Self-assembling tetrahedral protein cages

Abstract

Disclosed herein are self-assembling tetrahedral protein cages.

Description

SELF-ASSEMBLING TETRAHEDRAL PROTEIN CAGES

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application No.63 / 514,276, filed July 18, 2023, which is herein incorporated by reference in its entirety.

[0003] REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA PATENT CENTER

[0004] The content of the XML text file of the sequence listing named “20240630_034044_252WO1_seq_ST26” which is 45,732 bytes in size was created on June 30, 2024 and electronically submitted via PatentCenter herewith the application is incorporated herein by reference in its entirety.

[0005] ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with Government support under GM129854 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] 1. FIELD OF THE INVENTION

[0009] The field generally relates to self-assembling protein cages.

[0010] 2. DESCRIPTION OF THE RELATED ART

[0011] Self-assembling protein cages are a class of large supramolecular protein assemblies. See, for example, US8969521, US9066870, US9630994, US10248758, US10501733, US20200397886, US20210163540, and WO2020220044. Typically, protein cages are formed with (a) a plurality of subunits having the same amino acid sequence, or (b) a plurality of 2−3 different subunits (i.e., 2−3 different amino acid sequences). That is, protein cages typically comprise multiple copies (e.g., 12, 24, or 60 copies) of subunit proteins that self-assemble into a symmetric geometric shapes that mimic the 3D shape of any one of the Platonic solids: tetrahedron, cube, octahedron, icosahedron, and dodecahedron and the subunits are held together via non-covalent interactions that do not involve the sharing of electrons (e.g., electrostatic interactions, Van der Waals forces, π-effects, hydrophobic effects, etc.). Protein cages have a variety of uses as, e.g., therapeutics, diagnostics, and drug delivery agents.

[0012] SUMMARY OF THE INVENTION

[0013] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of asequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO: 39. In some embodiments, the first polypeptide comprises SEQ ID NO: 39. In some embodiments, the first polypeptide comprises SEQ ID NO: 39, wherein In some embodiments, X1 is A, E, or D; X2 is G or L; X3 is V or I; X4 is E or L; X5 is L or R; X6 is A or R; X7 is R or K; X8 is V or S; X9 is E, R, or L; X10 is K or M; X11 is C or N; X12 is E, K, or R; X13 is P or I; X14 is E or C; X15 is A or H; X16 is I or L; X17 is I, L, or M; X18 is R or T; X19 is E, R, or Q; X20 is P or V; X21 is E or A; X22 is A, D, or E; X23 is A or E; X24 is R, K, or E; X25 is M or Y; X26 is A, K, or R; X27 is E or T; X28 is T, V, or E; X29 is K, N, or G; X30 is A, K, or E; X31 is N or Q; X32 is I or L; X33 is D or A; X34 is H or S; X35 is A or V; X36 is G or P; X37 is R, A, or Y; X38 is I or A; X39 is P or E; X40 is A or Q; X41 is L or W; X42 is P or R; X43 is L or D; X44 is E or A; X45 is E or P; X46 is T or V; X47 is A, P, or V; X48 is P or D; X49 is P or A; X50 is I or L; X51 is D or E; X52 is E, R, or L; X53 is V or L; X54 is L or A; X55 is V or F; X56 is T or V; X57 is V or P; X58 is A or C; X59 is E or R; X60 is D, R, or K; X61 is V or A; X62 is R, E, or V; X63 is L, V, or R; X64 is R or D; X65 is S or A; X66 is E or I; X67 is H or G; X68 is G or N; X69 is Y, V, or R; X70 is V, A, or T; X71 is A, V, or E; X72 is D or K; X73 is E, P, or L; X74 is E, A, or V; X75 is A or D; X76 is R or L; X77 is K, R, or E; X78 is R or A; X79 is A or V; X80 is R, A, or I; X81 is A or E; X82 is E or D; X83 is K, Q, or D; X84 is I or L; X85 is D or S; X86 is L or I; X87 is R, A, or S; X88 is E, P, or D; X89 is A or E; X90 is R or L; and X91 is L, E, or W.

[0014] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. In some embodiments, the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO: 40. In some embodiments, the first polypeptide comprises SEQ ID NO: 40. In some embodiments, the first polypeptide comprises SEQ ID NO: 40, wherein X1 is L or M; X2 is E, R, or N; X3 is K or D; X4 is M or F; X5 is K, R, or E; X6 is Q, N, L, or K; X7 is I, A, or E; X8 is E, M, or A; X9 is F or Y; X10 is A or R; X11 is N, Q, E, or R; X12 is E, T, or N; X13 is T, I, or L; X14 is I or V; X15 is A or C; X16 is Y or F; X17 is T, Q, or S; X18 is K or L; X19 is N or K; X20 is E, N, or K; X21 is L, Y, or F; X22 is L or K; X23 is N, E, L, or K; X24 is E, R, or D; X25 is E, D, or K; X26 is E or K; X27 is Q, K, or E; X28 is E or M; X29 is E, N, K, or L; X30 is L, Y, or F; X31 is L or K; X32 is D or N; X33 is D, E, or K; X34 is N, S, or E; X35 is E or L; X36 is A or Y; X37 is F or Y; X38 is T, Q, or K; X39 is K or L; X40 is E or K; X41 is K, A, V, or E; X42 is T, E, or L; X43 is K or H; X44 is A, Q, or L; X45 is G, N, or A; X46 is K, A, or H; X47 is L, N, or K; X48 is E, Q, or K; X49 is K or L; and X50 is A or E.

[0015] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 17; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16 or SEQ ID NO: 18. In some embodiments, the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO: 41. In some embodiments, the first polypeptide comprises SEQ ID NO: 41. In some embodiments, the first polypeptide comprises SEQ ID NO: 41, wherein X1 is E or D; X2 is F or Q; X3 is K or R; X4 is E or L; X5 is N or D; X6 is V or T; X7 is I or V; X8 is A or C; X9 is A or E; X10 is L or I; X11 is K or W; X12 is I or V; X13 is A or K; X14 is L or R; X15 is A or R; X16 is A or R; X17 is E or R; X18 is S or A; X19 is T or S; X20 is Q or R; X21 is A or R; X22 is A or R; X23 is K or R; X24 is K or R; X25 is K or E; X26 is V or A; X27 is L or E; X28 isE or R; X29 is A or R; X30 is A or L; X31 is E or R; X32 is A or Y; X33 is A or E; X34 is P or E; X35 is V or L; and X36 is K or E.

[0016] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20; and the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 19. In some embodiments, the second polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO: 42. In some embodiments, the second polypeptide comprises SEQ ID NO: 42. In some embodiments, the second polypeptide comprises SEQ ID NO: 42, wherein X1 is T or A; X2 is E or G; X3 is V or T; X4 is V or A; X5 is L or V; X6 is E or L; X7 is F or G; X8 is L or D; X9 is P or E; X10 is A or V; X11 is P or K; X12 is P or K; X13 is A or P; X14 is R or E; X15 is F or G; X16 is P or E; X17 is K or L; X18 is A or P; X19 is V or G; X20 is F or L; X21 is P or E; X22 is K or L; X23 is E or L; X24 is F or L; X25 is P or E; X26 is P or E; X27 is I or V; X28 is P or E; X29 is A or P; X30 is R or K; X31 is K or E; X32 is Y or K; X33 is E or K; X34 is K or E; X35 is M or A; X36 is F or K; X37 is K or R; X38 is E or K; X39 is F or A; X40 is E or K; and X41 is N or H.

