reporter system with a pair of proteins known to bind serves as an excellent positive control to ensure that your setup works. The last thing you want is to run your lovely library screen only to find the system is not working because your reporter protein doesn'tdoesn't function in yeast!
Many false negatives stem from or are exacerbated by the expression of target proteins in heterologous systems.
While this may be difficult to resolve for a wide range of prey proteins, several yeast two-hybrid systems are available in model organisms, including bacteria, alternative fungi, and mammalian cells.
If the problem is that your proteins need to be post-translationally modified, co-expression of the enzyme responsible for the modification in the assay host strain can help.
To reduce the chance that the configuration of your fusion proteins physically blocks the binding sites for the protein partner or the UAS/reporter gene, the same bait and prey libraries can be screened using both N- and C-terminal fusions of these proteins. This way, both ''end'' of your protein is screened for binding.
Using a variety of expression levels and systems can alleviate false negatives that arise from low or faulty expression of your target proteins. A good overall strategy to reduce false negatives and produce a more vigorous screen is to use several different bait and prey vectors. This is as effective as using five independent protein interaction detection methods.
To bind DNA and activate a reporter gene in eukaryotic cells, fusion proteins must gain access to the nucleus. A split ubiquitin system has been devised to circumvent this requirement for transmembrane proteins.
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Tips: Above is part of the yeast two-hybrid analysis, yeast two-hybrid service, and yeast two-hybrid screen. You can also CONTACT US with any question or inquiry you may have. We will be happy to discuss your needs and design an appropriate action plan.
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