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WO2006040154A2 - Systeme procaryote a deux hybrides - Google Patents

Systeme procaryote a deux hybrides Download PDF

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WO2006040154A2
WO2006040154A2 PCT/EP2005/011019 EP2005011019W WO2006040154A2 WO 2006040154 A2 WO2006040154 A2 WO 2006040154A2 EP 2005011019 W EP2005011019 W EP 2005011019W WO 2006040154 A2 WO2006040154 A2 WO 2006040154A2
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protein
plasmid
bait
prey
mobilisation
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WO2006040154A3 (fr
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Michael O'connel
Paul Clarke
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Dublin City University
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Dublin City University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the invention relates to a method of detecting protein-protein, or protein-nucleic acid, interaction which employs a prokaryotic hybrid- system of the type in which a bait plasmid vector and a prey plasmid vector are introduced into a bacterial host, each of the bait and prey plasmid vectors comprising DNA that encodes a bait and prey fusion protein, respectively, wherein interaction of the bait and prey fusion proteins produces a screenable phenotype which is indicative of protein-protein or protein-nucleic acid interaction.
  • the invention also relates bait and prey plasmid vectors, and bacterial hosts transformed with bait and prey plasmid vectors.
  • the invention also relates to a high throughput method of mapping protein-protein interaction which employs a prokaryotic hybrid system of the invention.
  • Protein-protein interactions mediate virtually every cellular process and the detection of such interactions can be a critical step in the determination of protein function.
  • the specificity of protein-protein interactions means that they also represent potential targets for novel therapeutics.
  • the availability of a continuously expanding list of fully sequenced genomes provides new opportunities for the analysis of proteins and their interconnecting links. High throughput, easy to handle, technologies are required to facilitate the exploitation of the enormous volume of data available.
  • Y2H Yeast two-hybrid
  • the Y2H system employs hybrid proteins, generally termed Bait and Prey hybrid proteins, for the detection of protein-protein interactions via reporter gene activation resulting in a readily detectable phenotype (see Figure 1) .
  • the Y2H system is highly sensitive, often detecting interactions undetected by other techniques such as immunological co-precipitation and since its initial description has been spawned the development of a whole repertoire of similar genetic based systems for the detection of a wide range of bio- molecular interactions.
  • the Y2H system itself has been widely applied not only to detect novel interacting partners for a protein of interest (often termed a Bait protein) by performing library screens, but to study interactions between defined protein pairs to identify minimal domains and critical residues required for interaction.
  • Bait plasmids with cloned inserts are transformed into a haploid Yeast strain of one mating type (Mat a) and Prey plasmids with cloned inserts are transformed into a haploid reporter Yeast strain of opposite mating type (MAT ⁇ ) .
  • Specific Bait clones are then mated with either individual Prey clones within a matrix of Prey clones for exhaustive mapping, or pooled sets of Prey clones for high- throughput library screens, and the resulting diploid Yeast cells assayed for reporter gene activation to detect protein-protein interactions (see Figure 3A) .
  • a protein X is fused to the LexA wild type DBD (LexAWT-X) and a protein Y is fused to the mutant LexA408 DBD (LexA408-Y) and the two proteins are co-expressed in the SU202 reporter strain only heterologous interactions between X an Y will generate a heterodimeric protein, with one LexAWT DBD and one LexA408 DBD, capable of binding the hybrid op408/op + operator to repress expression of the lacZ reporter gene.
  • P2H systems hold distinct advantages over Yeast based systems. These systems function in E. coli, making them very amenable to molecular biology techniques and facilitating rapid screening of complex libraries due to the higher transformation efficiency and faster growth rate of E. coli.
  • the systems permit the investigation of prokaryotic protein-protein interactions in a prokaryotic background but they can also be used for the analysis of eukaryotic proteins in a prokaryotic background, which may be advantageous in circumstances where homologous Yeast proteins interfere with an interaction resulting in either false positives or false negatives.
  • the absence of such homologous eukaryotic proteins in E. coli may result in the observation of less false positives and negatives, commonly observed in Yeast two-hybrid assays, when performing library screens.
  • Prokaryotic based systems can also permit analysis of eukaryotic regulatory proteins, such as cell cycle checkpoint proteins and signal transduction pathway proteins, which may be toxic in Yeast because they interfere with the function of yeast homologues.
  • eukaryotic regulatory proteins such as cell cycle checkpoint proteins and signal transduction pathway proteins
  • These systems thus represent an important experimental alternative to the Yeast based system but despite their many proposed advantages P2H systems still have not been widely implemented for large scale or proteomic scale protein interacting mapping in the same way as the Y2H system and thus the potential of these systems, and their susceptibility to false positives or negatives, has not yet been properly evaluated.
