**Our Mission** Understanding how proteins interact with each other is key to deciphering their function in bacteria, animal and plant cells. Basic, pharmaceutical and agro-biotech research relies on protein interaction data to understand cell physiology and pathology, identify new therapeutic targets and uncover the mechanisms of action of drugs. Since its inception in 1998, Hybrigenics has been a major player in the field of protein interactions with landmark contributions and achievements. Hybrigenics scientists were among the first to provide the community with high quality, large-scale protein interaction maps for bacteria (Rain et al. Nature 2001) and eukaryotes such as Drosophila (Formstecher et al., Genome Research 2005) and Homo sapiens (Colland et al., Genome Research 2004). These published dataset can be browsed freely thanks to PIMRider software, our dedicated protein interaction database. The company was founded on its optimized yeast two-hybrid (Y2H) technology that enables researchers to discover novel protein interactions in any cell type or tissue in a comprehensive manner. Our patented protocol is backed by unique bioinformatics capabilities for traceability, storage of experimental data and time-saving analysis and presentation of the results. With over 10,000 screens performed on more than 45 species, 2,800 academic and industrial customers worldwide and the only ISO 9001 quality certification among Y2H providers, Hybrigenics Services enjoy an unparalleled commercial and scientific recognition.
ULTImate Y1H
ULTImate Y1H (yeast one-hybrid) uses a DNA sequence as a bait. ULTImate Y1H (yeast one-hybrid) uses a DNA sequence as a bait. Derived from our ULTImate Y2H™ technology, it allows to screen to saturation highly complex libraries to find the protein partners of a DNA bait.
Applications: Identify and characterize DNA-protein interactions
Molecules Tested: DNA sequences (e.g. promoter regions, responsive elements)
How does it work?
To conduct an ULTImate Y1H assay, a DNA sequence of interest, the ‘DNA bait’, is first cloned upstream of a reporter gene to create a ‘DNA bait - reporter’ construct.
The DNA bait attached to the reporter is integrated into the genome of a dedicated yeast strain by site-specific recombination. A cell-to-cell mating is then performed between the ‘DNA bait - reporter’ yeast strain and a yeast strain pre-transformed with a high complexity domain library. This allows screening for novel protein partners of this DNA bait in an exhaustive fashion.
Key-benefits
Fast and exhaustive screening thanks to Hybrigenics patented cell-to-cell mating protocol
Detection of even weak interactions and interactions with proteins whose transcripts are rare
Full sophisticated bioinformatics analysis of the results including confidence scores
Up to 380 positive clones analyzed (5’ and 3’ sequences)
Scientific assistance for the best outcome of your project
Drug Target Deconvolution
ULTImate Yeast Chemical Hybrid (YChemH)
Applications
Our ULTImate YChemH ™ technology is a target deconvolution platform for small bioactive molecules to identify its direct protein partners. At different stage of a drug discovery project, it allows to:
Decision making
Strength
ULTImate Yeast Chemical Hybrid (YChemH) is a unique screening platform based on the Y3H technique and adapted from the most comprehensive Y2H technology developed at Hybrigenics (ULTImate Y2H ™, 20+ years as a fee-for-service):
Standard YChemH platform Work Packages
Principle
The small molecule of interest is used as a bait to screen highly complex protein domain libraries. The small molecule - protein interactions are detected thanks to the reconstitution of an active transcription factor from DNA Binding Domain (DBD) and Activation Domain (AD) moieties.
Three components are used:
When a "small molecule – prey protein" interaction takes place, the bait derivative bridges the gap between the DBD and the AD thanks to the anchor-ABD interaction. This enables the expression of the reporter gene and subsequent yeast growth on a selective medium. Positive clones are then analyzed by sequencing to identify the protein partners.
Publications
Results from a YChemH target deconvolution study have recently been published (Mueller et al, Heptalogy, 2018, doi: 10.1002/hep.30329). https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.30329 . It describes the experiments we performed to identify the protein targets and fine-tune the interacting domain.
ULTImate Y2H™ is an optimized version of the yeast two-hybrid (Y2H) screening technique to discover novel protein interactions. Unlike classical Y2H sequential transformation protocols, ULTImate Y2H™ takes advantage of our patented cell-to-cell mating process to test on average 83 million interactions per screen (2011 average).
This guarantees that our highly complex domain libraries are screened to saturation for every project. As a consequence, even rare partners or transient interactions can be detected, with a high reproducibility. This enables the accurate comparison of protein interaction maps obtained from wild-type proteins or mutants.
Conversely, our unmatched experience and unique bioinformatics capabilities allow us to flag the technical false positives and compute a confidence score for each interaction.
Applications
Molecules tested
Highly complex domain libraries screened to saturation
ULTImate Y1H
ULTImate Y1H (yeast one-hybrid) uses a DNA sequence as a bait. ULTImate Y1H (yeast one-hybrid) uses a DNA sequence as a bait. Derived from our ULTImate Y2H™ technology, it allows to screen to saturation highly complex libraries to find the protein partners of a DNA bait.
