Targeted-assisted iterative screening (tais):a novel screening format for large molecular repertoires

• US 20060099713A1
• Filed: 10/01/2002
• Published: 05/11/2006
• Est. Priority Date: 10/01/2002
• Status: Abandoned Application

• Current Assignee: Buck Institute for Research on Aging
• Original Assignee: Buck Institute for Research on Aging
• Inventors: Bredesen, Dale, Kourakine, Alexei
• Application Number:
  US10/515,210
• Publication Number:
  US 20060099713A1
• Time in Patent Office: Days
• US Class Current: 436/86
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

This invention provides a new in vitro screening method for the detection of protein-protein and other interactions. The method has been developed and applied to a commercial cDNA library to search for novel protein-protein interactions. PDZ, WW and SH3 domains from PSD95, Nedd4, Src, Abl and Crk proteins were used as test targets. 12 novel putative and 2 previously reported interactions were identified for 6 protein interaction modules in test screens. The novel screening format, dubbed TAIS (target-assisted iterative screening), provides an alternative platform to existing technologies for a pair-wise characterization of protein-protein, and other, interactions.

Claims

1. A method of identifying interacting proteins from a plurality of potentially interacting proteins, said method comprising:

i) contacting one or more target proteins with a protein display library comprising a plurality of potential binding proteins for said one or more target proteins;

ii) selecting members of said protein display library that bind to said one or more target proteins to provide a preselected set of potential binding proteins;

iii) separating said members of said preselected set of potential binding proteins from the bound target protein and immobilizing said members on a solid support such that said members are spatially addressable; and

iv) contacting members of said preselected set of potential binding proteins with one or more target proteins; and

v) detecting specific binding of members of said preselected set of potential binding proteins with said one or more target proteins whereby binding of a member of said set of potential binding partners with a target protein indicates that said member and said target protein are interacting proteins.

2. The method of claim 1, wherein said one or more target proteins are attached to a solid support.

3. The method of claim 1, wherein said protein display library is a phage- or bacterial-display library.

4. The method of claim 3, wherein said phage- or bacterial-display library is a phage display library.

5. The method of claim 4, wherein said phage display library is a lytic phage library.

6. The method of claim 1, wherein said separating comprises amplifying members of said protein display library that bind to said one or more target proteins.

7. The method of claim 1, wherein said separating and/or immobilizing comprises amplifying members of said protein display library that bind to said one or more target proteins.

8. The method of claim 7, wherein said amplifying comprises amplification of said members when they are spatially separated and addressable.

9. The method of claim 3, wherein said phage- or bacterial-displayed library comprises a cDNA library.

10. The method of claim 1, wherein said protein display library comprises at least 100 different members.

11. The method of claim 10, wherein said protein display library comprises at least 1000 different members.

12. The method of claim 2, wherein said selecting comprises removing unbound members of said protein display library from said solid support.

13. The method of claim 1, wherein said selecting comprises capturing said one or more target proteins using an affinity matrix.

14. The method of claim 1, wherein contacting members of said preselected set of potential binding partners with one or more target proteins comprises adsorbing members of said preselected set of potential binding partners to a solid support.

15. The method of claim 14, wherein said solid support is a membrane.

16. The method of claim 1, wherein said detecting comprises detecting a label attached to said target protein.

17. The method of claim 16, wherein said label is selected from the group consisting of a fluorescent label, a radioactive label, an enzymatic label, a colorimetric label, and a magnetic label.

18. The method of claim 1, wherein:

said contacting of step (i) comprises contacting said one or more target proteins with a protein display library where said one or more target proteins are attached to a solid support;

said contacting of step (iv) comprises attaching members of said preselected set of potential binding proteins to a solid support to provide a set of attached preselected potential binding proteins and contacting the attached preselected potential binding proteins with the one or more target proteins.

19. The method of claim 18, where the one or more target proteins used in the contacting of step (iv) are labeled with a detectable label before the target proteins are contacted to the preselected potential binding proteins.