[0017] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 23; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 24. In some embodiments, the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO: 43. In some embodiments, the first polypeptide comprises SEQ ID NO: 43. In some embodiments, the first polypeptide comprises SEQ ID NO: 43, wherein X1 is I or V; X2is V or E; X3 is K or R; X4 is A or L; X5 is G or R; X6 is G or R; X7 is E or Q; X8 is L or N; X9 is I or V; X10 is A or G; X11 is L or S; X12 is Y or I; X13 is E or R; X14 is N or D; X15 is K or E; X16 is R or D; X17 is P or Q; X18 is V or E; X19 is Y or L; X20 is L or F; X21 is S or T; X22 is S or L; X23 is K or R; X24 is A or L; X25 is L or R; X26 is A or E; X27 is K or R; X28 is A or S; X29 is K or A; X30 is V or T; X31 is L or M; X32 is P or T; X33 is E or D; X34 is R or L; X35 is V or L; X36 is K or L; and X37 is L or E.

[0018] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27 or SEQ ID NO: 29; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO: 44. In some embodiments, the first polypeptide comprises SEQ ID NO: 44. In some embodiments, the first polypeptide comprises SEQ ID NO: 44, wherein X1 is A or P; X2 is D or Q; X3 is R or N; X4 is R or N; X5 is S or D; X6 is L or T; X7 is D or Q; X8 is R or N; X9 is E or V; X10 is E or D; X11 is I or E; X12 is A or H; X13 is E or L; X14 is A or T; X15 is N or R; X16 is K or M; X17 is D or L; X18 is A or D; X19 is A or D; X20 is F or W; X21 is L or A; X22 is A or E; X23 is T or A; X24 is L or D; X25 is C or R; X26 is T or F; X27 is L or K; X28 is G or Q; X29 is A or K; X30 is R or M; X31 is T or Q; X32 is G or L; X33 is L or A; X34 is E or Q; and X35 is K or N.

[0019] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3; and the second polypeptide comprises or consists of a sequence having atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 9; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 11; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 13; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 14; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ IDNO: 17; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 20; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 23; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 24; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 26; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 29; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 30;^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 31; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 32; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 33; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 34; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 35; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 36; or ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 38.

[0020] Provided herein are protein cages comprising or consisting of a first polypeptide and a second polypeptide, wherein ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ IDNO: 7; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 29; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 30; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 26; ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 20; or ^ the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 23; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 24.

[0021] The protein cages described herein may optionally include an expression tag, e.g., a histidine tag, at or near the N-terminus or C-terminus of one of their subunit amino acid chains.

[0022] Provided herein are compositions comprising or consisting essentially of one or more protein cages as described herein and a pharmaceutically acceptable carrier.

[0023] Provided herein are kits comprising one or more protein cages as described herein or a composition thereof, packaged together with a buffer, a reagent, a device, or a container for use with the one or more one or more protein cages.

[0024] Also provided are methods of using the one or more protein cages as described herein as a carrier molecule for a given compound of interest (e.g., a given therapeutic agent of interest or a given diagnostic agent of interest). In some embodiments, the given compound of interest is carried within the interior of the one or more protein cages or on the surface of the one or more protein cages. In some embodiments, the given compound of interest is non-covalently bound to the first polypeptide and / or the second polypeptide. In some embodiments, the given compound of interest is covalently bound to the first polypeptide and / or the second polypeptide. In some embodiments, the given compound of interest is recombinantly fused to the first polypeptide and / or the second polypeptide.

[0025] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention.

[0026] DESCRIPTION OF THE DRAWINGS

[0027] This invention is further understood by reference to the drawings wherein:

[0028] FIG.1 is a sequence alignment showing that the “A” subunits of protein cages F2, D5, and C5 (i.e., SEQ ID NOs: 1, 3, and 5) have significant sequence identity with each other. * = identical residues, + = conservative substitutions. The identical residues are enumerated in SEQ ID NO: 39.

[0029] FIG.2 is a sequence alignment showing that the “A” subunits of protein cages D4, A4, B4, and C4 (i.e., SEQ ID NOs: 7, 9, 11, and 13) have significant sequenceidentity with each other. * = identical residues, + = conservative substitutions. The identical residues are enumerated in SEQ ID NO: 40.

[0030] FIG.3 is a sequence alignment showing that the “A” subunits of protein cages E3 and G4 (i.e., SEQ ID NOs: 15 and 17) have significant sequence identity with each other. * = identical residues, + = conservative substitutions. The identical residues are enumerated in SEQ ID NO: 41.

[0031] FIG.4 is a sequence alignment showing that the “B” subunits of protein cages G4 and G3 (i.e., SEQ ID NOs: 18 and 20) have significant sequence identity with each other. * = identical residues, + = conservative substitutions. The identical residues are enumerated in SEQ ID NO: 42.

[0032] FIG.5 is a sequence alignment showing that the “A” subunits of protein cages A1 and F1 (i.e., SEQ ID NOs: 21 and 23) have significant sequence identity with each other. * = identical residues, + = conservative substitutions. * = identical residues, + = conservative substitutions. The identical residues are enumerated in SEQ ID NO: 43.

[0033] FIG.6 is a sequence alignment showing that the “A” subunits of protein cages F3 and E12 (i.e., SEQ ID NOs: 27 and 29) have significant sequence identity with each other. * = identical residues, + = conservative substitutions. The identical residues are enumerated in SEQ ID NO: 44.

[0034] FIG.7 is an exemplary SDS-PAGE gel of the IMAC chromatography fraction for protein cage F2.

[0035] FIG.8 to FIG.15 are electron micrographs of exemplary protein cages. Electron Micrograph of F2 (FIG.8); Electron Micrograph of B3 (FIG.9); Electron Micrograph of D4 (FIG.10); Electron Micrograph of D5 (FIG.11); Electron Micrograph of F3 (FIG. 12); Electron Micrograph of G4 (FIG.13); Electron Micrograph of E4 (FIG.14); Electron Micrograph of E12 (FIG.15).

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] Disclosed herein are protein cages having a symmetric tetrahedral three- dimensional shape, which comprise two different subunit protein chains (subunit A and subunit B) with a total subunit stoichiometry of A12B12.