How does it work?
To conduct an ULTImate Y1H assay, a DNA sequence of interest, the ‘DNA bait’, is first cloned upstream of a reporter gene to create a ‘DNA bait - reporter’ construct.
The DNA bait attached to the reporter is integrated into the genome of a dedicated yeast strain by site-specific recombination. A cell-to-cell mating is then performed between the ‘DNA bait - reporter’ yeast strain and a yeast strain pre-transformed with a high complexity domain library. This allows screening for novel protein partners of this DNA bait in an exhaustive fashion.
Key-benefits
ULTImate RNA Y3H
ULTImate RNA Y3H (RNA yeast three-hybrid) for RNA bait screening. Derived from our ULTImate Y2H technology, it allows to screen highly complex domain-based libraries to identify protein partners of any short RNA.
How does it work?
ULTImate RNA Y3H is based on ULTImate Y2H, with modifications to use a small RNA as a bait. It consists of the detection of ‘bait-prey’ interactions thanks to the reconstitution of a full transcription factor from DNA Binding Domain and Activation Domain moieties. In the RNA Y3H approach, three hybrid molecules are used:
Upon interaction of the RNA bait with the prey, the gap between the DBD and the AD is bridged, enabling the transcription of the reporter gene in yeast cells. The DNA of the positive clones is then sequenced and analyzed to identify the protein partners.
Key-benefits
ULTImate Yeast Chemical Hybrid (YChemH)
ULTImate Yeast Chemical Hybrid (YChemH) is a compound profiling technique adapted from our ULTImate Y2H™ technology to identify the protein partners of a given bioactive small molecule.
ULTImate YChemH is a direct target identification method based on the established yeast two-hybrid (Y2H) technique. The small molecule of interest is used as a bait to screen highly complex protein domain libraries. The small molecule - protein interactions are detected thanks to the reconstitution of an active transcription factor from DNA Binding Domain (DBD) and Activation Domain (AD) moieties.
Three components are used:
When a "small molecule – prey protein" interaction takes place, the bait derivative bridges the gap between the DBD and the AD thanks to the anchor-ABD interaction. This enables the expression of the reporter gene and subsequent yeast growth on a selective medium. Positive clones are then analyzed by sequencing to identify the protein partners.
MBMATE Y2H FOR MEMBRANE PROTEINS
We can even screen your transmembrane baits!
To identify the interacting partners of an integral transmembrane (TM) protein, you can take advantage of our exclusive MBmate Y2H. This is a modified Yeast Two-Hybrid technique in which the bait and its partners are expressed at the yeast's plasma membrane.
Based on a split-ubiquitin system, MBmate Y2H is the only service allowing to screen full TM baits rather than cytoplasmic, extracellular or lumenal fragments.
Applications
Molecules Tested
Highly complex domain libraries screened to saturation
How does it work?
Your membrane protein of interest is fused to the C-terminal half of ubiquitin (Cub) linked to a transcription factor (TF), LexA-VP16. Each prey in the library is fused to the N-terminal half of ubiquitin (Nub) bearing a point mutation resulting in a low affinity for Cub. Upon interaction between the bait and a prey, Cub and Nub re-associate to form an ubiquitin molecule, which is then recognized by endogenous proteases releasing the TF. The transcription of the His3 reporter gene is activated and allows yeast cells to grow on a medium lacking histidine. The DNA of the positive clones is then sequenced and analyzed to identify the protein partners.
1-BY-1 Y2H & INTERACTION DOMAIN MAPPING
1-by-1 Y2H and Interaction Domain Mapping (IDM) are robust and versatile techniques to investigate interactions and delineate interacting domains. Get insights into your favorite protein interactions and receive publication-grade figures.
Applications
Molecules Tested
How does it work?
1-by-1 Y2H and Interaction Domain Mapping take advantage of our ULTImate Y2H™ Yeast Two-Hybrid technology.
The two proteins or protein fragments to be tested are cloned in frame with LexA or Gal4 DNA binding domain and Gal4 activation domain.
In yeast, the interaction between the two fragments leads to the reconstitution of a functional transcription factor and activates the transcription of HIS3 and LacZ reporter genes.
HIS3 allows yeast cells to grow on a medium lacking histidine, whereas LacZ offers quantitative measurement of the interaction’s strength thanks to colorimetric or luminescent ß-galactosidase substrates.
To delineate the interacting domain on each protein, multiple fragments are cloned by gap repair in yeast and tested for their interaction with the protein partner.
For integral membrane proteins the process is adapted and takes advantage of our MBmate Y2H* protocol.
Key-benefits
Deliverables
Application examples
PIMRider® Software
PIMRider® is a functional proteomics software for the exploration of Protein Interaction Maps (PIM®).
