Resources

Protein-protein interactions are the specific physical contacts established between two or more proteins. Protein-protein interactions fundamentally underlie the function of all biological systems.

Measurements of PPIs are central to understanding the molecular basis of disease phenotypes, and for enabling biotechnology to produce novel products. These products range from effective therapeutics to next-generation foods, materials, and fuels.

It depends on if they measure binary protein interaction (only two proteins) or a protein complex (three or more proteins). Binary interactions are easily determined by genetic selection systems such as yeast two-hybrid and display technologies used in vivo. Protein complexes are most often isolated and analyzed by affinity purifications followed by mass spectrometry.

The determination of PPI motifs is important to generate loss-of-function mutations or assist structural and drug discovery studies. With our precision mapping of interaction sites, we can define the exact binding domains for two interacting proteins. Scientists can then use this information to generate and test drugs that disrupt and alter binding. We can also consider using the methodology to define novel interaction sites on target proteins that are induced by drugs.

These are complex systems of tens of thousands of protein-protein interactions in the cell. We can envision cells as complex webs of macromolecular interactions. There are three interactome network types: metabolic, protein-protein interaction, and gene regulatory networks. These are composed of physical or biochemical interactions between macromolecules. Binary interactome empirical frameworks using the yeast two-hybrid offers a way to estimate the global size of interactomes. Next Interactions is offering high-throughput screening technology for >100 Y2H screens that allow for a comprehensive overview of interactome networks (e.g. disease pathways and host-pathogen interactions).

Yes, Next Interactions has methods available that allow compound testing. To learn more contact us.

Yeast two-hybrid screening (often called Two-Hybrid system or Y2H) is a classic molecular biology technique to discover protein-protein interactions and/or protein-DNA interactions. Y2H tests for physical interactions or binding between two proteins (binary interactions).Most of the classic pathway discoveries were made by Y2H. The transcription factor of the yeast is split into two separate fragments, called the DNA-binding domain (DBD or BD) and activating domain (AD). The DBD is cloned to the known “bait” protein and the AD is cloned to the “prey” protein or a library of many prey proteins (possible interactors).  During the interaction of the two proteins (bait and pre), these domains get into physical proximity and transcription occurs. A reporter gene is also inserted right next to it, and its transcription signals that a successful interaction of two proteins (bait and prey) took place. 

The prey protein is fused to the activation domain AD of the yeast’s transcription factor. We can either test a single known protein as prey, or we can screen many known or unknown proteins, which is then a  library of different prey. A cDNA (complementary DNA) library is a collection of protein-encoding sequences that represent all the proteins expressed in a tissue or an organism. Screening libraries can also be generated from assembled ORF libraries and genomic DNA (gDNA).These protein-encoding sequences are inserted into the prey plasmid. This plasmid is then transfected into yeast cells. Each cell ultimately expresses no more than a single member from the protein library, therefore no multiple copies of the same protein will be expressed. At Next Interactions we use a modified protocol for preparing our libraries. This enables us to carry out efficient next-generation sequencing readouts followed by bioinformatics. To learn more, please contact us.

Conventional Y2H is the original simple method. As the readout, it is using  Sanger AKA first-generation sequencing or capillary sequencing. The NGS-Y2H is an innovation that relies on next-generation sequencing, which enables several levels of measurement with complex bioinformatics analysis. Visit our services page to learn more.

You need to send the extracted RNA from the tissue for the library that you would like us to prepare. We are not specialized in extraction from all the various tissue sources. You need to measure and send us the RIN scores so that we know what quality RNA we are working with.

Best is an RNA Integrity (RIN) value of 8-10. If that is not possible, for RNAs from certain sources (plant samples), a lower amount may also work, we just need higher amounts of it.

RIN stands for RNA Integrity Number, and is a metric of the quality of RNA, scaled from 0-10. It is based upon the relative size and ratio of the 18S and 28S peaks in a given RNA sample. If the RNA is more degraded, one or both of those peaks will be smaller than expected and the background noise will be higher, resulting in a lower RIN score.

With our technology, we can rapidly generate cDNA libraries from any source, and we have readily available libraries from different sources: Arabidopsis thaliana, Zea mays, Glycine max, human heart, lung, kidney, yeast genomic libraries and more. Contact us to ask about your project needs.

The complexity of the library means:  the greater the diversity of clones, the more likely the library contains sequences that will bind a given target with sufficient affinity. With a low complexity library there is low coverage of the template DNA or RNA with library fragments, and hence a significant proportion of reads share identical start sites or go even undetected in a screen. In contrast, a high complexity library contains many unique fragments and a minimum of identical fragments. With our platform, we can easily generate highly complex libraries consisting of 5-10 millions of unique cDNA or gDNA fragments that represent primary clones in the original transformation. Contact us to see what is needed in your case.

Low complexity problems for a library can arise when the starting material is low, i.e. when multiple amplification cycles are required to generate the library that has then to be transformed into yeast. When too few copies of the sample sequence were used to construct the library, resulting in random sampling bias. For NGS, input requirements are usually dictated by the nature of the sample and the amount that is available. Please contact us with your specific project parameters, so we can answer your question.

With our NGS-Y2H approach, we can detect all potential hits in a Y2H screen. So there is less or no need for normalizing the library separately.

In general, soluble proteins and protein fragments that form soluble domains are the easiest to screen. Transmembrane domains and hydrophobic domains may cause improper localization and folding in the Y2H context. A frequent problem is posed by self-activating bait proteins, which cause the improper activation of Y2H reporters in the absence of an interaction with an activation domain linked prey protein. Transcription factors as baits tend to self-activate Y2H reporters as it is their natural function to activate transcription. But self-activation is also often caused by exposed acidic patches in proteins that are not transcription factors. 

To evaluate bait proteins for problematic sequences that may affect proper expression and function in yeast we developed the Baitshop application (manuscript in preparation) which is a specialized bioinformatics tool for this purpose. 

Besides self-activation, other problems are also observed in bait fusion expression. For example, difficult sequences (repeats) and bait toxicity are the main sources of bait failure of a yeast fusion protein. Fusion proteins are not properly expressed, rapidly degraded or they cause toxicity in yeast upon high-level expression which leads to diminished yeast cell growth. Our Baitshop application has functions that allow us to determine if a protein has difficult structures such as repeat sequences that may hamper its expression. We also look for homologies to yeast proteins and to known apoptotic factors that could interfere with yeast cell growth and viability.

Self-activating baits activate the transcriptional reporters without any interaction with a prey protein. These proteins are either not screenable or need more careful adjustment.  We apply Baitshop procedures to determine self-activating sequences with sequence scanning to predict activation motifs, and evaluation of modeled protein structures to determine if such motifs are exposed on the protein surface.

We take great care using Baitshop bioinformatics to generate function bait proteins and to find good conditions to screen even for the most difficult sequences. With this approach, we minimize the occurrence of bait proteins that have significant problems in the screening procedures.

We perform the NGS for you and we will determine what is best for your project.

The conventional Y2H screening can result in up to 50-80% false positives therefore you need to spend a lot of time validating hits by other methods and rule them out one by one. With the NGS-Y2H the false positives are ruled out at the same time performing the bioinformatics analysis when we have data also from the background controls (level 3 NGS-Y2H). As a result, you need to spend less time (and money) for validation experiments.