Hybrigenics Services SAS

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:

• Identify the protein target of a compound, following a phenotypic drug discovery approach, and elucidate the mechanism of action (MOA)
• Explore and manage the polypharmacology of the molecule of interest.
• Identify the off-targets that could potentially leads to side effects
• Evaluate safety of chemicals (toxico-proteomics)
• Support drug repositioning in new therapeutic areas

Decision making

• Chemical series lead optimization
• Select the best candidate
• De-risk molecules
• Better design the preclinical and clinical studies
• Reduce and optimize animal testing
• Discover opportunities for drug repositioning

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):

• Exhaustive screening of the most complex protein domain libraries available (120+ libraries from more than 35 species available as of today). https://www.hybrigenics-services.com/library/1 . We construct new libraries upon request.
• Unbiased approach as it screens libraries for all proteome from a given tissue or cell lines of interest.
• High sensitivity (transcriptional read-out, no washing steps)
• Simplified and fast analysis of the results thanks to sophisticated bioinformatics (Interacting domain, Confidence score, ...)

Standard YChemH platform Work Packages

• WP1: Chemistry (Design and synthesis of the tagged compound(s))
• WP2: YChemH screening part (2-3 months)
  -- WP2a: Permeability in yeast and cell viability
  -- WP2b: YChemH Screen
• WP3: Specific Library construction if needed (optional)
• Validation studies (optional):
  -- WP4: In cell validation of selected targets - 1-by-1 YChemH assays
  -- WP4a: validation of experimental fragments
  -- WP4b : validation of corresponding full length proteins
  -- WP5: In cell validation of selected targets - competition with free small molecules

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:

• A hybrid protein containing a DBD fused to an Anchor Binding Protein (ABP)
• A hybrid protein containing a transcriptional AD fused to a ‘prey’ protein fragment from the library
• A bait derivative (Anchor – Linker – Small Molecule Bait).

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.

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.

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.

1-by-1 Y2H and Interaction Domain Mapping (IDM) are robust and versatile techniques to investigate interactions and delineate interacting domains.

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

• Discover novel protein partners for your favorite protein
• Elucidate mechanisms of action
• Ascribe functions to uncharacterized proteins
• Unravel pathways and molecular machines

Molecules tested

• Full-length proteins or fragments, peptides
• Cytoplasmic or extracellular proteins
• Loops and tails of membrane 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

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

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.

• Applications: Identify and characterize RNA-protein interactions
• Molecules Tested: 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:

• A hybrid protein containing a DNA Binding Domain (DBD) fused to a RNA Binding Domain (RBD)
• A hybrid protein containing a transcriptional Activation Domain (AD) fused to a ‘prey’ protein fragment from the library
• A hybrid RNA molecule consisting of two RNA fragments, the RNA anchor interacting with RBD and the RNA bait.

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

• 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

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.

• Identify hit targets and decipher drug mechanisms of action following a phenotypic screening (drug profiling, target deconvolution)
• Study the off-target effects of an active compound
• Support drug repositioning in new therapeutic areas
• Evaluate safety of chemicals (toxico-proteomics)
• Molecules Tested: Small bioactive molecules, Drugs under development or marketed

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:

• A hybrid protein containing a DBD fused to an Anchor Binding Protein (ABP)
• A hybrid protein containing a transcriptional AD fused to a ‘prey’ protein fragment from the library
• A bait derivative (Anchor – Linker – Small Molecule Bait).

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

• Discover novel integral membrane protein partners for your favorite membrane protein
• Elucidate mechanisms of action
• Ascribe functions to uncharacterized membrane proteins
• Unravel pathways and molecular machines

Molecules Tested

• Full-length membrane proteins or transmembrane fragments

Highly complex domain libraries screened to saturation

• Choose from a unique collection of membrane protein domain libraries, or request a custom one
• All libraries are screened to saturation by covering 10 times their complexity on average

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

• Test the interaction between two candidate proteins
• Compare the binding of wild-type and mutant proteins or between isoforms
• Map the exact interacting domain on a protein

Molecules Tested

• Full- length wild-type or mutant protein
• Protein fragments or peptides
• Cytoplasmic or extracellular proteins
• Loops, tails or whole membrane proteins

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

• Simple, fast and robust pairwise interaction test
• Quantification of the strength of the interactions
• Deeper characterization of your interaction and the domains involved
• Publication-grade illustrations including all positive and negative controls
• Scientific assistance to select the domains to be tested

Deliverables

• Electronic pictures of solid growth assays
• Graphs for the quantitative LacZ assays
• Detailed experimental methods

Application examples

• Mirouse et al., Development (2006)
• Vielemeyer et al., BMC Biotechnology (2010)
• Burgo et al., Developmental Cell (2012)

PIMRider® Software

PIMRider® is a functional proteomics software for the exploration of Protein Interaction Maps (PIM®).

