Self-assembling two-dimensional protein arrays

Patent metadata

Field	Value
Publication number	US-2016369264-A1
Application number	US-201615187599-A
Country	US
Kind code	A1
Filing date	Jun 20, 2016
Priority date	Jun 19, 2015
Publication date	Dec 22, 2016
Grant date	—

Abstract

This document relates to two dimensional (2D) protein arrays can be used in biotechnology applications, as well as methods of making and using 2D protein arrays. In some cases, a 2D protein array can be used to evaluate (e.g., image) a structure (e.g., a three dimensional (3D) structure) of a protein of interest. In some cases, a 2D protein array can be used to evaluate (e.g., characterize) protein-protein interactions (e.g., stable interactions vs. transient interactions). In some cases, a 2D protein array can be used to evaluate a binding domain in a protein of interest. In some cases, a 2D protein array can be used to evaluate (e.g., identify) binding targets and/or partners of a protein of interest.

First claim

What is claimed is:

1 . A two-dimensional (2D) protein array comprising:
a plurality of oligomeric protein unit cells, wherein each oligomeric protein unit cell comprises at least one axis of rotational symmetry, and wherein each oligomeric protein unit cell comprises a plurality of self-assembling proteins; wherein said plurality of oligomeric protein unit cells interact with one another at one or more symmetrically repeated protein-protein interfaces.

2 . The 2D protein array of claim 1 , wherein said axis of rotational symmetry is cyclic or dihedral.

3 . The 2D protein array of claim 1 , wherein said one or more symmetrically repeated protein-protein interfaces comprises two, three, or four symmetrically repeated protein-protein interfaces.

4 . The 2D protein array of claim 1 , wherein said oligomeric protein unit cell is selected from the group consisting of a dimeric protein unit cell, a trimeric protein unit cell, a tetrameric protein unit cell, a pentameric protein unit cell, or a hexameric protein unit cell.

5 . The 2D protein array of claim 1 , wherein said at least one axis of rotational symmetry comprises the z axis.

6 . The 2D protein array of claim 1 , wherein said oligomeric protein unit cell comprises a surface area of greater than 400 Å2.

7 . The 2D protein array of claim 1 , wherein said oligomeric protein unit cell comprises a shape complementarity of about 0.1 Sc to about 10 Sc.

8 . The 2D protein array of claim 7 , wherein said oligomeric protein unit cell comprises a shape complementarity of about 0.5 Sc to about 1.8 Sc.

9 . The 2D protein array of claim 1 , wherein said plurality of self-assembling proteins comprises a self-assembling protein selected from the group consisting of: p3Z_11 (SEQ ID NO: 1); p3Z_42 (SEQ ID NO: 2); p4Z_9 (SEQ ID NO: 3); p6_9H (SEQ ID NO: 4); or p6_9H_KDKCKXX (SEQ ID NO: 5).

10 . The 2D protein array of claim 1 , wherein said plurality of self-assembling proteins comprises a self-assembling protein about 25 to about 500 amino acids in length.

11 . The 2D protein array of claim 10 , wherein said self-assembling protein is about 200 to about 250 amino acids in length.

12 . The 2D protein array of claim 1 , wherein at least one of said plurality of self-assembling proteins is a self-assembling fusion protein.

13 . The 2D protein array of claim 12 , wherein said self-assembling fusion protein comprises a self-assembling protein fused to a protein of interest.

14 . The 2D protein array of claim 13 , wherein said self-assembling fusion protein further comprises a linker between said self-assembling protein and said protein of interest.

15 . The 2D protein array of claim 14 , wherein said linker comprises a glycine-glycine or a glycine-serine.

16 . The 2D protein array of claim 13 , wherein said protein of interest is a protein with an unknown three dimensional (3D) structure.

17 . The 2D protein array of claim 13 , wherein said protein of interest is a protein with an unknown binding partner.

18 . The 2D protein array of claim 1 , wherein said interaction between said oligomeric protein unit cells is a non-covalent interaction.

19 . The 2D protein array of claim 1 , wherein said 2D protein array has a thickness of about 0.1 nM to about 100 nM.

20 . The 2D protein array of claim 19 , wherein said 2D protein array has a thickness of about 3 nM to about 8 nM.

21 . The 2D protein array of claim 1 , wherein said 2D protein array has a length of about 0.05 μm to about 5 μm.

22 . The 2D protein array of claim 21 , wherein said 2D protein array has a length of about 1 μm.

23 . A method of assembling a two-dimensional (2D) protein array comprising:
providing a plurality of self-assembling proteins under conditions that allow said self-assembling proteins to interact with one another to form a plurality of oligomeric protein unit cells, wherein each oligomeric protein unit cell comprises at least one axis of rotational symmetry; wherein said plurality of oligomeric protein unit cells interact with each other at one or more symmetrically repeated protein-protein interfaces to form said 2D protein array.

24 . The method of claim 23 , wherein said providing comprises expressing said plurality of self-assembling proteins from a cell-based expression system.

25 . The method of claim 24 , wherein said cell-based expression system is a bacterial expression system.

26 . The method of claim 25 , wherein said bacterial expression system is an Escherichia coli expression system.

27 . The method of claim 20 , wherein said 2D protein array is formed intracellularly.

28 . A method for determining a three dimensional (3D) structure of a protein of interest, said method comprising:
providing a plurality of self-assembling fusion proteins under conditions that allow said self-assembling fusion proteins to interact with one another to form a plurality of oligomeric protein unit cells, wherein at least one of said self-assembling fusion proteins comprises a self-assembling protein fused to the protein of interest, wherein each of said plurality of oligomeric protein unit cells comprises at least one axis of rotational symmetry; wherein said plurality of oligomeric protein unit cells interact with each other at one or more symmetrically repeated protein-protein interfaces to form a 2D protein array, wherein said 2D protein array presents the protein of interest on its surface; and determining the 3D structure of the protein of interest present on the surface of the 2D protein array.

29 . The method of claim 28 , wherein said determining comprises X-ray crystallography, NMR spectroscopy, or dual polarisation interferometry.

30 . A method for determining a binding partner of a protein of interest, said method comprising:
providing a plurality of self-assembling fusion proteins, wherein each of said self-assembling fusion proteins comprises a self-assembling protein fused to the protein of interest, under conditions that allow said self-assembling fusion proteins to interact with each other to form a plurality of oligomeric protein unit cells, wherein each oligomeric protein unit cell comprises at least one axis of rotational symmetry; wherein said plurality of oligomeric protein unit cells interact with each other at one or more symmetrically repeated protein-protein interfaces to form said 2D protein array; wherein said 2D protein array presents the protein of interest on its surface; providing at least one potential binding target; and determining if the at least one potential binding target is a binding partner of the protein of interest present on the surface of the 2D protein array.

31 . The method of claim 30 , wherein said determining comprises fluorescence resonance energy transfer (FRET).

32 . The method of claim 31 , wherein said protein of interest is labeled with a first detectable label, and wherein said at least one potential binding target is labeled with a second detectable label.

33 . The method of claim 32 , wherein said first detectable label comprises a first fluorescent label and said second detectable label comprises a second fluorescent label.

Assignees

• Hughes Howard Med Inst
• Univ Washington

Inventors

• Gonen Tamir
• Gonen Shane
• Lionnet Timothee
• Baker David
• DiMaio Frank
• English Brian
• Rouault Harve

Classifications

• G06F19/16

  Physics · mapped topic

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• G01N2610/00

  Assays involving self-assembled monolayers [SAMs] · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title