  • a method of detecting protein-protein, or protein- nucleic acid, interaction which employs a prokaryotic hybrid-system of the type in which a bait plasmid vector and a prey plasmid vector are introduced into a bacterial host, each of the bait and prey plasmid vectors comprising DNA that encodes a bait and prey fusion protein, respectively, wherein interaction of the bait and prey fusion proteins produces a screenable phenotype which is indicative of protein-protein or protein-nucleic acid interaction, characterised in that one of the bait or prey plasmid vectors is carried in a donor bacterium and the other of the bait or prey plasmid vectors is carried in a recipient bacterium, wherein the bait or prey plasmid vector carried by the donor bacterium comprises an origin of transfer site which is capable of facilitating the mobilisation of said bait or prey plasmid and wherein the donor bacterium includes plasmid mobilisation genes, which method comprises
  • the invention also provides a bait or prey plasmid vector comprising DNA encoding a fusion protein, wherein the bait or prey plasmid vector includes an origin of transfer site which is capable of facilitating the mobilisation of the plasmid by plasmid mobilisation genes.
  • the invention also provides a plasmid vector comprising:
  • - DNA encoding one or other part of a pair of functionally complementing polypeptides - an origin of transfer site which is capable of facilitating the mobilisation of the plasmid vector by plasmid mobilisation genes; and, optionally, - a site, typically a restriction site, which allows incorporation into the plasmid vector of DNA encoding for a protein X such that expression of the plasmid results in the expression of a fusion protein comprising protein X fused to one or the other part of the pair of functionally complementing polypeptides.
  • the invention also relates to a use of the plasmid vector of invention in constructing a library of bait, and/or prey, plasmid vectors for use in detecting protein-protein or protein-nucleic acid interaction in prokaryotic hybrid systems.
  • the invention provides a high throughput method of mapping protein-protein (or protein-nucleic acid interaction) which ideally employs a prokaryotic two-hybrid system, comprising the steps of:
  • the term "bait or prey fusion protein” should be taken to mean a chimeric protein produced by expression of a bait or prey plasmid vector, and in which the chimeric protein comprises a test protein fused to one or other part of a pair of functionally complementing polypeptides.
  • the pair may be separate parts of a protein which, when reconstituted, restore a detectable activity of the protein.
  • the pair may comprise parts of a transcription activator which, when brought into the proximity of each other, function to activate a gene under the control of the transcriptional activator.
  • the pair of functionally complementing polypeptides comprise a DNA binding domain (DBD) and a domain that provides a transcription of a gene, for example, a reporter gene such as LexA, Gal4, or His3.
  • the domain that promotes transcription is a fragment of a DNA polymerose enzyme, or an activation domain (AD) proper.
  • the pair of functionally complementing polypeptides comprise first and second fragments of a reporter protein which when reconstituted produces, or causes to be produced, a detectable signal.
  • the pair of fragments may comprise the T25 and T18 polypeptides that constitute the catalytic domain of Bordella pertussis adenylate cyclase.
  • the pair of fragments may comprise separate (poly)peptides which together form a visually detectable protein, such as green fluorescent protein (GFP) .
  • GFP green fluorescent protein
  • the pair of functionally complementing polypeptides together constitute a functional repressor of a constitutively expressed gene, such as LacZ.
  • a functional repressor of a constitutively expressed gene such as LacZ.
  • prokaryotic hybrid system should be taken to include one-hybrid systems, two-hybrid systems, reverse two-hybrid systems, and other multi-hybrid systems, useful in the detection of protein-protein, or protein-nucleic acid, interaction in a bacterial host.
  • the systems are useful for detection of interaction of both prokaryotic, and eukaryotic, proteins in a bacterial setting.
  • International Patent Application No. 96/29429 describes a one-hybrid system of detecting protein-RNA interactions which employs a first hybrid protein comprising a DNA binding protein and a first RNA-binding domain, a second hybrid protein comprising a transcriptional activation domain and a second RNA-binding domain, and a hybrid RNA.
  • nucleic acid should be understood to include DNA and RNA.
  • organ of transfer site should be taken to mean the site on the plasmid at which a DNA strand is broken for the purpose of plasmid mobilisation. Details of such sites (i.e. ori-T site) will be well known to those skilled in the art.
  • plasmid mobilisation genes refers to the genes required to achieve mobilisation of a plasmid from a donor bacterium to a recipient bacterium.
  • the plasmid mobilisation genes are the IncP-group plasmid mobilisation genes.
  • the IncP group plasmid mobilisation genes are RP4 plasmid mobilisation genes.
