Please find below a summary of Hybrigenics differentiating features regarding our Y2H screening services:
1. Bait design: Investigators have an option of expressing their “bait” either as a N- or C-terminal fusion protein relative to the DNA-binding domain (DBD).
2. Bait testing: An initial test screen will be performed to evaluate whether your “bait” autoactivates a HIS3 gene reporter. The “bait” will be tested in the absence or presence of a dose-range of 3-aminotriazol (3-AT), which is used to minimize background levels. If your “bait” displays some degree of autoactivation, then the amount of 3-AT will be adjusted accordingly.
If the “bait” exhibits some degree of toxicity in yeast, then your cDNA insert will be transferred into an inducible vector.
3. Library construction: We offer high complexity domain-enriched libraries. Inserts are between 800-1000 bp, which corresponds to an average size of a protein domain. Our random-primed libraries contain several independent fragments coding for the same protein. I have included for you a link to a complete list of our cDNA libraries (90+) for Y2H screens: (http://www.hybrigenics-services.com/files/medias/hybrigenics-list-of-libraries-ultimate-screen.pdf).
The use of domains is one of the key features of our libraries. Why is this important? When expressed in yeast, full-length proteins, especially if they are large, can be misfolded, mislocalized or toxic. Construction of highly complex random-primed libraries overcomes these barriers. Random oligonucleotide priming generates overlapping fragments of a given protein domain thereby increasing the chances of proper folding. Moreover, overlapping fragments span the entire length of the protein sequence, which increases the likelihood of detecting a protein interaction. Importantly, fragments of membrane, secreted and toxic proteins are well represented in our libraries and are routinely identified in our Y2H screens. Lastly, the identification of several independent fragments in a Y2H screen allows us to easily determine a minimal interacting domain of a prey protein.
4. Y2H screening: Large-scale Y2H screens are performed using our LexA and Gal4 systems. We use a patented cell-to-cell yeast-mating assay in order to obtain and subsequently test on average 83 million interactions, which corresponds to a 8-fold in library coverage.
5. Turnaround time: Approximately 3 months
6. Bioinformatics analysis: Included in our Y2H results package is a comprehensive bioinformatics analysis of protein interactors. Hybrigenics’ bioinformatics algorithms compute a numerical rank for each protein interaction, which is then assigned a statistical confidence score. This information helps guide investigators to identify the most relevant protein binding partners as well as to exclude false positives.
7. Deliverables: Results from your ULTImate Y2H screen are reported on 3 separate files:
a) Results Summary File: Includes the technical parameters of your screen, a Predicted Biological Score (PBS), which is computed to assess the reliability of each interaction and prey fragment analysis.
b) DomSight file: Compares the bait fragment and the Selected Interaction Domain (SID) of the prey proteins with the functional and structural domains (PFAM, SMART, TMHMM, SignalP, Coil algorithms) of these proteins.
c) Excel worksheet file: Contains raw data, in particular 5’ and 3’ experimental sequences of the preys and bioinformatics analysis.
8. Expertise and scientific assistance: You will be assigned a dedicated scientific project leader, who will be in charge of your project and will be working with you throughout the course of your Y2H screen. They will help you with the design of your “bait”, provide regular updates on your Y2H screen and thoroughly review all results with you.
For more information about getting Hybrigenics to complete your Y2H screen, contact Brent Passer.