[0038] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and subunit B comprises a sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, subunit A is a species that falls within a genus sequence that comprises SEQ ID NO: 39 as follows: PX1RPALX2X3LX4LKSYAX5GVX6VADAALX7AAPX8X9LLX10X11X12X13VX14PGKX15L X16MX17X18GX19X20X21X22VX23X24AX25X26AALX27X28AX29X30GSGX31LX32X3 3X34X35FIX36X37X38HX39X40LX41X42FLX43X44X45X46X47AX48X49X50EX51PD X52AX53X54X55VEX56KTX57X58AAIX59AADAALX60X61APVX62LX63X64MRLX65X6 6X67IX68GKAX69FX70LX71GX72X73X74X75VX76X77AAX78X79VX80X81VAGX82X8 3LX84X85X86X87IIPRPHX88X89LX90GRX91FF (SEQ ID NO: 39) wherein each X is independently any amino acid. In some embodiments, X1 is A, E, or D; X2 is G or L; X3 is V or I; X4 is E or L; X5 is L or R; X6 is A or R; X7 is R or K; X8 is V or S; X9 is E, R, or L; X10 is K or M; X11 is C or N; X12 is E, K, or R; X13 is P or I; X14 is E or C; X15 is A or H; X16 is I or L; X17 is I, L, or M; X18 is R or T; X19 is E, R, or Q; X20 is P or V; X21 is E or A; X22 is A, D, or E; X23 is A or E; X24 is R, K, or E; X25 is M or Y; X26 is A, K, or R; X27 is E or T; X28 is T, V, or E; X29 is K, N, or G; X30 is A, K, or E; X31 is N or Q; X32 is I or L; X33 is D or A; X34 is H or S; X35 is A or V; X36 is G or P; X37 is R, A, or Y; X38 is I or A; X39 is P or E; X40 is A or Q; X41 is L or W; X42 is P or R; X43 is L or D; X44 is E or A; X45 is E or P; X46 is T or V; X47 is A, P, or V; X48 is P or D; X49 is P or A; X50 is I or L; X51 is D or E; X52 is E, R, or L; X53 is V or L; X54 is L or A; X55 is V or F; X56 is T or V; X57 is V or P; X58 is A or C; X59 is E or R; X60 is D, R, or K; X61 is V or A; X62 is R, E, or V; X63 is L, V, or R; X64 is R or D; X65 is S or A; X66 is E or I; X67 is H or G; X68 is G or N; X69 is Y, V, or R; X70 is V, A, or T; X71 is A, V, or E; X72 is D or K; X73 is E, P, or L; X74 is E, A, or V; X75 is A or D; X76 is R or L; X77 is K, R, or E; X78 is R or A; X79 is A or V; X80 is R, A, or I; X81 is A or E; X82 is E or D; X83 is K, Q, or D; X84 is I or L; X85 is D or S; X86 is L or I; X87 is R, A, or S; X88 is E, P, or D; X89 is A or E; X90 is R or L; and X91 is L, E, or W.

[0039] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. In some embodiments, subunitA is a species that falls within a genus sequence that comprises SEQ ID NO: 40 as follows: MNX1AX2X3X4YX5AGX6X7X8X9X10KGX11YX12X13AIX14X15X16X17LALLX18DPX19 NAEAWYNLGX20AYX21AX22GX23YX24X25AIX26AYX27KALX28LDPNNAEAWYNLGX29A YX30AX31GX32YX33X34AIX35X36X37X38X39ALX40LDPNNX41X42AX43X44X45LX4 6X47AKX48KX49X50LE (SEQ ID NO: 40) wherein each X is independently any amino acid. In some embodiments, X1 is L or M; X2 is E, R, or N; X3 is K or D; X4 is M or F; X5 is K, R, or E; X6 is Q, N, L, or K; X7 is I, A, or E; X8 is E, M, or A; X9 is F or Y; X10 is A or R; X11 is N, Q, E, or R; X12 is E, T, or N; X13 is T, I, or L; X14 is I or V; X15 is A or C; X16 is Y or F; X17 is T, Q, or S; X18 is K or L; X19 is N or K; X20 is E, N, or K; X21 is L, Y, or F; X22 is L or K; X23 is N, E, L, or K; X24 is E, R, or D; X25 is E, D, or K; X26 is E or K; X27 is Q, K, or E; X28 is E or M; X29 is E, N, K, or L; X30 is L, Y, or F; X31 is L or K; X32 is D or N; X33 is D, E, or K; X34 is N, S, or E; X35 is E or L; X36 is A or Y; X37 is F or Y; X38 is T, Q, or K; X39 is K or L; X40 is E or K; X41 is K, A, V, or E; X42 is T, E, or L; X43 is K or H; X44 is A, Q, or L; X45 is G, N, or A; X46 is K, A, or H; X47 is L, N, or K; X48 is E, Q, or K; X49 is K or L; and X50 is A or E.

[0040] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 17; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16 or SEQ ID NO: 18. In some embodiments, subunit A is a species that falls within a genus sequence that comprises SEQ ID NO: 41 as follows: MSISYRKLDIALSADGEEVLVX1GX2VLPTX3FFX4X5X6X7VTTMLNAX8GTDEENINX9X10L ADVHAAGLDVSNYGX11ASEX12YX13KGDPEKRAEAEARRAEAEARRAEX14X15X16X17LX1 8X19PEAX20X21EX22X23X24EX25X26X27X28X29X30X31LX32X33RFGX34AGX35X3 6AGL (SEQ ID NO: 41) wherein each X is independently any amino acid. In some embodiments, X1 is E or D; X2 is F or Q; X3 is K or R; X4 is E or L; X5 is N or D; X6 is V or T; X7 is I or V; X8 is A or C; X9 is A or E; X10 is L or I; X11 is K or W; X12 is I or V; X13 is A or K; X14 is L or R; X15 is A or R; X16 is A or R; X17 is E or R; X18 is S or A; X19 is T or S; X20is Q or R; X21 is A or R; X22 is A or R; X23 is K or R; X24 is K or R; X25 is K or E; X26 is V or A; X27 is L or E; X28 is E or R; X29 is A or R; X30 is A or L; X31 is E or R; X32 is A or Y; X33 is A or E; X34 is P or E; X35 is V or L; and X36 is K or E.

[0041] In some embodiments, subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20; and subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 19. In some embodiments, subunit B is a species that falls within a genus sequence that comprises SEQ ID NO: 42 as follows: MTX1EX2EX3X4X5AIAX6LX7X8X9DPHX10RAEAAKKLGKIGDX11EAVX12X13LIX14ALX 15DX16DPAVRAAAAX17ALGKIGDKEAVPX18LIX19ALX20DX21DPAVRVAAAX22ALGKIG DKEAVPALIX23ALX24DX25DX26AVRVAAAX27ALGKIGDKEAVX28X29LVX30ALX31X32 EEGLVRX33AAAIALX34KIGGEEVKKAX35EELAKX36GEGEAX37X38X39AEEYLKX40X41 (SEQ ID NO: 42) wherein each X is independently any amino acid. In some embodiments, X1 is T or A; X2 is E or G; X3 is V or T; X4 is V or A; X5 is L or V; X6 is E or L; X7 is F or G; X8 is L or D; X9 is P or E; X10 is A or V; X11 is P or K; X12 is P or K; X13 is A or P; X14 is R or E; X15 is F or G; X16 is P or E; X17 is K or L; X18 is A or P; X19 is V or G; X20 is F or L; X21 is P or E; X22 is K or L; X23 is E or L; X24 is F or L; X25 is P or E; X26 is P or E; X27 is I or V; X28 is P or E; X29 is A or P; X30 is R or K; X31 is K or E; X32 is Y or K; X33 is E or K; X34 is K or E; X35 is M or A; X36 is F or K; X37 is K or R; X38 is E or K; X39 is F or A; X40 is E or K; and X41 is N or H.

[0042] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 23; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 24. In some embodiments, subunit A is a species that falls within a genus sequence that comprises SEQ ID NO: 43 as follows:VVLX1TVPSEEX2ARX3IARAX4VEX5X6LAAX7VX8IX9PX10LTX11X12YRX13X14GX15V VEX16X17X18X19LLLVX20X21TEX22X23FPX24LX25X26LVX27X28LHPEX29X30PX3 1IVALPVVDGNX32X33YLX34WX35X36X37NTG (SEQ ID NO: 43) wherein each X is independently any amino acid. In some embodiments, X1 is I or V; X2 is V or E; X3 is K or R; X4 is A or L; X5 is G or R; X6 is G or R; X7 is E or Q; X8 is L or N; X9 is I or V; X10 is A or G; X11 is L or S; X12 is Y or I; X13 is E or R; X14 is N or D; X15 is K or E; X16 is R or D; X17 is P or Q; X18 is V or E; X19 is Y or L; X20 is L or F; X21 is S or T; X22 is S or L; X23 is K or R; X24 is A or L; X25 is L or R; X26 is A or E; X27 is K or R; X28 is A or S; X29 is K or A; X30 is V or T; X31 is L or M; X32 is P or T; X33 is E or D; X34 is R or L; X35 is V or L; X36 is K or L; and X37 is L or E.