  • the RP4 plasmid mobilisation genes are chromosomally incorporated in the donor bacteria.
  • the donor bacteria is E. CoIi S 17-1.
  • the plasmid mobilisation genes are carried on an extra-chromosomal plasmid.
  • the donor bacterium carries a chromosomally integrated RP4 plasmid, and a bait plasmid vector modified with an origin of transfer site, and wherein the second bacteria is a strain of E. coli carrying a prey plasmid vector.
  • screenable phenotype includes activation of a reporter gene, reconstitution of a functional repressor of a constitutively expressed reporter gene, reconstitution of an enzyme, or reconstitution of a protein which is capable of producing a visual signal.
  • the constitutively expressed reporter gene is lacZ.
  • the terms “protein”, “peptide” and “polypeptide” are used interchangeably to refer to amino acid chains in which the amino acid residues are linked by covalent peptide bonds.
  • the set of proteins may comprise two subsets of proteins, wherein one set is used to generate a library of bait plasmid vectors, and the other set is used to generate a library of prey plasmid vectors.
  • One set may comprise proteins from one cell type, and the other set may comprise proteins from a second cell type.
  • the mapping method may employ either the pooled library approach or the matrix library approach, the details of which will be known to those skilled in the art.
  • Figure 1 Schematic diagram depicting the activation of transcription in a typical eukaryotic system and in the Yeast two-hybrid system.
  • A Activation of transcription by a typical eukaryotic transcriptional activator with separable DNA binding domain (DBD) and activation domain (AD) .
  • DBD DNA binding domain
  • AD activation domain
  • X represents a given protein fused to a specific DBD. In library screens this protein is termed the 'Bait'.
  • Y represents a given protein, or a pool of proteins encoded by a DNA library fused to a transcriptional AD. This fusion protein is often termed the 'Prey'. If X and Y interact, the AD is brought to the vicinity of the DNA bound DBD and transcription is activated from the adjacent promoter resulting in a clearly detectable phenotype.
  • Figure 2 The LexA Repressor Based P2H System For The Detection Of Homologous And Heterologous Protein-Protein Interactions:
  • (A) Depicts binding of the wild type LexA repressor protein to wild type lexA operator sequences (op +) in a sulA promoter thus repressing expression of an adjacent lacZ reporter gene.
  • the LexA repressor can only bind to operator sequences efficiently as a dimer and dimerization is mediated by its C- terminal dimerization domain (DD WT ) . Binding of operator sequences is mediated by its N- terminal DNA binding domain (DBD WT ) .
  • the wild type LexA dimerization domain can be replaced by a heterologous dimerization domain, such as the leucine zipper motif of the eukaryotic protein Jun, and the resulting hybrid LexA protein (DBD WT -Jun) can still bind to wild type lexA operator to repress expression of lacZ. Making fusions to the wild type LexA DBD can thus be used to study of the homo-dimerizing properties of the fused moieties.
  • C Depicts the detection of heterologous protein-protein interactions between the Fos and Jun leucine zippers using fusions to a wild type LexA DBD and a mutated LexA DBD, called LexA408 (DBD 408 ), with an altered DNA binding specificity for a mutated operator sequence (op408) .
  • Jun is capable of homodimerizing but only heterologous protein- protein interactions between Jun and Fos will result in the formation of a heterodimer capable of binding to a hybrid lexA operator (op408-op + ) to repress lacZ expression.
  • FIG. 3 Mating Based Protein-Protein Interaction Mapping
  • A Application of Yeast mating for protein- protein interaction mapping: The Bait vector carrying a cloned gene of interest is transformed into a haploid Yeast reporter strain of one mating type (MAT a) . This is then mated either with individual Prey clones, or pools of Prey clones, maintained in a haploid Yeast reporter strain of opposite mating type (MAT ⁇ ) .
  • Diploid yeast reporter strains carrying both a Bait and a Prey vector are selected for and then assayed for reporter gene activation to detect protein-protein interactions
  • B Application of bacterial mating for protein- protein interaction mapping using the LexA repressor based P2H system: This is analogous to Yeast mating strategies.
  • a mobilisable Bait vector carrying a cloned gene of interest is transformed into E. coli strain S17-1. This is then mated either with individual Prey clones, or pools of Prey clones, maintained in the E. coli reporter strain for the P2H system.
  • Transconjugant E. coli reporter strain cells carrying both a Bait and a Prey vector are selected for and then assayed for reporter gene repression to detect protein-protein interactions.
  • Fig. 4 A strategy of high-throughput protein- protein interaction mapping in E. coli using a protein two-hybrid system and bacterial conjugation.
  • Fig. 5 The construction of vectors for the LexA repressor based prokaryotic two hybrid system.
