Polypeptide assemblies and methods for the production thereof

Patent metadata

Field	Value
Publication number	US-10501733-B2
Application number	US-201615541201-A
Country	US
Kind code	B2
Filing date	Feb 29, 2016
Priority date	Feb 27, 2015
Publication date	Dec 10, 2019
Grant date	Dec 10, 2019

Abstract

The application discloses multimeric assemblies including multiple oligomeric substructures, where each oligomeric substructure includes multiple proteins that self-interact around at least one axis of rotational symmetry, where each protein includes one or more polypeptide-polypeptide interface (“O interface”); and one or more polypeptide domain that is capable of effecting membrane scission and release of an enveloped multimeric assembly from a cell by recruiting the ESCRT machinery to the site of budding by binding to one or more proteins in the eukaryotic ESCRT complex (“L domain”); and where the multimeric assembly includes one or more subunits comprising one or more polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”), as well as membrane-enveloped versions of the multimeric assemblies.

First claim

We claim:

1. A multimeric assembly, comprising a plurality of oligomeric substructures, wherein each oligomeric substructure comprises a plurality of proteins that self-interact around at least one axis of rotational symmetry, wherein each protein comprises:
(a) one or more polypeptide-polypeptide interface (“O interface”), wherein the one or more O interfaces comprise the amino acid sequence of SEQ ID NO: 1 or 4, or wherein the one or more O interfaces comprise the amino acid sequence having at least 75% sequence identity along its full length the to the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-9, 20, and 304; and
(b) one or more polypeptide domain that is capable of effecting membrane scission and release of an enveloped multimeric assembly from a cell by recruiting the ESCRT machinery to the site of budding by binding directly or indirectly to one or more ESCRT or ESCRT-associated proteins (“L domain”);
wherein one or more protein in the multimeric assembly further comprises one or more polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”);

wherein the M domain, L domain, and O interface are not each present in a single naturally occurring protein, wherein the plurality of oligomeric substructures interact with each other at the one or more O interfaces.

2. The multimeric assembly of claim 1 , wherein at least one protein in each oligomeric structure comprises one or more M domain, or wherein each protein comprises one or more M domain.

3. The multimeric assembly of claim 1 , wherein the one or more O interfaces of different proteins in the oligomeric substructure orient the plurality of oligomeric substructures such that their symmetry axes are aligned with symmetry axes of the same kind in a designated mathematical symmetry group.

4. The multimeric assembly of claim 1 , wherein the one or more O interfaces in each protein of each oligomeric substructure are identical.

5. The multimeric assembly of claim 1 , wherein the one or more M domains are capable of non-covalently interacting with a lipid bilayer.

6. The multimeric assembly of claim 1 , wherein the one or more L domains are capable of non-covalently interacting with one or more proteins in the ESCRT pathway.

7. The multimeric assembly of claim 1 , wherein the one or more M domains are selected from the group consisting of SEQ ID NOs: 52-15 1 and 280-300.

8. The multimeric assembly of claim 1 , wherein the one or more O interfaces comprise the amino acid sequence having at least 75% sequence identity along its full length to the amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 304.

9. The multimeric assembly of claim 8 wherein the one or more O interfaces comprise the amino acid sequence having at least 85% sequence identity along its full length to the amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 304.

10. The multimeric assembly of claim 9 , wherein the one or more O interfaces comprise the amino acid sequence having at least 95% sequence identity along its full length to the amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 304.

11. The multimeric assembly of claim 10 , wherein the one or more O interfaces comprise the amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 304.

12. The multimeric assembly of claim 1 , wherein the one or more L domains comprise a linear amino acid sequence motif selected from the group consisting of SEQ ID NOs: 152-197 or 305-306, or overlapping combinations thereof.

13. The multimeric assembly of claim 1 , further comprising a packaging moiety present in one or more of the proteins.

14. The multimeric assembly of claim 13 , further comprising a cargo interacting with the packaging moiety.

15. The multimeric assembly of claim 1 , wherein each protein in the plurality of proteins comprises or consists of the amino acid sequence of SEQ ID NOs: 227-269 or 317.

16. The multimeric assembly of claim 1 , further comprising a lipid bilayer enveloping the multimeric assembly, wherein one or more of the M domains is bound to the lipid bilayer.

17. The multimeric assembly of claim 16 , further comprising one or more transmembrane protein or membrane-anchored protein embedded in the lipid bilayer.

18. The multimeric assembly of claim 1 , wherein the multimeric assembly comprises a cargo, wherein the cargo is not bound to the multimeric assembly.

19. A recombinant polypeptide comprising (a) a polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”); (b) a polypeptide-polypeptide interface (“O interface”), wherein the one or more O interfaces comprise the amino acid sequence of SEQ ID NO: 1 or 4, or wherein the one or more O interfaces comprise the amino acid sequence having at least 75% sequence identity along its full length to the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-9, 20, and 304; and (c) a polypeptide domain that is capable of effecting membrane scission and release of an enveloped multimeric assembly from a cell by recruiting the ESCRT machinery to the site of budding by binding to one or more proteins in the eukaryotic ESCRT complex (“L domain”); wherein the M domain, the L domain, and the 0 interface are not each present in a single naturally occurring protein.

20. The recombinant polypeptide of claim 19 , wherein the one or more O interfaces comprise the amino acid sequence having at least 75% sequence identity a long its full length to the amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 304.

21. The recombinant polypeptide of claim 20 , wherein the one or more O interfaces comprise the amino acid sequence having at least 85% sequence identity a long its full length to the amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 304.

22. The recombinant polypeptide of claim 21 , wherein the one or more O interfaces comprise the amino acid sequence having at least 95% sequence identity along its full length to the amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 304.

23. A recombinant nucleic acid encoding the recombinant polypeptide of claim 19 .

24. A recombinant expression vector comprising the recombinant nucleic acid of claim 23 operatively linked to a promoter.

25. A recombinant host cell comprising the recombinant expression vector of claim 24 .

Assignees

• Univ Washington
• Univ Utah
• Univ Utah Res Found

Inventors

• King Neil
• Sundquist Wesley
• Votteler Joerg
• Hsia Yang
• Baker David
• Bale Jacob
• Lajoie Marc
• Butterfield Gabriel
• Gray Elizabeth
• Stetson Daniel

Classifications

• C12Y401/02014

  2-Dehydro-3-deoxy-phosphogluconate aldolase (4.1.2.14) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C07K2319/06

  containing a lysosomal/endosomal localisation signal · CPC title