[0043] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27 or SEQ ID NO: 29; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, subunit A is a species that falls within a genus sequence that comprises SEQ ID NO: 44 as follows: LRLERDGAVARLLIDRX1X2X3X4NAFX5X6X7MWQX8LPX9LLAX10ASGDDALRVLVVKSANG GAFCAGADX11X12X13LLX14X15X16X17DX18X19X20HX21X22NQQAINRAQYELARFRL PTVAMVEGDCIGGGCGIALACDMRIAAPAARFGITPAKLGLVYPLHDVKLLVDLVGPGQARRLMF TGGLIDANEAHRIGLVELLGESEDALVGQLATVSSFSTQAIKSFVRRVLDGQVADDX23X24SLX 25VFASAX26X27X28X29DFX30EGX31X32AFX33X34X35RPPVF (SEQ ID NO: 44) wherein each X is independently any amino acid. In some embodiments, X1 is A or P; X2 is D or Q; X3 is R or N; X4 is R or N; X5 is S or D; X6 is L or T; X7 is D or Q; X8 is R or N; X9 is E or V; X10 is E or D; X11 is I or E; X12 is A or H; X13 is E or L; X14 is A or T; X15 is N or R; X16 is K or M; X17 is D or L; X18 is A or D; X19 is A or D; X20 is F or W; X21 is L or A; X22 is A or E; X23 is T or A; X24 is L or D; X25 is C or R; X26 is T or F; X27 is L or K; X28 is G or Q; X29 is A or K; X30 is R or M; X31 is T or Q; X32 is G or L; X33 is L or A; X34 is E or Q; and X35 is K or N.

[0044] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2.

[0045] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4.

[0046] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6.

[0047] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8.

[0048] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 9; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10.

[0049] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 11; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12.

[0050] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 13; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 14.

[0051] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16.

[0052] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18.

[0053] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 20.

[0054] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22.

[0055] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 23; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 24.

[0056] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 26.

[0057] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28.

[0058] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 29; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 30.

[0059] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 31; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 32.

[0060] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 33; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 34.

[0061] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 35; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 36.

[0062] In some embodiments, subunit A comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37; and subunit B comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 38.

[0063] The protein cages and their subunit chains may be made using methods known in the art including chemical synthesis, biosynthesis (in vitro and in vivo synthesis) using recombinant DNA methods, and solid phase synthesis. See, e.g., Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol. 12, J. K. Setlow ed., Plenum Press, NY, pp 1-19; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2ed., Pierce, Rockford, IL, which are herein incorporated by reference. The proteins may be purified using protein purification techniques known in the art such as reverse phase high-performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See, e.g., Olsnes and Pihl (1973) Biochem 12(16):3121-3126; and Scopes (1982) Protein Purification, Springer-Verlag, NY, which are herein incorporated by reference. Alternatively, the proteins may be made by recombinant DNA techniques known in the art.

[0064] Uses

[0065] The protein cages described herein have physical characteristics, e.g., size and shape, that make them useful for various applications. For example, the protein cages may be employed in chromatography and / or light scattering methodologies for dynamic range standardization and / or instrument calibration. Native-PAGE or Size Exclusion Chromatography are specific examples. The sizes and shapes of the protein cages are observable using microscopy and, as such, the protein cages may be employed as fiducial markers, stain contrast agents, and labeling reagents. The protein cages have an interior cavity wherein a cargo of interest, e.g., a given compound or biomolecule (protein, nucleic acid, lipid, carbohydrate, etc.) may be packaged. Alternatively, or in addition to a cargo within the interior cavity, one or more passenger molecules, e.g., a given compound or biomolecule (protein, nucleic acid, lipid, carbohydrate, etc.), which may bethe same or different from any cargo in the interior cavity, may be covalently or non- covalently attached to the exterior surface of the protein cages. As such, the protein cages are particularly useful as drug delivery agents.

[0066] Kits

[0067] In some embodiments, the present invention provides kits comprising one or more protein cages described herein packaged together with one or more reagents for using the protein cages, e.g., buffers and reagents for labeling and detecting the protein cages, and / or devices for using the protein cages, e.g., syringes pre-filled with the protein cages, In some embodiments, the kits comprise the one or more protein cages, a carrier, package, or container for receiving and / or mixing the protein cages. In some embodiments, the kits optionally include an identifying description or label or instructions relating to its use. In some embodiments, the kits include information prescribed by a governmental agency that regulates the manufacture, use, or sale of compounds and compositions as contemplated herein. In some embodiments, the kits optionally include a given compound or biomolecule (protein, nucleic acid, lipid, carbohydrate, etc.) that is desired to be packaged within the interior cavity and / or attached, covalently or non-covalently, to the exterior surface of the protein cages.

[0068] Compositions

[0069] Compositions, including pharmaceutical compositions, comprising, consisting essentially of, or consisting of one or more protein cages are contemplated herein. For example, such compositions comprise one or more one or more protein cages as described herein and a pharmaceutically acceptable vehicle.

[0070] The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject. Pharmaceutical compositions may be formulated for the intended route of delivery and administered to subjects accordingly using methods in the art. Pharmaceutical compositions may include one or more of the following: a pharmaceutically acceptable vehicle, pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. The compositions and formulations may be optimized for increased stability and efficacy using methods in the art. See, e.g., Carra et al., (2007) Vaccine 25:4149-4158.

[0071] As used herein, a “pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, for pharmaceutical administration. Thus, for example, unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration. Pharmaceutically acceptable vehicles include those known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th ed (2000) Lippincott Williams & Wilkins, Baltimore, MD.

[0072] The following examples are intended to illustrate but not to limit the invention.

[0073] EXAMPLES

[0074] The protein cages exemplified herein were designed, produced, and assayed as described in Meador, et al. (2024) “A suite of designed protein cages using machine learning and protein fragment-based protocols” Structure 32(6):751-765.e11 and the Supplemental Information thereto, which are herein incorporated by reference in their entirety.

[0075] The amino acid sequences of the protein cages are as follows:

[0076] Example 1 − Protein Cage F2

[0077] Subunit A: MGDPARPALGVLELKSYALGVAVADAALRAAPVELLKCEPVEPGKALIMIRGEPEAVARAMAAAL ETAKAGSGNLIDHAFIGRIHPALLPFLLEETAAPPIEDPDEAVLVVETKTVAAAIEAADAALDVA PVRLLRMRLSEHIGGKAYFVLAGDEEAVRKAARAVRAVAGEKLIDLRIIPRPHEALRGRLFF (SEQ ID NO: 1)

[0078] Subunit B: MPIALTVVPPEEAEPLARELVEAGLAAEVLLVPVRRIYREKGKVREEEVTLLLILVSREGVPALR AWIEARHPDDIPLFIVLAVDEEASNKRYLGYIAAETHLYSA (SEQ ID NO: 2)

[0079] Example 2 − Protein Cage D5

[0080] Subunit A: MGPDEPERPALLILELKSYARGVRVADAALKAAPVRLLKCKIVEPGKALIMLTGRPEDVEKAYKA ALTVANKGSGNLIDSVFIPAIHPALLPFLLEETPAPPLEDPDRALLFVEVKTVAAAIRAADAALRAAPVELVRMRLSEHIGGKAVFALVGDPADVLRAAAVVAEVAGDQLLDIAIIPRPHPALLGREFF (SEQ ID NO: 3)