  • Plasmids pMS604 and pDP804 are designated in this work as Prey and Bait vectors respectively (see Figure 5) .
  • Plasmid pMS604 (Porte et al. , 1995) expresses a chimeric protein comprising of the wild type LexA DNA binding domain (DBD) fused to the Fos leucine zipper (LexAWT-Fos) .
  • Plasmid pDP804 (Dmitrova et al., 1998) expresses a chimeric protein consisting of a mutated LexA DBD, called LexA408, fused to the Jun leucine zipper (LexA408-Jun) .
  • LexA408-Jun Jun leucine zipper
  • the hybrid proteins on both of these ' plasmids are expressed from UV ⁇ lac promoters and thus expression can be induced using ImM IPTG.
  • Plasmid pJQ200-sk (Quandt & Hynes, 1993) carries an RP4 mob site and can thus be mobilized with high efficiency from an E. coli strain such as S17-1 described below.
  • Plasmid pJQ200-NS (see Figure 5) was derived from plasmid pJQ200-sk.
  • the NcoI-SacII- Sacl restriction sites of the multiple cloning site (MSC) of pJQ200-sk were eliminated by restricting the vector sequentially with Ncol and then Sacl, filling in the resulting 5' cohesive ends with klenow and then re-ligating the blunt ends. This was done to permit the incorporation of these restriction sites as unique sites within a newly designed MCS.
  • the pJQ200-sk plasmid also carries a sacB gene that is toxic in E. coli when expressed in the presence of 5% sucrose. E. coli strains can be therefore be easily cured of this plasmid by culturing in the absence of antibiotic selection for the plasmid followed by plating on media containing 5% sucrose.
  • the E. coli strain S17-1 (Simon et al., 1983) was used as a donor strain for bacterial matings. This strain harbors a chromosomally integrated IncP group plasmid RP4 encoding all of the transfer functions necessary for plasmid mobilization. These products can function in trans mediating the mobilization of any plasmid carrying a recognizable RP4 mobilizable element called a mob site.
  • the E. coli strain SU202 (Dmitrova et al., 1998) was the reporter strain used for the detection of heterologous protein-protein interactions.
  • the strain is both kanomycin resistant (due to the chromosomalIy inserted Tn5) and chloramphenicol resistant (due to the Tn9 inserted on the F factor) but kanamycin was preferentially used for transconjugant selection. This is because the F factor does not carry a mutation in the traD gene and thus was found to be transferable during bacterial matings complicating transconjugant selection.
  • the strain also carries a chromosomally integrated reporter gene consisting of a lacZ gene placed under the transcriptional control of a modified sulA promoter bearing a hybrid op408/op + LexA operator sequence (see Figure 1C) .
  • E. coli strains were routinely cultured on LB medium (Sambrook et al. , 1989) . Transformation was by the method of Inoue et al (Inoue et al. , 1990) and E. coli XLl-Blue was used for routine transformation and plasmid preparation. Antibiotics were used at the following concentrations: 30 ⁇ g kanamycin ml "1 , 20 ⁇ g tetracycline ml "1 and 20 ⁇ g gentamycin ml "1 50 ⁇ g streptomycin ml "1 .
  • IPTG was used at a concentration of ImM for induction of hybrid protein expression and ⁇ -galactosidase activity was assayed by plating onto LB agar containing the chromogenic substrate X-gal at a concentration of 50 ⁇ gml " x .
  • Bacterial conjugation was carried out essentially using the protocol described by O'Connell et al (O'Connell et al. , 1987) .
  • Plasmid pDL804 was linearized by digestion with EcoRI and then treated with klenow to fill in the 5' cohesive ends generated. The plasmid was then digested with BgIII. The 524bp blunt-Bglll lexA408-jun fragment was then gel purified. Plasmid pJQ200-NS was linearized by digestion with Xhol, the 5' cohesive ends were filled by klenow treatment and the vector was subsequently digested with BamHI. The blunt-BamHI pJQ200-NS plasmid was then ligated with the gel purified lexA408-jun blunt-Bglll and the resulting vector was called pPC810 (see Figure 5A) .
  • the reverse primer was designed to incorporate a multiple cloning site (MCS) , terminating in a BgIII site, C-terminally to the lexA408 sequence.
  • MCS multiple cloning site
  • the resulting 717bp PCR product was cloned into the pCR2.1 vector of Invitrogen' s original TA cloning kit and verified by sequencing.
  • the fragment was then sub-cloned as a SacII-Bglll fragment back into the original pDP804 plasmid replacing the original lexA408-jun construct with a lexA408-MCS construct and the resulting plasmid was called pPC807 (see Figure 5B) .
