Claims
- 1. A method for identifying a compound capable of modulating activity of a target active domain, comprising:
(a) generating a first fusion protein, wherein the first fusion protein comprises an anchor component and a variable component; (b) generating a second fusion protein, wherein the second fusion protein comprises a docking domain and an active domain, wherein the anchor component of the first fusion protein and the docking domain of the second fusion protein are binding partners; (c) contacting the first and second fusion proteins under conditions in which the anchor component and the docking domain bind; wherein the binding of the anchor component and docking domain do not affect the activity of the target domain; and (d) determining the activity of the target domain relative to the activity of the target domain in the absence of the first fusion protein, wherein increased or decreased activity of the target domain in the presence of the first fusion protein indicates that the variable component of the first fusion protein is a modulator of the target domain.
- 2. The method of claim 1, wherein the first fusion protein is a library of fusion proteins comprising the same anchor component and different variable components.
- 3. The method of claim 1, wherein the binding partners are selected from a group consisting of (i) the Fc portion of an immunoglobulin and the Fc-binding portion of an Fc receptor; (ii) a protein domain and a antibody specific for the protein domain; (iii) a small molecule and a protein domain capable of binding the small molecule (iv) the Fc portion of an immunoglobulin and protein A or protein G; (v) a ligand and the ligand-binding domain of its cognate receptor; (vi) a pair of interacting leucine zippers; and (vii) fos and jun.
- 4. The method of claim 3, wherein the binding affinity of the binding partners is at least 1 μM.
- 5. The method of claim 3, wherein the binding partners bind to each other with an affinity at least 10 times higher than the variable component and active domain.
- 6. The method of claim 3, wherein The above method, wherein the protein domain and small molecule capable of binding the protein domain are selected from the group consisting of (i) a small molecule and a single-chain or multi-chain antibody immunospecific for the small molecule, (ii) fluorescein and an anti-fluorescein single-chain or multi-chain antibody; (iii) dinitrophenyl (DNP), or a DNP derivative and an anti- DNP single-chain or multi-chain antibody; (iv) novobiocin or a novobiocin derivative and a novobiocin-binding domain of gyrase B; (v) biotin, or a biotin derivative and avidin, streptavidin or neutravidin; (vi) FK506, or an FK506 derivative, and FKBP.
- 7. The method of claim 1, conducted in a cell.
- 8. The method of claim 1, wherein the activity of the active domain is determined by a means selected from the group consisting of signal transduction, signal transduction inhibition, a change in the level of cAMP, a calcium flux, a change in cell migration, the phosphorylation state of an indicator molecule, the rate of transcription of a reporter gene, channel dilation, ion gate opening or closure, change in extracellular or intracellular pH, translocation of a molecule within the cell, apoptosis, change in cell growth or change in metabolism.
- 9. The method of claim 1, wherein the compound identified is an activator and the activity of the target domain is increased in the presence of the first fusion protein.
- 10. The method of claim 1, wherein the active domain is selected from the group consisting of a membrane channel, a symporter transproter; an antiporter transporter; an ATPase; an enzyme; or a receptor.
- 11. The method of claim 10, wherein the receptor is a G-protein coupled receptor (GPCR).
- 12. A library of anchored fusion proteins, wherein each fusion protein comprises a constant anchor component and a variable component, wherein the anchor component is capable of binding a target molecule without modulating activity of the target molecule.
- 13. A method of identifying a constitutively activated target molecule, the method comprising:
(a) constructing a fusion protein comprising a variable or test compound fused to an active target domain; and (b) measuring the activity of the fusion protein, wherein a fusion protein exhibiting an increased activity relative to the active target domain is a constitutively activated target molecule.
- 14. A library of fusion proteins, wherein each fusion protein comprises a variable compound fused to a target domain.
- 15. A method for identifying a compound capable of modulating activity of a target active domain, comprising:
(a) generating an anchor molecule comprising an anchor component and a variable component; (b) generating a target molecule comprising a docking domain and a potentially active domain, wherein the anchor component of the anchor molecule and the docking domain of the target molecule are binding partners; (c) contacting the anchor and target molecules under conditions in which the anchor component and the docking domain bind; wherein the binding of the anchor component and docking domain do not affect the activity of the potentially active domain, (d) determining the activity of the target domain relative to the activity of the target domain in the absence of the anchor molecule, wherein increased or decreased activity of the target domain in the presence of the anchor molecule indicates that the variable component of the anchor molecule is a modulator of the target domain.
- 16. The method of claim 15, wherein the variable component is a small molecule.
- 17. A method of identifying a compound capable of binding a known protein, comprising:
(a) generating a first fusion protein comprising a test component and an active component; (b) generating a second fusion protein comprising a docking domain and an active domain, wherein the active component of the first fusion protein binds the active domain with low affinity and wherein the docking domain is a known protein or fragment thereof; (c) contacting the first and second fusion proteins; and (d) determining the activity of the active domain relative to the activity of the active domain in the absence of the first fusion protein, wherein increased or decreased activity of the active domain in the presence of the first fusion protein indicates that the test component of the first fusion protein is capable of binding the docking domain.
- 18. The method of claim 17, wherein the test and active components and/or the active and docking domains are connected by a spacer of 1-15 amino acids.
- 19. The method of claim 18, wherein the spacer is 10-15 amino acids.
- 20. An assay kit for identifying a compound capable of binding a target active domain, comprising:
(a) a first fusion protein, wherein the first fusion protein comprises an anchor component and a variable component; (b) a second fusion protein, wherein the second fusion protein comprises a docking domain and an active domain, wherein the anchor component of the first fusion protein and the docking domain of the second fusion protein are binding partners; (c) means for measuring activity of the target domain; and (d) instructions for conducting the assay.
STATEMENT OF RELATED APPLICATIONS
[0001] This application claims priority under 35 USC § 119(e) to provisional application U.S. Ser. No. 60/423,767 filed 5 Nov. 2002, which application is herein specifically incorporated by reference in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60423767 |
Nov 2002 |
US |