[0081] Subunit B: MPMLVVYVPEGYSEAQKRALLFRLAAAVVEATGTPLENVRIILTTYAPADVLLGGAIGVPLVVIL VYLLEGLSPEQKAALVKALTAAAAEALGVDPENIRVILVPVPPENFGVGNGKTAAEA (SEQ ID NO: 4)

[0082] Example 3 – Protein Cage C5

[0083] Subunit A: MGPAGEPDRPALGVLLLKSYARGVAVADAALKAAPSLLLMNRPVCPGKHLLMMRGQVAEVEEAMR AALEEAGEGSGQLLASAFIPYAHEQLWRFLDAPVVADAIEEPDLAVAVVETKTPCAAIRAADAAL KAAPVVLRDMRLAIGINGKARFTLEGKLVDVLEAAAVVIEVAGDDLISLSIIPRPHDELRGRWFF (SEQ ID NO: 5)

[0084] Subunit B: MADFHEQMATMFKNLAKILKAKNAAEVKDALKEMRKAALAAHKEVPPSLKDKPLNSQEMIEFHDE MLELAWAIHDAAHLAKEGKIEEAKKKAEEILKMVSRLVSLY (SEQ ID NO: 6)

[0085] Example 4 − Protein Cage D4

[0086] Subunit A: MNLAEKMYKAGQIEFAKGNYETAIIAYTLALLKDPNNAEAWYNLGEAYLALGNYEEAIEAYQKAL ELDPNNAEAWYNLGEAYLALGDYDNAIEAFTKALELDPNNKTAKAGLKLAKEKKALE (SEQ ID NO: 7)

[0087] Subunit B: MTDLSSLIETADLRLLLTTVPTETEALYLALAAVEKGLAAEVLITPVTRVRRENGKLVVEDVYRL SFKTTRERLDALVAWLQRRHPLALPECLVLTPIASSVAYRDWLRSSLQ (SEQ ID NO: 8)

[0088] Example 5 − Protein Cage A4

[0089] Subunit A: MNLAEKMYKAGNAMYRKGQYTIAIIAYSLALLLDPKNAEAWYNLGNAYYAKGEYDDAIKAYEKAL MLDPNNAEAWYNLGNAYYAKGDYESAILAYQLALKLDPNNAEAKQNLANAKQKLALE (SEQ ID NO: 9)

[0090] Subunit B: MKILIVVTHGPEDLDRTYAPLFLAVVAAERGYKTSVFFMIKGPLLLNRDYIAKVALEGGNPYLEY LYKAKQLGVEIYVCVQSLRDMCHLKEEDIIGGVKLVGGSTLIDLTLEADRTLFF (SEQ ID NO: 10)

[0091] Example 6 − Protein Cage B4

[0092] Subunit A:MNMARDFYRAGLIAYAKGEYETAIVAFQLALLLDPNNAEAWYNLGKAYYALGLYREAIEAYKKAL ELDPNNAEAWYNLGKAYYALGDYESAIEAYKKALELDPNNVEAHANLHKAKKKLALE (SEQ ID NO: 11)

[0093] Subunit B: MPSYAVSSRAGLIDQERRAAVADLITALHSEILKIPRYLVQVIFNDLDAGALFLAGREAPEGHVW IHADIISGRTKEQKKAFLQALTVEVARVLGLPEEQVWVYVNEIPGENMTLFGQILPAPGEEEAWF ATLPEELQKRLADLR (SEQ ID NO: 12)

[0094] Example 7 − Protein Cage C4

[0095] Subunit A: MNLANDFYEAGKEEFAKGRYNLAIVCFSLALLKDPNNAEAWYNLGKAYFALGKYDKAIEAYQKAL ELDPNNAEAWYNLGLAYFALGNYKEAIEYYKKALELDPNNELAKLALKLAKEKLELE (SEQ ID NO: 13)

[0096] Subunit B: MAMPAVKLVIVTEKILLKDITRIILESGAKGFTVMNTGGIGSRERAGEGEPDIDKIRANIKFEVL CESRELAELIAEAIASKFFDKYAGIIYTCSAEVLYGHDFCGPEGS (SEQ ID NO: 14)

[0097] Example 8 − Protein Cage E3

[0098] Subunit A: MSISYRKLDIALSADGEEVLVEGFVLPTKFFENVIVTTMLNAAGTDEENINALLADVHAAGLDVS NYGKASEIYAKGDPEKRAEAEARRAEAEARRAELAAELSTPEAQAEAKKEKVLEAAELAARFGPA GVKAGL (SEQ ID NO: 15)

[0099] Subunit B: MYPVDLHMHTIKNDASFSTLSDYIARAKEKGIKLIAITDHGPSHPIAPHPEYYVRMKELPDVVDG VGVLRGVEANILDTNGNIDVTPEMEKSLDLILAGLYESVYPPQSRAENTKALINAIASGKVHVIS HPADPRYPVDYRALAGAAAVAGVALEITEHAFGEEFPGAEPRARELARAVKEAGGYVALGSDAHH AWHLGRFEHAERVLREVGFPEERVLNRSPEKLLAFLESRGVPKKPAFADL (SEQ ID NO: 16)

[0100] Example 9 − Protein Cage G4

[0101] Subunit A: MSISYRKLDIALSADGEEVLVDGQVLPTRFFLDTVVTTMLNACGTDEENINEILADVHAAGLDVS NYGWASEVYKKGDPEKRAEAEARRAEAEARRAERRRRLASPEARRERRREEAERRLRLYERFGEA GLEAGL (SEQ ID NO: 17)

[0102] Subunit B: MTTEEEVVLAIAELFLPDPHARAEAAKKLGKIGDPEAVPALIRALFDPDPAVRAAAAKALGKIGD KEAVPALIVALFDPDPAVRVAAAKALGKIGDKEAVPALIEALFDPDPAVRVAAAIALGKIGDKEA VPALVRALKYEEGLVREAAAIALKKIGGEEVKKAMEELAKFGEGEAKEFAEEYLKEN (SEQ ID NO: 18)

[0103] Example 10 − Protein Cage G3

[0104] Subunit A: MKLPNKVSLVAGTAEGATPENALHGARLNAGIGDVNLVPVSGIAPAGAEIVPLPELPPGALLPTA EASIVSDVPGKTIAAAVAVGIPKDPSLPGIIATYAGEMSAEEARRRVELIVIEQFLQRGWELESI HSVAVEHTVKRLGAALAAAVLWYK (SEQ ID NO: 19)

[0105] Subunit B: MTAEGETAVAIALLGDEDPHVRAEAAKKLGKIGDKEAVKPLIEALGDEDPAVRAAAALALGKIGD KEAVPPLIGALLDEDPAVRVAAALALGKIGDKEAVPALILALLDEDEAVRVAAAVALGKIGDKEA VEPLVKALEKEEGLVRKAAAIALEKIGGEEVKKAAEELAKKGEGEARKAAEEYLKKHKLE (SEQ ID NO: 20)

[0106] Example 11 − Protein Cage A1

[0107] Subunit A: MIVVLITVPSEEVARKIARAAVEGGLAAEVLIIPALTLYYRENGKVVERPVYLLLVLSTESKFPA LLALVKALHPEKVPLIVALPVVDGNPEYLRWVKLNTG (SEQ ID NO: 21)