  • the lexA408-MCS construct was then sub- cloned from plasmid pPC807 onto pJQ200-NS. Plasmid pPC807 was linearized by digestion with EcoRI and then treated with klenow to fill in the 5' cohesive ends generated. The plasmid was then digested with Xbal. The resulting 426bp blunt-Xbal lexA408-MCS fragment was then gel purified. Plasmid pJQ200-NS was linearized by digestion with Xhol, the 5' cohesive ends were filled by klenow treatment and the vector was subsequently digested with Xbal. The blunt-Xbal pJQ200-NS plasmid was then ligated to the gel purified lexA408-MCS blunt-Xbal fragment and the resulting vector was called pPC811 (see Figure 5C) .
  • the reverse primer was designed to incorporate a multiple cloning site (MCS) , terminating in an Xhol site, C-terminally to the lexA408 sequence.
  • MCS multiple cloning site
  • the resulting 465bp PCR product was cloned into the pCR2.1 vector of invitrogens original TA cloning kit and sequenced. The fragment was then sub-cloned as a Hindlll-Xhol fragment back into the original pMS604 plasmid replacing the original lexAWT-fos construct with a lexAWT-MCS construct and the resulting plasmid was called pPC605 (see Figure 5D) .
  • the pellet was resuspended in 40 ⁇ l of LB and spotted onto 0.45 ⁇ m filter paper placed on an LB agar plate. After a 4 to 5 hour incubation at 37 0 C the filter paper was immersed in 2ml of LB broth, agitated, the resulting cell suspension diluted and plated on selective media.
  • Total recipient and total transconjugant counts were determined by plating on LB agar supplemented with kanamycin, tetracycline and LB agar supplemented with kanamycin, tetracycline and gentamycin respectively.
  • the transfer frequency (Tf) was then calculated according to the equation;
  • Tf Total Number Of Transconjugants Total Number Of Recipients
  • Recipient SU202 strains carrying either pMS604 or pPC605 were inoculated into LB broth with appropriate antibiotic selection and incubated overnight at 37°C. These overnight cultures were then distributed in 150 ⁇ l aliquots into separate wells of a 96 well plate to prepare a matrix of recipient Prey clones which was stored at 4°C. Liquid matings were carried out using a modification of the protocol described by O'Connell et al (O'Connell et al., 1987) . The Prey matrix of recipient clones was replica inoculated into a 96 well plate with 50 ⁇ l of fresh LB broth in each well.
  • the plate was then sealed using sterile pierceable adhesive aluminium foil and incubated overnight at 37°C on a microtitre plate shaker.
  • a donor S17-1 strain carrying either pPC810 or pPC ⁇ ll, was inoculated into- 5ml of LB with appropriate antibiotic selection, and incubated overnight with shaking at 37°C.
  • the overnight donor culture was then used to inoculate 30ml of fresh LB broth and incubated at 37°C until mid log phase (OD 600 0.3- 0.5) .
  • Cells were pelleted by centrifugation and then resuspended in 6ml of fresh LB broth.
  • Matings were prepared by transferring 50 ⁇ l of the concentrated donor culture to the wells of the Prey matrix of recipient clones.
  • the 96 well mating plate was incubated at 37 0 C without shaking for 4 hours and then a 2 ⁇ l aliquot from each of the wells was transferred to the corresponding well in a fresh transconjugant selection plate.
  • the transconjugant selection plate contained 150 ⁇ l of fresh LB media, with appropriate antibiotics for transconjugant selection (kanamycin, tetracycline and gentamycin for selection of SU202 harbouring both a Prey and a Bait vector) , in each well.
  • the initial transconjugant selection plate was then incubated overnight at 37 0 C with shaking.
  • the initial transconjugant selection plate was used to replica inoculate a second fresh transconjugant selection plate to further purify transconjugants by transferring a l ⁇ l aliquot from each well of the initial transconjugant selection plate to its corresponding position in the second transconjugant selection plate.
  • the media used in this round of selection also incorporated ImM IPTG to induce expression of hybrid genes from the Prey and Bait vectors. Following incubation at 37°C overnight a sample of culture from each well was spotted onto the surface of an indicator LB agar plate incorporating appropriate antibiotic selection, IPTG and X-gal to test for protein- protein interactions.
  • coli plasmids carrying a cis-acting Mob region can be transferred between E. coli strains, and into many other gram-negative bacteria, using the RP4-specific mobilization system with transfer efficiencies approaching unity when matings are carried out under optimal conditions.
  • These mobilizable plasmids have mainly been used as suicide vectors for introducing transposons and cloned genes into non-E. coli hosts for mutagenesis and expression studies.