Applications

• Explore and analyze your protein interaction data in an intuitive format
• Combine your interaction data with other biological information (e.g. functional annotations, protein interactions from the literature, genetics, expression profiles)

Key-benefits

• Straightforward analysis and navigation inside complex networks
• Immediate overview of biological connections involving your favorite proteins
• Direct access to protein annotations and publications
• Comparison of interacting and functional domains

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

• Identify critical amino acids required for your favorite interaction
• Correlate loss of interaction with loss of function in cells

Molecules tested

• Full-length proteins or fragments, peptides
• Cytoplasmic or extracellular proteins
• Loops and tails of membrane proteins

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

• Powerful point mutant screening method based on ULTImate Y2H™ yeast two-hybrid
• Deeper understanding of your interaction and the amino acids involved
• Generation of invaluable tools for functional studies in cells or in vitro
• Rapidly conducted further to an ULTImate Y2H™ screen or a 1-by-1 Y2H assay

Deliverables

• Pictures of the solid LacZ assay
• Nucleic and proteic sequences of 192 mutant clones
• Quantitative colorimetric LacZ results for 25 selected mutants, ready for publication
• Delivery of DNA plasmids of up to 25 mutants
• Detailed experimental methods

Application examples

• Emiliani et al., JBC (2005)
• Maroun et al., JBC (2006)

1-BY-1 WITH HTRF®

Use a powerful and highly sensitive interaction assay based on TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer). This is the perfect in vitro tool to test a direct interaction between two proteins.

Applications

• Test the direct interaction in vitro of two candidate proteins
• Compare the binding of wild type and mutant proteins

**Molecules tested

• Full-length proteins or fragments, peptides
• Cytoplasmic or extracellular proteins
• Loops and tails of membrane proteins

How does it work?

The two proteins or fragments tested are fused to different tags and produced in bacteria. Antibodies coupled to a fluorescence donor (Europium cryptate, EuK) or acceptor (XL665, d2), recognize the tags. If the two candidate proteins interact, energy is transferred from the donor to the acceptor upon excitation at 337 nm, and an emission at 665 nm is detected.

Because Europium cryptate has a much longer half-life than standard fluorescence donors, the specific FRET signal can be monitored several dozens of μs after excitation, once the fluorescence background has collapsed. This accounts for 1-by-1 with HTRF unprecedented sensitivity and robustness.

The assay is miniaturized in a 384-well homogeneous format, requiring only minute amounts of each candidate protein and no washing steps. As a consequence, multiple tags and TR-FRET configurations can be investigated simultaneously for each protein pair, to achieve proper recombinant protein folding and fluorescence transfer.

Interaction assays set up in this miniaturized format can subsequently be screened against libraries of small molecules to identify bioactive compounds disrupting or modulating the protein interaction.

Key benefits

• Highly sensitive homogeneous assay – no washing steps required
• Miniaturized 384-well format requiring only minute amount of proteins
• Simultaneous evaluation of multiple tags and FRET configurations for each protein pair, to achieve proper protein folding and fluorescence transfer
• Confirmation of your ULTImate Y2H interaction in an in vitro system
• Full compatibility of Y2H and HTRF* plasmids for fast and efficient subcloning

Deliverables

• SDS-PAGE gel pictures
• Raw fluorescence data and publication-grade graphs
• Detailed experimental procedures

Application example

• Lemay et al., Retrovirology (2008)

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:

• We verify a complexity of at least 10 million independent, primary fragments in yeast for our random-primed cDNA libraries. The complexity of the genomic fragment libraries constructed for organisms that contain no or few introns is defined by the size of the genome
• We sequence hundreds of clones to check the fragment size distribution and ensure that ribosomal RNA, mitochondrial DNA and empty vectors represent less than 10% of all sequences.
• We conduct validation screens with several bait proteins to assess the library quality and recapitulate published interactions.

Proteomics Services

Selection and validation of VHH nanobody from a unique synthetic library.
Link to our manuscript (eLife, 2016 Jul 19;5) describing the first fully synthetic phage display library of a humanized llama single-domain antibody as well as proof-of-concept of VHH selection & validation using various types of target Ag’s: http://www.ncbi.nlm.nih.gov/pubmed/27434673
The pure in vitro selection allows for selection on non-immunogenic antigen.
It is an open system we can easily for you modify:

• Temperature
• Buffer
• Use a protein complex as antigen
• Use an antibody/antigen complex as antigen
  Pure in vitro selection identify conformational VHH antibody with strong binding and selectivity
  See more about our Humanized synthetic single-domain selection and validation of antibodies (https://www.hybribody.com).

ULTImate Y2H™ is an optimized version of the yeast two-hybrid (Y2H) screening technique to discover novel protein interactions.

More than 400 published scientific paper with our results. More than 2000 Customer

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.

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.

1-by-1 Y2H and Interaction Domain Mapping (IDM) are robust and versatile techniques to investigate interactions and delineate interacting domains.