Applications
Key-benefits
A Loss of Affinity Mutants (LAM) screening yields interaction-deficient point mutants of your bait protein for a given partner. LAM takes advantage of our ULTImate Y2H™ technique. Use LAM screening to map interaction hotspots and obtain dominant negatives to speed up your functional studies!
Applications
Molecules tested
How does it work?
Mutants are generated by PCR and the experimental conditions are adapted to each target gene to get on average a single mutation per clone. The mutant library is then screened against the interacting partner by yeast two-hybrid using the LacZ reporter gene. White or light blue yeast clones in which no or a weak interaction occurs are selected. 192 clones are fully sequenced to identify their mutation. Their phenotypes are confirmed in a secondary, semi-quantitative LacZ assay. You select up to 25 point mutants which are further characterized in a quantitative colorimetric interaction assay. You then receive DNA plasmids for these 25 mutants for your own susbsequent functional studies.
Key benefits
Deliverables
Application examples
We are proud to have the largest collection of libraries ready to be screened.
With over 90 libraries from more than 35 species, Hybrigenics Services is proud to have the largest collection available for screening. From human to plants, from bacteria to drosophila, the diversity of our highly complex libraries allows us to serve you whatever your research area is.
We keep constructing new libraries to meet the needs of the research community. We value your feedback, please let us know what you want in our contact form or on Twitter with the hashtag #HGXlibraries.
Our libraries are also the most complex ones on the market and ULTImate Y2H™ cell-to-cell mating is the only technology to screen them exhaustively with an average 10-fold coverage of their complexity.
These protein fragment libraries can be used for all ULTImate screens to discover the partners of a protein, a DNA sequence, a short RNA or a small molecule of interest.
Each ULTImate library we construct is validated by stringent quality controls:
Proteomics Services
We offer a fully synthetic single-domain antibody selection & validation service called Hybribody (https://www.hybribody.com).
Please find below some general information about our synthetic nanobody capabilities & services.
Advantages of our in vitro VHH selection system include: 1) VHH’s are selected against conformationally-sensitive epitopes, 2) Antigens need not be immunogenic since VHH selection takes place in vitro (no immunizations), 3) A negative or counter-selection step may be performed prior to positive selection in order to remove unwanted VHH’s recognizing a related antigen(s), 4) VHH’s can be selected against membrane-associated antigens either by whole-cell biopanning, embedding antigens in nanodiscs or proteoliposomes.
Highlighted below are some of our Hybribody capabilities:
*Antibody selection by Phage Display on a Synthetic Humanized Single Domain Ab (hs2dAb) library coding for 3 x109 llama VHH’s
*The VHH scaffold from which the library was derived was optimized for high intracellular bacterial expression, thermostability, solubility and non-aggregating properties.
*Antigen requirements for VHH selection: 100 -150 ug of antigen (>85% purity) in a PBS buffer at a concentration of 0.5 to 1ug/ul.
*Choice of various fusion tags (i.e., fluorescent markers, enzymes, multi-Fc species) resulting in fully functional recombinant antibodies.
*Enrichment of functional intracellular antibodies or “intrabodies” for in vivo applications.
*Deliverables: Full report containing the description of selection/validation process, DNA sequences analysis of VHH clones and up to 10 validated VHH Abs in the pHEN2 bacterial expression vector.
*Number of validated VHH clones: 4-20.
*Timeline: ~ 3 months.
Our Elife paper (2016): describing the first fully synthetic phage display library of a humanized llama single-domain antibody as well as proof-of-concept VHH selection & validation projects using various types of antigens. http://www.ncbi.nlm.nih.gov/pubmed/27434673
ULTImate Y2H+Chem™ "molecular glue" system is an optimized version of the yeast two-hybrid (Y2H) screening technique to discover chemically induced protein-protein interactions. Unlike classical Y2H sequential transformation protocols, ULTImate Y2H+Chem™ takes advantage of our patented cell-to-cell mating process to test on average 83 million interactions per screen (2011 average). This guarantees that our highly complex domain libraries are screened to saturation for every project. As a consequence, even rare partners or transient interactions can be detected, with a high reproducibility.
In the Y2H+Chem system, a bait protein/small molecule pair is screened against one of our high complexity domain-enriched libraries (120+ libraries; 35+ species): https://www.hybrigenics-services.com/files/medias/libraries/old/ultimate_libraries.pdf
Molecular dependency assays are performed to determine whether preys from the Y2H+Chem screen are uniquely dependent on the presence (not the absence) of the bait/small molecule pair.
**Applications** - Discover novel chemically induced PPI's - Elucidate mechanisms of action - Ascribe functions to uncharacterized proteins - Unravel pathways and molecular machines **Molecules tested** - Full-length proteins or fragments, peptides screened in the presence of a small molecule compound - Cytoplasmic or extracellular proteins - **Highly complex domain libraries screened to saturation** - Choose from the largest collection of cDNA or genomic libraries, or request a custom library - Benefit from the most complex random-primed cDNA libraries, with 10 million primary clones in yeast - All libraries are screened to saturation by covering 10 times their complexity on average
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