[0108] Subunit B: MLDEIAFADARILTPFTEADIERLLDALELEPGTRVVDLGCGTGYFLVLGAERKGITATGIDISE LAIEKARELARERGVEDRVEFIHGDVSTYVAEEKVDVAACIGAEAYFGGIEGALKALEKSLKEGG IILLGVPYWRTRPATEAEARACGFDSIDDLDTLAETVAKLEALGYRVIQIVLADEHGMEELYYGR LVQLDRWLREHPDHPFAPALEAELATLAARYEKYVRRHLGYGVFALRKRLE (SEQ ID NO: 22)

[0109] Example 12 − Protein Cage F1

[0110] Subunit A: MPLVVLVTVPSEEEARRIARALVERRLAAQVNIVPGLTSIYRRDGEVVEDQELLLLVFTTELRFP LLRELVRSLHPEATPMIVALPVVDGNTDYLLWLLENTG (SEQ ID NO: 23)

[0111] Subunit B: MPFEKALYFLTYLSYTIDIAELSILIKKGDKSIIIVDVRDAEAYKECHIPTAISIPGNKINEEST KDLPKDKTIITYCWGPACNGATRASRKFAELGFDVKRLIGGIEYWRKENGEVEGTLGAKADLFWN MKKESLE (SEQ ID NO: 24)

[0112] Example 13 − Protein Cage D1

[0113] Subunit A: MLAFEFLHEEKELGKIIVVDTGTSPHHLKGQLETVGDFIDYVKFAGMTAAVMPKKVVCEKILIYH KHGIKVMPGGTLFEKAVSKGKEKEFLLECRELGFDAIEISDLNIDLSDEELKKLIKMAKEEGFEV FTKVGRADKERDAKLTVEDIIAKINFYLEAGADYVIIYGGASGKGIGLYDENGKLKKEWLDEIKK NVDMSKIIFEAPLPEQQKELLDKFGPSANLAEISLHDVARLAEMRYGLRGDTFGKV (SEQ ID NO: 25)

[0114] Subunit B:MAQAQTQGQEEEQKKKIVIIIRHGPEEPIYCVTPLRLAVVAAEQGYETTIVFTELGPELLNKLYW IEEMAKGGNPVTKYLLKAREKGVKIYVCEWSLEEICKLKKEDIIPGVEIIDDKDIIKLMLEADVV IFF (SEQ ID NO: 26)

[0115] Example 14 − Protein Cage F3

[0116] Subunit A: MSLRLERDGAVARLLIDRADRRNAFSLDMWQRLPELLAEASGDDALRVLVVKSANGGAFCAGADI AELLANKDDAAFHLANQQAINRAQYELARFRLPTVAMVEGDCIGGGCGIALACDMRIAAPAARFG ITPAKLGLVYPLHDVKLLVDLVGPGQARRLMFTGGLIDANEAHRIGLVELLGESEDALVGQLATV SSFSTQAIKSFVRRVLDGQVADDTLSLCVFASATLGADFREGTGAFLEKRPPVF (SEQ ID NO: 27)

[0117] Subunit B: MKVVVQIKDFDKVPQALQSVLNLFLDLGNAEIEVVLHQSAIKALLLNSPTRSIIEELIKLNILIV GCEHSIRSQNLDHRQLIDGIKIVRSGVGEIVRKQSEGWIYLAL (SEQ ID NO: 28)

[0118] Example 15 − Protein Cage E12

[0119] Subunit A: MSDLRLERDGAVARLLIDRPQNNNAFDTQMWQNLPVLLADASGDDALRVLVVKSANGGAFCAGAD EHLLLTRMLDDDWHAENQQAINRAQYELARFRLPTVAMVEGDCIGGGCGIALACDMRIAAPAARF GITPAKLGLVYPLHDVKLLVDLVGPGQARRLMFTGGLIDANEAHRIGLVELLGESEDALVGQLAT VSSFSTQAIKSFVRRVLDGQVADDADSLRVFASAFKQKDFMEGQLAFAQNRPPVF (SEQ ID NO: 29)

[0120] Subunit B: MYETIRYEVKGQVAWLTLNRPDQLNAFTEQMNAEVTKALKQAGADPNVRCVVITGAGEAFCAGED LSGVTEEMDHGDVLRSRYAPMMKALHHLEKPVVAAVNGKAAGAGMSLALACDFRLLSETASFAPA FISVGLVPDAGHLYYLPRLVGRAKALELAVLGVRVTALQAAKLGLATAVIPKHLWELAVKAYASA LSNMPTKAIGLIKRLLRESEETTFDRYLEREAECQRIAGLTSDHREGVKARNESRKPLFQGN (SEQ ID NO: 30)

[0121] Example 16 − Protein Cage B3

[0122] Subunit A: MAIIETTTPTEEEAKAIAKKLLENRLIAEAIITPALTKIYRENGEIKSETVTRVTLYTEEENVPK AVTYIKAIHPDPIPPIIVITPTDANPAYKGWVAFET (SEQ ID NO: 31)

[0123] Subunit B: MGDPERPALGILELSSYARGVKVADAALKAAPVKLLKCEPVEPGRALIMLLGEPEDVAKAMIAAL DVAGLGSGNLIDYALIPEIHPQLLPFLKEYKKSEPIKDPNKAIIVAEVSTVAAAIEAADVALRLA NVELTSMRLAEHIGGRASFTLIGDKEDVEKAARAIRGVAGERLLDLEIIEKPVEALIGNEFF (SEQ ID NO: 32)

[0124] Example 17 − Protein Cage E4

[0125] Subunit A: MTAVFAAIVGFLDKKIEELKKIQKHKTLPKMSGGWELELNGTEAKLVREVDGYKVTVTFNINNSI PPTFDGEEEPSQGQPVEEQPPELTSTPNFVVEIVKASQPDTALVFDCYYPEDEVGQEEEEEKPLF EIKEVSFQSTGESEWKDTNYTLNTDNLPEDLLLAFYALLAALGVDNDFAKELIELSTALEHQERI TFFEQLRDFIA (SEQ ID NO: 33)

[0126] Subunit B: MNLAEKMYEAGKYFAAQGNYELAIIAYTLALLKDPNNAEAWYNLGKAYAALGKYEEAIEAYKKAL ALDPNNAEAWYNLGGAYAGLGKYEEAIEYLEKALALDPNNELAKLMLKFAKLQLELEG (SEQ ID NO: 34)

[0127] Example 18 − Protein Cage B1

[0128] Subunit A: MPMVIFECSDNIREEAKFEELFARLNPALASTGLFPLEEIVGRVHWVDTWQFADGQHDYAFVHVT IDVPAGLSEEDRLLVLNGVFALLLGHLEPLMKEHLLYLSLELRVLPATLSRRWNNAIELFK (SEQ ID NO: 35)

[0129] Subunit B: MSVKIDVIRVEIPEGTWVIIGQSHSRSIVFDLSQTLQSASGRLRFGIAYCEASGKRLILHDGNDP ALVELAKETALKIGAGHTFVIYIRNGRPEDILNRIKNIESVVRIFAATANPLQVLVAETDQGRGV IGVVDGYTPLGVATAEDRAALAAALRAEGYKR (SEQ ID NO: 36)

[0130] Example 19 − Protein Cage A3

[0131] Subunit A: MAEVSIKTKIKAKHRLYSKNLSEEENKMLFGSAAKKGGHEHNYTITVTVKGEIDPTTGLVINGTD LRIWIEKAIILPLDNKNLNEDVPYFKTNVPTTENIAKYIKENLEKVLPKGLLSKVVVEETEEHKV TIKGE (SEQ ID NO: 37)

[0132] Subunit B: MPAIILTTTPTEADARALAEGLLEKRLIAEAIITPNVTRIYLENGEIKSEKVVRMELYTVEEKVE AAMTYIEAHHPDPIPPIIVIKPDKVSPKYKKWILEQTAL (SEQ ID NO: 38)

[0133] The above subunit sequences may optionally include a histidine tag at or near their N-terminus or C-terminus.

