Reversed mammalian protein-protein interaction trap

a technology of protein and trap, which is applied in the field of reversed mammalian proteinprotein interaction trap, can solve problems such as inability to modify, and achieve the effects of facilitating identification and/or isolation, negative feedback, and optimizing the flexibility of proteins

Inactive Publication Date: 2004-08-26
VLAAMS INTERUNIVERSITAIR INST VOOR BIOTECHNOLOGIE VZW
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0029] FIG. 1: Principle of the reversed mammalian protein-protein interaction trap. B: bait; K: modifying enzyme activity; L: ligand; M: possible modification of the bait; P: prey. F and Y represent the amino acids that function as a mutated (F) or functional (Y) receptor activation site or inhibitor recruitment site. The example illustrates a tyrosine phosphorylation site. As is shown in the figure, the bait-prey interaction may be modification dependent. The inhibitor, such as SOCS or a functional part thereo...

Problems solved by technology

Preferably, the inhibitor may inhibit directly the modifying enzyme activity that is activating the activation site of the receptor, or it may bind to the activation site and block the modificati...

Method used

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  • Reversed mammalian protein-protein interaction trap
  • Reversed mammalian protein-protein interaction trap
  • Reversed mammalian protein-protein interaction trap

Examples

Experimental program
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Effect test

example 1

Specific Inhibition of Activation of the EpoR-LepRFFY-EpoR by the SOCS3 CISSH2 Chimera is Disrupted by Overexpression of SOCS2

[0110] The following combinations of plasmids were transfected in 4.times.10.sup.5 HEK293T cells.

[0111] a. pSV-EpoR-LepR FFY-EpoR+pMET7mcs+pXP2d2-rPAP1-luci+pUT651

[0112] b. pSV-EpoR-LepR FFY-EpoR+pMET7-fSOCS3 CISSH2+pXP2d2-rPAP1-luci+pUT-651

[0113] c. pSV-EpoR-LepR FFY-EpoR+pMET7-fSOCS3 CISSH2+pEF-FLAG-I / SOCS2+pXP2-d2-rPAP1-luci+pUT651

[0114] d. pSV-EpoR-LepR FFY-EpoR+pMET7-fCIS+pXP2d2-rPAP1-luci+pUT651

[0115] DNA amounts in a 300 .mu.l precipitation mixture were: 1 .mu.g pSV-EpoR-LepRFFY-EpoR chimeric receptor construct, 1 .mu.g of pXP2d2-rPAP1-luci reporter construct, 25 ng of pUT651 for normalization and 100 ng of the other plasmids. Additional pMET7mcs vector was added to keep DNA amounts constant in the transfections. 200 .mu.l of this precipitation mixture was added to 4.times.10.sup.5 HEK293T cells in 6-well plates. Twenty-four hours after transfection, c...

example 2

Disruption of the ALK4-FKB12 Interaction by FK506

[0117] To demonstrate the feasibility of the technique, a normal ALK4-FKB12 interaction, measured with a positive read-out receptor-based interaction trap (i.e., induction of the luciferase activity, by activation of a recombinant receptor comprising a ligand-binding domain and a cytoplasmic domain that comprises a heterologous bait polypeptide, whereby the receptor is activated by binding of a ligand to the ligand-binding domain and by binding of a prey polypeptide, fused to an activation domain to the heterologous bait peptide, as described in the European patent application 00201771.3) was inhibited by the addition of FK506.

[0118] The ALK4-FKBP12 interaction has been described by Wang et al. (1994).

[0119] To test the ALK4-FKBP12 interaction in the positive read-out receptor-based interaction trap, 400,000 HEK293T cells (6-well plate) with a combination of the following plasmids:

[0120] 1. pSEL1-ALK4 (1 .mu.g)+pMET7mcs (1 .mu.g)+pXP2...

example 3

Expression of the pMET7-fPPD1-FKBP 12 Inhibitory Prey Leads to Inhibition of Signaling via the pSELFFY-ALK4 Bait and can be Blocked by FK506

[0127] In a first experiment, it was demonstrated that the signaling activity of pSELFFY bait constructs can be inhibited by the inhibitory prey pMET7-fPPD1-FKBP12, comprising the PTP-1B phosphatase domain. 400,000 HEK293T cells (6-well plate) were transfected with a combination of following plasmids:

[0128] 1. pSELFFY-ALK4 (1 .mu.g)+pMET7mcs (1 .mu.g)+pXP2d2-rPAP1-luci (200 ng)+pUT651 (25 ng)

[0129] 2. pSELFFY-ALK4 (1 .mu.g)+pMET7-fPPD1-FKBP12 (500 ng)+pMET7mcs (500 ng)+pXP2d2-rPAP1-luci (200 ng)+pUT651 (25 ng)

[0130] 3. pSELFFY-EpoR (1 .mu.g)+pMET7mcs (1 .mu.g)+pXP2d2-rPAP1-luci (200 ng)+pUT651 (25 ng)

[0131] 4. pSELFFY-EpoR (1 .mu.g)+pMET7-fPPD1-FKBP12 (500 ng)+pMET7mcs (500 ng)+pXP2d2-rPAP1-luci (200 ng)+pUT651 (25 ng)

[0132] pMET7mcs was added as indifferent plasmid to transfect each time with an equal amount of DNA.

[0133] Two days after transfe...

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Abstract

The present invention relates to a recombinant receptor, comprising a ligand-binding domain and a signaling domain that comprises a heterologous bait polypeptide, which receptor is inactivated by binding of a prey polypeptide to the heterologous bait peptide, either in presence or absence of a ligand binding to the ligand-binding domain. The receptor is activated by addition of a compound that disrupts the bait-prey interaction. The present invention also relates to a method of screening compounds that disrupt compound-compound-binding using the recombinant receptor.

Description

[0001] This application is a continuation of PCT International Patent Application No. PCT / EP02 / 07419, filed on Jul. 2, 2002, designating the United States of America, and published, in English, as PCT International Publication No. WO 03 / 004643 A2 on Jan. 16, 2003, the contents of the entirety of which is incorporated by this reference.[0002] The present invention relates generally to biotechnology, and more particularly to a recombinant receptor comprising a ligand-binding domain and a signaling domain that comprises a heterologous bait polypeptide, which receptor is inactivated by binding of a prey polypeptide to the heterologous bait peptide, either in presence or absence of a ligand binding to the ligand-binding domain. The receptor is activated by addition of a compound that disrupts the bait-prey interaction. The present invention also relates to a method of screening compounds that disrupt compound-compound binding using the recombinant receptor.[0003] Protein-protein interact...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K14/705C12N15/10
CPCC07K14/4703C07K14/4718C12N15/1055C07K2319/00C07K14/705
Inventor EYCKERMAN, SVENTAVERNIER, JANVANDEKERCKHOVE, JOEL
Owner VLAAMS INTERUNIVERSITAIR INST VOOR BIOTECHNOLOGIE VZW
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