  • two novel mobilizable Bait vectors for use with the LexA repressor based P2H system have been constructed.
  • Plasmid pJQ200-NS was derived from pJQ200-sk and therefore carries an RP4 mobilizable element, the mob site.
  • This site permits the efficient mobilization of this plasmid, and therefore the new Bait plasmids pPC810 and pPC ⁇ ll, from a donor strain of E. coli such as S17-1.
  • This strain of E. coli carries a chromosomally integrated RP4 encoding all of the required transfer functions and its use ensures that once a plasmid has been mobilized to a recipient strain it cannot be further mobilized.
  • An alternative strategy for mobilizing plasmids would be to use a helper plasmid such as pRK ⁇ OO.
  • the helper plasmid also carries all of the transfer functions required to mobilize plasmids and can be used to mobilize plasmids out of any E. coli strain.
  • the S17-1 strain was preferred as a donor because it avoided any complications arising as a result of the helper plasmid entering the recipient.
  • the Bait Plasmids pPC810 And pPC811 Can Be Mobilized With High Frequency Into SU202 Reporter Cells Already Harboring A Prey Vector
  • the pPC ⁇ ll plasmid was mated in the same manner to verify all four possible combinations of the Bait and Prey vectors.
  • Initially surface matings were prepared using fresh donor and recipient cultures grown without antibiotic selection to mid log phase (OD ⁇ oo 0.3 - 0.5). Matings were undertaken by mixing donor and recipient culture in a ratio of 4:1 based on the culture OD 60 O values. It was expected that the use of excess donor in this way would maximize the transfer frequencies ensuring that every recipient cell would receive a copy of a Bait vector.
  • Matings were prepared in duplicate with one mating of each duplicate being removed and plated on selective media after 4 hours incubation at 37 0 C while and the second mating was incubated overnight. Very high transfer efficiencies of 3.5 - 5 x 10 "1 were achieved for the 4-hour matings. From this experiment it was concluded that a 4-hour incubation was sufficient for carrying out highly efficient bacterial matings.
  • libraries of Prey clones were prepared.
  • the resulting 10 ""3 dilution therefore represents a library of SU202 Prey clones in which an interacting partner, LexAWT-Fos encoded by plasmid pMS604, occurs at a known frequency of 1:1000 among non-interacting species represented by clones harboring the pPC605 plasmid and thus only expressing the LexAWT DBD.
  • This library was screened by carrying out a surface mating with an S17-1 donor strain carrying plasmid pPC810. After a 4-hour incubation transconjugants were recovered, dilutions were prepared and plated to determine an optimal dilution factor that would give approximately 1000 transconjugants per plate and to estimate the transfer frequencies for the matings.
  • Plasmid preps were prepared for each of the clones and the identity of the plasmid was verified by restriction with Pstl that cuts within the fos gene of plasmid pMS604 and does not cut plasmid pPC605. All of the white clones were verified as having the pMS604 Prey plasmid.
  • the mobilizable Bait vectors will also permit the implementation of the LexA based P2H system for automated exhaustive protein- interaction mapping strategies analogous to those developed for Y2H systems.
  • Libraries of Prey and Bait clones can be constructed, the Prey constructs being transformed into and maintained in the E. coli SU202 reporter strain and Bait constructs being transformed into and maintained in E. coli S17-1.
  • Each of these clone libraries can be maintained in a matrix format, either in 96 well or 384 well formats, where the identity of each clone at each position in the matrices is known.
  • Each clone in the Bait library matrix can then be mated with each clone in the Prey library in a matrix and the resulting transconjugants tested for reporter gene expression to detect protein-protein interactions (see Figure 4) .
  • This exhaustive screening in a matrix fashion reveals all possible interactions between proteins in the Prey and Bait libraries.
  • To demonstrate the feasibility of this strategy we again prepared a matrix of Prey clones by distributing cultures of SU202 harbouring either pMS604 or pPC605, grown with appropriate antibiotic selection, into individual wells of a 96 well plate. This plate was retained as a stock plate.
  • the matrix was replica inoculated into a fresh 96 well plate with LB broth (using commercially available replication tools available from www.genetix.com), and incubated overnight. An aliquot of a concentrated logarithmic phase S17-l-pPC810 donor culture (grown in LB without antibiotic selection) was added to each well. After 4-hour incubation an aliquot from each well was transferred to a fresh 96 well plate with LB broth and appropriate antibiotic selection for transconjugants. A further round of transconjugant selection was then carried out in the presence of IPTG to induce hybrid protein expression.