[0134] Protein expression

[0135] Plasmids containing two genes of interest, but constituting a single design, were acquired from Twist Biosciences. Each plasmid contains a Kanamycin resistance gene for selection as well as a pET-Duet type interspatial expression cassette cloned between the genes of interest to enable bicistronic expression of the two proteins under the controlof a LacO inducible T7 promoter. Plasmids were transformed into LOBSTR BL21(DE3)-RIL competent cells (Cat# EC1002, Kerafast, Boston, MA) and always supplemented with 50 µg / ml of Kanamycin during growth. First, cells were grown to saturation overnight in 20 ml of PG media (Studier 2005). The following day, 10 ml of saturated culture was used to inoculate 1L ZYM-5052 cultures (Studier 2005) which was left to grow for either about 66 hours at 18°C, about 42 hours at 25°C, or 16 hours at 37°C. Cells were harvested by centrifugation at 4,000 x g for 10 − 15 minutes and quickly moved to freezing conditions at approximately −80°C. Pellets were transferred to −20°C after 24 hours at −80°C.

[0136] Immobilized metal affinity chromatography (IMAC)

[0137] Purification of proteins from cell pellets was performed with a 1:4 ratio of grams of pellet to milliliters of purification buffer (e.g., 10 g / 40 ml). Purification buffer contained 50 mM Tris-HCl, pH 7.5 at 4°C, 300 mM KCl, and 30 mM imidazole, while lysis buffer (utilized during cell lysis) was supplemented with 0.5 mM EDTA, 300 µg / ml lysozyme, 100 µg / ml benzonase nuclease, and one tablet of protease inhibitor cocktail EDTA-free (Thermo Scientific) per 50 ml of cellular suspension. Pellets were resuspended in lysis buffer with end over end rotation for about 30 minutes at 4°C until no visible chunks of cell pellet remained. Lysis of cellular resuspension proceeded with three passes of high pressure homogenization in a EmulsiFlex C3 homogenizer (Avestin, Ottawa, Canada) pressurized to > 15,000 psi. Lysate was then clarified using centrifugation at 20,000 x g for 30 minutes or 10,000 x g for 45 minutes at 4°C. The soluble supernatant fraction was collected and either incubated with 2 ml Ni-NTA resin per 50 ml of soluble lysate (batch binding) or applied to a 5 ml HisTrap pre-equilibrated with purification buffer.

[0138] For batch binding, side over side rotation of the soluble lysate and Ni-NTA resin proceeded for 30 minutes at 4°C, after which the solution was allowed to stand for five minutes at 4°C, enough to separate the Ni-NTA resin and the supernatant by gravitational forces. Then, the supernatant was removed and the remaining Ni-NTA resin was collected and loaded into a filtered column where remaining soluble lysate was cleared from the Ni-NTA resin by gravity. The Ni-NTA column was washed by gravity flow with 10 column volumes (CV) of purification buffer supplemented to 60 mM imidazole (wash buffer). Finally, protein of interest was eluted from the resin with gravity flow using 9 CV of purification buffer supplemented with 250 mM imidazole (elution buffer).

[0139] For HisTrap binding, the soluble lysate was applied to the 5 ml column at 1 ml / min until all soluble lysate had passed through the column. Next, 10 CV of purification buffer were applied, followed by 2 CV of wash buffer and then a gradient from 60 mM imidazole to 300 mM imidazole (elution buffer concentration) was carried out for 3 CV. Finally, 10 CV of elution buffer was passed over the column.

[0140] Fractions corresponding to the lysate, the clarified lysate, the insoluble lysate, the unbound Ni-NTA supernatant (aka the flow through), the wash, and the elution were analyzed by SDS-PAGE for bands at the hypothesized size for the proteins of interest and classified according to one of insoluble, soluble, or co-eluting based on the presence of both bands.

[0141] Size Exclusion Chromatography (SEC)

[0142] After IMAC purification, fractions containing proteins of interest were concentrated using Amicon® Ultra 10 kDa MWCO concentrators until samples reached a volume of about 500 µl or precipitation was visibly observed at which point concentration was immediately stopped. After concentration, samples were clarified by centrifugation for 10 minutes at 16,900 x g to remove any precipitation from soluble protein. All chromatography was performed using the Bio-Rad NGC HPLC system. Analytical chromatography was performed using a 10 / 300 gl size column with either Superose® 6 Increase or Superdex® 200 Increase resin with flow at 0.3 ml / minute. For preparative scale chromatography, 16 / 60 scale columns with either Superose® 6 or Superdex® 200 residue was used while flowing at 0.9 ml / minute. Peaks in the chromatograms observed by absorbance at 230 / 280 nm indicate the self-assembly of the subunit proteins into protein cages consistent with their predicted 3D tertiary structures using 3D protein modeling. Fractions corresponding to the peaks in the chromatograms were analyzed by SDS-PAGE to confirm that the protein cages of each fraction were composed of their respective subunit proteins.

[0143] Electron Microscopy (EM)

[0144] After either IMAC or SEC purification, fractions visibly containing proteins of interest were further measured for protein concentration using a spectrophotometer with absorbance at 280 nm. Using a linear combination of extinction coefficients and molecular weights from both protein subunits of the described cages, concentration values were calculated and samples were diluted to about 0.1 mg / ml of protein using the purification buffer in which they resided. Ted Pella Formvar Copper grids (prod#01754-F) were glow discharged for 30 seconds with a Pelco easiGlow glow discharger using 15 mA and a negative polarity. Protein samples were applied to the polarized grids, allowed to rest for 1 minute, then blotted to a thin film of liquid with Whatman filter paper. Immediately after blotting, the grid was incubated with a 2% uranyl acetate solution. Blotting and uranyl acetate proceeded for three iterations, where the final application of uranyl acetate was left to rest for 1 minute, and finally all the liquid was blotted from the grid with filter paper. The prepared specimens were completely dried through evaporation and loaded into a transmission electron microscope to view protein specimens using negative stain.

[0145] Results of Co-Elution by IMAC: ^ Protein cage A1: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 11.14 kDa and 28.87 kDa. ^ Protein cage B1: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 16.06 kDa and 17.48 kDa. ^ Protein cage D1: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 28.12 kDa and 16.99 kDa. ^ Protein cage F1: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 11.63 kDa and 17.07 kDa. ^ Protein cage F2: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 21.80 kDa and 11.84 kDa. ^ Protein cage A3: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 16.8 kDa and 11.72 kDa. ^ Protein cage B3: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 11.22 kDa and 21.97 kDa. ^ Protein cage E3: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 14.43 kDa and 28.36 kDa. ^ Protein cage G3: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 17.43 kDa and 19.61 kDa. ^ Protein cage A4: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 15.27 kDa and 13.33 kDa. ^ Protein cage B4: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 13.79 kDa and 17.76 kDa.^ Protein cage C4: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 13.90 kDa and 13.48 kDa. ^ Protein cage D4: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 13.55 kDa and 14.30 kDa. ^ Protein cage E4: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 23.31 kDa and 15.19 kDa. ^ Protein cage G4: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 17.15 kDa and 19.58 kDa. ^ Protein cage C5: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 22.22 kDa and 12.03 kDa. ^ Protein cage D5: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 20.58 kDa and 14.23 kDa. ^ Protein cage F3: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 27.64 kDa and 12.11 kDa. ^ Protein cage E12: On SDS-PAGE analysis of IMAC, bands were observed at molecular weights 27.05 kDa and 29.00 kDa.