20. The method of claim 18, where the one or more target proteins used in the contacting of step (iv) are labeled with a detectable label simultaneous with or after the target proteins are contacted to the preselected potential binding proteins.

21. The method of claim 18, further comprising sequencing the nucleic acid encoding the displayed protein on a member of the preselected display library that binds to the target protein.

22. The method of claim 1, wherein:

said contacting of step (i) comprises contacting said one or more target proteins with a protein display library where said one or more target proteins and said protein display library are in solution.

23. The method of claim 22, wherein said selecting comprises capturing target proteins bound to members of said protein display library using an affinity matrix that specifically binds the target proteins or a tag attached to the target proteins.

24. The method of claim 23, wherein said contacting of step (iv) comprises attaching members of said preselected set of potential binding proteins to a solid support to provide a set of attached preselected potential binding proteins and contacting the attached preselected potential binding proteins with the one or more target proteins.

25. The method of claim 24, where the one or more target proteins used in the contacting of step (iv) are labeled with a detectable label before the target proteins are contacted to the preselected potential binding proteins.

26. The method of claim 24, where the one or more target proteins used in the contacting of step (iv) are labeled with a detectable label simultaneous with or after the target proteins are contacted to the preselected potential binding proteins.

27. The method of claim 1, wherein, said detecting comprises determining the amino acid sequence of a member of said set of potential binding partners that binds a target protein.

28. The method of claim 1, further comprising recording the amino acid sequence or identity of a member of said set of potential binding partners that binds a target protein in a database of proteins that interact with the target.

30. The method of claim 26, wherein said library is selected from the group consisting of a phage display library, a bacterial display library, a yeast display library, a eukaryotic virus library, a direct encoded plasmid library.

31. The method of claim 26, wherein said library is an in vitro display library selected from the group consisting of a covalent display technology (CDT) library, a polysome display library, and an RNA-peptide fusion library.

35. A kit for identifying interacting proteins from a plurality of potentially interacting proteins, said kit comprising:

a protein display library; and

instructional materials providing protocols for the method of claim 1.

36. The kit of claim 35, wherein said protein display library is a bacterial or phage display library.

37. The kit of claim 36, wherein said bacterial or phage display library comprises a cDNA library.

29. A method of identifying proteins or nucleic acids that interact with target moieties from a nucleic acid or protein library comprising a plurality of nucleic acids or proteins, said method comprising:

i) contacting one or more target moieties with said library;

ii) selecting members of said library that bind to said one or more target moieties to provide a preselected set of potential binding partners;

iii) separating said members of said preselected set of potential binding partners from the bound target and immobilizing said members on a solid support such that said members are spatially addressable;

iv) contacting members of said preselected set of potential binding partners with one or more target moieties; and

v) detecting binding of members of said set of potential binding partners with said one or more target moieties whereby binding of a member of said set of potential binding partners with a target binding moiety indicates that said member is a binding partner that interacts with the target moiety.

32. A method of identifying proteins that interact with target moieties from a plurality of potentially interacting proteins, said method comprising:

i) contacting one or more target moieties with a protein display library comprising a plurality of potential binding partners for said target moieties;

ii) selecting members of said protein display library that bind to said one or more target moieties to provide a preselected set of potential binding partners;

iii) separating said members of said preselected set of potential binding proteins from the bound target protein and immobilizing said members on a solid support such that said members are spatially addressable; and

iv) contacting members of said preselected set of potential binding partners with one or more target moieties; and

v) detecting binding of members of said set of potential binding partners with said one or more target moieties whereby binding of a member of said set of potential binding partners with a target binding moiety indicates that said member is a protein that interacts with the target moiety.

33. The method of claim 32, wherein said target moiety is selected from the group consisting of a nucleic acid, a lipid, a carbohydrate, a glycoprotein, a small organic molecule, and an inorganic molecule.

34. The method of claim 32, wherein said target moiety is a DNA or an RNA.