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Abstract

La présente invention concerne un procédé permettant de détecter une interaction entre deux protéines ou entre une protéine et un acide nucléique, lequel procédé consiste à utiliser un système procaryote à deux hybrides du type de ceux dans lesquels un vecteur plasmidique appât et un vecteur plasmidique proie sont introduits dans un hôte bactérien; chacun de ces vecteurs plasmidiques comprenant un ADN codant pour une protéine hybride proie et appât, respectivement. L'interaction des protéines hybrides proie et appât produit un phénotype détectable qui indique une interaction entre deux protéines ou entre une protéine et un acide nucléique. L'un des deux vecteurs plasmidiques est transféré vers une bactérie donneuse et l'autre vecteur est transféré dans une bactérie receveuse. Le vecteur plasmidique proie ou appât transféré vers la bactérie donneuse comprend un site de transfert d'origine qui permet de faciliter la mobilisation dudit vecteur, et la bactérie donneuse comprend des gènes de mobilisation des plasmides. Le procédé décrit dans cette invention comprend les étapes qui consistent à incuber les bactéries donneuse et receveuse pendant un laps de temps suffisant pour permettre l'appariement entre elles, à sélectionner des bactéries de transconjugaison transportant à la fois le vecteur plasmidique appât et le vecteur plasmidique proie, puis à mettre en oeuvre un dosage biologique des bactéries de transconjugaison pour le phénotype détectable qui indique les interactions entre deux protéines ou entre une protéine et un acide nucléique.
PCT/EP2005/011019 2004-10-14 2005-10-13 Systeme procaryote a deux hybrides Ceased WO2006040154A2 (fr)

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WO2006040154A3 WO2006040154A3 (fr) 2006-08-17

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WO2009138519A1 (fr) 2008-05-16 2009-11-19 Ablynx Nv Séquences d'acides aminés dirigées contre cxcr4 et autres gpcr et composés renfermant ces dernières
WO2011073180A1 (fr) 2009-12-14 2011-06-23 Ablynx N.V. Anticorps à domaine variable unique dirigés contre ox4ql, produits de recombinaison et utilisation thérapeutique
WO2011083140A1 (fr) 2010-01-08 2011-07-14 Ablynx Nv Domaines variables simples d'immunoglobuline dirigés contre le cxcr4 doués d'une meilleure activité thérapeutique et produits de recombinaison les comprenant
WO2011098520A1 (fr) 2010-02-10 2011-08-18 Novartis Ag Polypeptides agonistes de liaison à dr5
WO2012062713A1 (fr) 2010-11-08 2012-05-18 Novartis Ag Polypeptides se liant aux récepteurs de chimiokines
EP2514767A1 (fr) 2006-12-19 2012-10-24 Ablynx N.V. Séquences d'acides aminés dirigées contre une métalloprotéinase de la famille ADAM et polypeptides les comprenant pour le traitement de maladies et troubles liés à ADAM
WO2012156219A1 (fr) 2011-05-05 2012-11-22 Ablynx Nv Séquences d'acides aminés dirigées contre il-17a, il-17f et/ou il17-a/f et polypeptides comprenant ces séquences
EP2557090A2 (fr) 2006-12-19 2013-02-13 Ablynx N.V. Séquences d'acides aminés dirigées contre les GPCR et polypeptides les comprenant pour le traitement de maladies et de troubles liés au GPCR
EP2650311A2 (fr) 2007-11-27 2013-10-16 Ablynx N.V. Séquences d'acides aminés dirigées contre des cytokines hétérodimériques et/ou leurs récepteurs et polypeptides les comprenant
US8568717B2 (en) 2008-04-03 2013-10-29 Vib Vzw Single domain antibodies capable of modulating BACE activity
WO2013168108A2 (fr) 2012-05-09 2013-11-14 Novartis Ag Polypeptides de liaison de récepteur de chimiokine
EP2947097A1 (fr) 2008-04-07 2015-11-25 Ablynx N.V. Séquences d'acides aminés dirigées contre les voies Notch et leurs utilisations
US9908943B2 (en) 2008-04-03 2018-03-06 Vib Vzw Single domain antibodies capable of modulating BACE activity
EP3424526A1 (fr) 2008-06-05 2019-01-09 Ablynx NV Domaines variables uniques d'immunoglobuline contre la protéine g d'enveloppe du virus de la rage et leurs utilisation pour le traitement et la prévention de la rage