[0146] FIG.7 is an exemplary SDS-PAGE gel of protein cage F2. Electron microscopy was performed on selected protein cages to further confirm the subunit proteins self- assembled into protein cages. See FIG.8 to FIG.15.

[0147] REFERENCES

[0148] The following references are herein incorporated by reference in their entirety with the exception that, should the scope and meaning of a term conflict with a definition explicitly set forth herein, the definition explicitly set forth herein controls: Meador K, et al. A suite of designed protein cages using machine learning and protein fragment-based protocols. Structure.2024 Jun 6;32(6):751-765.e11. doi: 10.1016 / j.str.2024.02.017. Yeates TO. Geometric Principles for Designing Highly Symmetric Self-Assembling Protein Nanomaterials. Annu Rev Biophys.2017 May 22;46:23-42 McConnell SA, Cannon KA, Morgan C, McAllister R, Amer BR, Clubb RT, Yeates TO. Designed Protein Cages as Scaffolds for Building Multienzyme Materials. ACS Synth Biol.2020 Feb 21;9(2):381-391.Yeates TO, Agdanowski MP, Liu Y. Development of imaging scaffolds for cryo-electron microscopy. Curr Opin Struct Biol.2020 Feb;60:142-149. Miller JE, Srinivasan Y, Dharmaraj NP, Liu A, Nguyen PL, Taylor SD, Yeates TO. Designing Protease-Triggered Protein Cages. J Am Chem Soc.2022 Jul 20;144(28):12681-12689.

[0149] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

[0150] The use of the singular can include the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” can include plural referents unless the context clearly dictates otherwise.

[0151] As used herein, “and / or” means “and” or “or”. For example, “A and / or B” means “A, B, or both A and B” and “A, B, C, and / or D” means “A, B, C, D, or a combination thereof” and said “A, B, C, D, or a combination thereof” means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).

[0152] As used herein, the phrase “one or more of”, e.g., “one or more of A, B, and / or C” means “one or more of A”, “one or more of B”, “one or more of C”, “one or more of A and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”.

[0153] The phrase “comprises or consists of A” is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue: comprises A or consists of A. For example, the sentence “In some embodiments, the composition comprises or consists of A” is to be interpreted as if written as the following two separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists of A.”

[0154] Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C.” As another example, the sentence “In some embodiments, the composition comprises atleast A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C.”

[0155] As used herein, a “protein cage” refers to a plurality of polypeptides (“subunits” or “oligomeric protein units”) which collectively form a three-dimensional structure having an outer surface, which may have an interior cavity.

[0156] As used herein, the terms “protein”, “polypeptide” and “peptide” are used interchangeably to refer to two or more amino acids linked together. Groups or strings of amino acid abbreviations are used to represent peptides. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequence is written from the N-terminus to the C-terminus. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequences are written from the N-terminus to the C-terminus. Similarly, except when specifically indicated, nucleic acid sequences are indicated with the 5’ end on the left and the sequences are written from 5’ to 3’.

[0157] As used herein, a given percentage of “sequence identity” refers to the percentage of nucleotides or amino acid residues that are the same between sequences, when compared and optimally aligned for maximum correspondence over a given comparison window, as measured by visual inspection or by a sequence comparison algorithm in the art, such as the BLAST algorithm, which is described in Altschul et al., (1990) J Mol Biol 215:403-410. Software for performing BLAST (e.g., BLASTP and BLASTN) analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The comparison window can exist over a given portion, e.g., a functional domain, or an arbitrary selection over a given number of contiguous nucleotides or amino acid residues of one or both sequences. Alternatively, the comparison window can exist over the full length of the sequences being compared. For purposes herein, where a given comparison window (e.g., over 80% of the given sequence) is not provided, the recited sequence identity is over 100% of the given sequence. Additionally, for the percentages of sequence identity of the proteins provided herein, the percentages are determined using BLASTP 2.8.0+, scoring matrix BLOSUM62, and the default parameters available at blast.ncbi.nlm.nih.gov / Blast.cgi. See also Altschul, et al., (1997) Nucleic Acids Res 25:3389-3402; and Altschul, et al., (2005) FEBS J 272:5101-5109.

[0158] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.

[0159] As provided herein, a sequence that falls within a genus sequence means that the sequence must have amino acid residues that are identical to those specifically enumerated for the genus sequence when optimally aligned thereto. That is, the sequence must comprise, when optimally aligned with the genus sequence, the same amino acid residues as the genus sequence except for Xaa’s that are “any amino acid”.

[0160] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

[0161] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

What is claimed is:

1. A protein cage comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:

6.

2. The protein cage according to claim 1, wherein the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO:

39.

3. A protein cage comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:

14.

4. The protein cage according to claim 3, wherein the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO:

40.

5. A protein cage comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 17; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16 or SEQ ID NO:

18.

6. The protein cage according to claim 5, wherein the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO:

41.

7. A protein cage comprising or consisting of a first polypeptide and a second polypeptide, wherein the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20; and the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO:

19.

8. The protein cage according to claim 7, wherein the second polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO:

42.

9. A protein cage comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 23; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22 or SEQ ID NO:

24.

10. The protein cage according to claim 9, wherein the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO:

43.

11. A protein cage comprising or consisting of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 27 or SEQ ID NO: 29; and the second polypeptide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28 or SEQ ID NO:

30.

12. The protein cage according to claim 11, wherein the first polypeptide is a species that falls within a genus sequence that comprises SEQ ID NO:

44.

13. The protein cage according to any one of claims 1 − 12, wherein a) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2; b) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4; c) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6; d) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8; e) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 9; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10; f) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 11; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12;g) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 13; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 14; h) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16; i) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18; j) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 20; k) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22; l) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 23; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 24; m) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 26; n) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28; o) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 29; and thesecond polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 30; p) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 31; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 32; q) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 33; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 34; r) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 35; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 36; s) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

38.

14. The protein cage according to claim 13, wherein 1) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2; 2) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28; 3) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8; 4) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3; and thesecond polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4; 5) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18; 6) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 29; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 30; 7) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 26; 8) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 20; or 9) the first polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 23; and the second polypeptide comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

24.

15. The protein cage according to any one of claims 1 − 14, wherein the first polypeptide or the second polypeptide comprises a histidine tag at or near its N-terminus or its C-terminus.

16. A composition comprising or consisting essentially of one or more protein cages according to any one of claims 1 − 15 and a pharmaceutically acceptable carrier.

17. A kit comprising one or more protein cages according to any one of claims 1 − 15 or the composition according to claim 16, packaged together with a buffer, a reagent, a device, or a container for use with the one or more one or more protein cages.

18. Use of one or more protein cages according to any one of claims 1 − 15 as a carrier molecule for a given compound of interest (e.g., a given therapeutic agent of interest or a given diagnostic agent of interest).

19. The use according to claim 18, wherein the given compound of interest is carried within the interior of the one or more protein cages or on the surface of the one or more protein cages.

20. The use according to claim 18 or claim 19, wherein the given compound of interest is non- covalently bound to the first polypeptide and / or the second polypeptide.

21. The use according to claim 18 or claim 19, wherein the given compound of interest is covalently bound to the first polypeptide and / or the second polypeptide.

22. The use according to claim 18 or claim 19, wherein the given compound of interest is recombinantly fused to the first polypeptide and / or the second polypeptide.