EP3470425A2 (fr) 2008-12-19 2019-04-17 Ablynx N.V. Immunoglobulines contre des antigènes associés à la cellule tels que p2x7
WO2021156316A1 (fr) 2020-02-04 2021-08-12 The University Of Bath Dosage de dimérisation
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WO2024083843A1 (fr) 2022-10-18 2024-04-25 Confo Therapeutics N.V. Séquences d'acides aminés dirigées contre le récepteur de la mélanocortine 4 et polypeptides les comprenant pour le traitement de maladies et de troubles liés à mc4r
WO2025068399A1 (fr) * 2023-09-29 2025-04-03 Univerza V Ljubljani Nouveaux dosages de criblage pour la détection d'homodimérisation de protéines

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US6332897B1 (en) * 1998-03-27 2001-12-25 Glaxo Wellcome Inc. Assay methods

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EP2557090A2 (fr) 2006-12-19 2013-02-13 Ablynx N.V. Séquences d'acides aminés dirigées contre les GPCR et polypeptides les comprenant pour le traitement de maladies et de troubles liés au GPCR
EP2650311A2 (fr) 2007-11-27 2013-10-16 Ablynx N.V. Séquences d'acides aminés dirigées contre des cytokines hétérodimériques et/ou leurs récepteurs et polypeptides les comprenant
US8568717B2 (en) 2008-04-03 2013-10-29 Vib Vzw Single domain antibodies capable of modulating BACE activity
US10377834B2 (en) 2008-04-03 2019-08-13 Vib Vzw Single domain antibodies capable of modulating BACE activity
US9908943B2 (en) 2008-04-03 2018-03-06 Vib Vzw Single domain antibodies capable of modulating BACE activity
EP2947097A1 (fr) 2008-04-07 2015-11-25 Ablynx N.V. Séquences d'acides aminés dirigées contre les voies Notch et leurs utilisations
WO2009138519A1 (fr) 2008-05-16 2009-11-19 Ablynx Nv Séquences d'acides aminés dirigées contre cxcr4 et autres gpcr et composés renfermant ces dernières
EP3424526A1 (fr) 2008-06-05 2019-01-09 Ablynx NV Domaines variables uniques d'immunoglobuline contre la protéine g d'enveloppe du virus de la rage et leurs utilisation pour le traitement et la prévention de la rage
EP3470425A2 (fr) 2008-12-19 2019-04-17 Ablynx N.V. Immunoglobulines contre des antigènes associés à la cellule tels que p2x7
WO2011073180A1 (fr) 2009-12-14 2011-06-23 Ablynx N.V. Anticorps à domaine variable unique dirigés contre ox4ql, produits de recombinaison et utilisation thérapeutique
EP3309176A1 (fr) 2009-12-14 2018-04-18 Ablynx N.V. Immunoglobulin anticorps à domaine variable unique contre ox40l, constructions et utilisation thérapeutique
WO2011083140A1 (fr) 2010-01-08 2011-07-14 Ablynx Nv Domaines variables simples d'immunoglobuline dirigés contre le cxcr4 doués d'une meilleure activité thérapeutique et produits de recombinaison les comprenant
WO2011098520A1 (fr) 2010-02-10 2011-08-18 Novartis Ag Polypeptides agonistes de liaison à dr5
EP3575321A1 (fr) 2010-11-08 2019-12-04 Ablynx N.V. Polypeptides se liant aux récepteurs de cxcr2
WO2012062713A1 (fr) 2010-11-08 2012-05-18 Novartis Ag Polypeptides se liant aux récepteurs de chimiokines
EP3578568A2 (fr) 2010-11-08 2019-12-11 Ablynx N.V. Polypeptides se liant aux récepteurs de cxcr2
EP3363815A1 (fr) 2011-05-05 2018-08-22 Merck Patent GmbH Séquences d'acides aminés dirigées contre il-17a, il-17f et/ou il17-a/f et polypeptides les comprenant
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EP4105231A1 (fr) 2011-05-05 2022-12-21 Merck Patent GmbH Séquences d'acides aminés dirigées contre il-17a, il-17f et/ou il17-a/f et polypeptides les comprenant
WO2013168108A2 (fr) 2012-05-09 2013-11-14 Novartis Ag Polypeptides de liaison de récepteur de chimiokine
WO2021156316A1 (fr) 2020-02-04 2021-08-12 The University Of Bath Dosage de dimérisation
CN116024670A (zh) * 2022-09-19 2023-04-28 西南大学 一种柑橘溃疡病诱导转录因子酵母文库、构建方法及其应用
WO2024083843A1 (fr) 2022-10-18 2024-04-25 Confo Therapeutics N.V. Séquences d'acides aminés dirigées contre le récepteur de la mélanocortine 4 et polypeptides les comprenant pour le traitement de maladies et de troubles liés à mc4r
WO2025068399A1 (fr) * 2023-09-29 2025-04-03 Univerza V Ljubljani Nouveaux dosages de criblage pour la détection d'homodimérisation de protéines

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