CN117203225A

CN117203225A - Artificial TRAP cage, use thereof and preparation method thereof

Info

Publication number: CN117203225A
Application number: CN202280030041.8A
Authority: CN
Inventors: 乔纳森·赫德尔; 阿图尔·比拉; 东佑翼; 伊扎贝拉·斯图普卡; 卡罗利娜·马吉斯特基维奇
Original assignee: Uniwersytet Jagiellonski
Current assignee: Uniwersytet Jagiellonski
Priority date: 2021-02-24
Filing date: 2022-02-24
Publication date: 2023-12-08
Also published as: PL437115A1; CN117203224A; CN117157311A

Abstract

The present invention provides an artificial TRAP cage comprising a selected number of TRAP loops held in place by a cross-linking agent, wherein the cross-linking agent is selected according to its particular characteristics, whereby the cage is programmably opened or kept closed as required under particular conditions.

Description

Artificial TRAP cage, use thereof and preparation method thereof

Technical Field

The present invention belongs to the field of biochemistry. To an artificial protein cage called a "TRAP cage" comprising a selected number of TRAP loops held in place by molecular cross-linking agents, wherein the cross-linking agents are selected according to their specific characteristics, whereby the cage can be programmably opened or kept closed as required under specific conditions.

Background

Protein complexes in nature appear as important and highly complex biological nanomachines and nanostructures. Large protein complexes in nature are typically composed of many individual proteins held together by non-covalent interactions (i.e., hydrogen bonding, hydrophobic packing). This is particularly evident in protein cages such as capsids, where multiple copies of the same protein subunit are held together in this way. In synthetic structural biology, the ability to design and construct artificial protein assemblies may be useful, potentially allowing the introduction of properties and capabilities not found in nature. For this reason, a new method of joining individual proteins together in a defined manner is needed.

Recently, the present inventors have studied this possibility of using TRAP (TRP RNA-binding attenuated protein) from geobacillus stearothermophilus (Geobacillus stearothermophilus) as a nanostructural block. The TRAP employs an oligomeric ring structure of 11 subunits in its natural state and, along with many other cyclic proteins, has proven to be a useful biological nanobuilding block.

In view of the shortcomings of the known methods, the inventors have sought to find other methods of ligating protein subunits. Although there are some disclosures of binding two or other amounts of proteins via their cysteine SH groups, the inventors focused on this area, considering the use of gold as a "suturing" reagent.

The incorporation of gold particles into nanostructures or the provision of nanoparticles as nanoclusters using gold compounds is well described in the literature and patent documents, which are prior art for the present invention, protein cages for a variety of applications, in particular as target molecules in delivery systems. For example, international application No. PCT/KR2013/004454 describes a method for preparing a hyaluronic acid-gold nanoparticle/protein complex, which can be used as a liver-targeted drug delivery system, by surface-modifying gold nanoparticles having excellent stability in vivo with hyaluronic acid having biocompatibility, biodegradability and liver tissue-specific delivery properties, and binding a protein drug for treating liver diseases to the unmodified surface of the gold nanoparticles.

U.S. patent application Ser. No. 10/142,838 discloses the incorporation of noble metal atoms such as gold into caged proteins such as apoferritin by modifying the internal structure of the caged proteins to form noble metal-recombinant caged protein complexes suitable for use in a variety of microstructures.

International application No. PCT/US2011/034190 discloses antibody-nanoparticle conjugates comprising two or more nanoparticles (such as gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof) directly linked to an antibody or fragment thereof via a metal-thiol bond.

Another example is U.S. patent application No. US14/849,379, which discloses a recombinant self-assembling protein comprising a targeting peptide fused to a self-assembling protein and a self-assembled gold ion reducing peptide.

International application No. PCT/IB2018/056150 discloses a method for conjugating biomolecules comprising one or more free thiol groups to form a biomolecular complex comprising a reaction of linking biomolecules using a gold donor reagent, wherein a-S-Au-S-bond is formed, characterised in that the Jin Gongti reagent is halogen (triarylphosphine) gold (I). Furthermore, the use of halogen (triarylphosphine) gold (I) molecules as gold donor reagents in biomolecular complex formation methods has been disclosed.

Publication of Malay et al: "An ultra-stable gold-coordinated protein cage displaying reversible assembly"; nature 569 (2019): 438-442, which is incorporated herein by reference, discloses TRAP cages bound together by a single Jin Yuanzi (i.e., au (I) ion) atom that forms a linear coordination bond between the sulfhydryl groups of a cysteine pair.

In the present invention, a new approach is achieved-the TRAP ring forming the artificial TRAP cage is held in place not by S-Au-S-bonds, but by a cross-linking agent not made of metal atoms, the cross-linking agent being selected according to its specific characteristics, whereby under specific conditions the cage is programmable to open or remain closed as required. This method allows for controlling the assembly and disassembly of the capsid-like protein complex, which is innovative from the point of view of the prior art. A novel method of using metal ions as cross-linking agents is also presented which also allows for controlled assembly and disassembly of capsid-like protein complexes, which is innovative in the prior art.

Disclosure of Invention

The subject of the first aspect of the invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by molecular cross-linking agents, wherein the cross-linking agents are molecules rather than individual atoms, e.g. not metal atoms, the cross-linking agents being selected according to their specific characteristics, whereby the cage can be programmably opened or kept closed as required under specific conditions.

Preferably the specific conditions correspond to specific cleavage characteristics of the molecular crosslinker.

The use of molecular cross-linking agents, rather than monoatomic cross-linking agents, provides a greater degree of design choice and flexibility in designing the cage. These allow for enhanced programmability and control of the cleavage characteristics of the crosslinker. The range of alternative molecular cross-linking agents is much broader than (metal) atom cross-linking agents.

Heretofore, all known TRAP cage syntheses utilized atomic gold or mercury crosslinking, and no work has been considered to use molecular crosslinkers. The larger size of the molecules and the different chemistries they require to crosslink the TRAP ring means that it is not obvious that the molecular crosslinking agent is able to perform a crosslinking function to form ordered cages, as all previous teachings focus on gold atoms.

Preferably the specific cleavage characteristics of the molecular crosslinker are selected from the group comprising:

(i) A reduction-resistant/insensitive molecular crosslinker, whereby the cage remains closed under reducing conditions;

(ii) A reduction responsive/sensitive molecular crosslinker whereby the cage opens under reducing conditions; and

(iii) The molecular cross-linking agent may be photoactivated whereby the cage opens upon exposure to light.

Preferably, the reduction-resistant/insensitive molecular crosslinker may be selected from the group consisting of: bismaleimidohexane (BMH), dibromodioxane and dibromoxylene.

Preferably the reduction responsive/sensitive molecular crosslinker may be selected from the group consisting of: a group of dithiobismaleimide ethanes (DTMEs).

Preferably the reduction responsive/sensitive molecular crosslinker may be selected from the group comprising: dihalomethylbenzenes and derivatives thereof, including 1, 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2, 4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

Preferably the molecular cross-linking agent is a homobifunctional molecular moiety and derivatives thereof.

Preferably the homobifunctional molecular crosslinker is Bismaleimidohexane (BMH).

Preferably the cage is resistant/insensitive to reducing conditions.

Preferably the homobifunctional molecular crosslinker is dithiobismaleimide ethane (DTME).

Preferably the cage is responsive/sensitive to reducing conditions.

Preferably, the molecular crosslinker is dihalomethylbenzene and its derivatives.

Preferably, the molecular crosslinker is selected from the group comprising: 1, 2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

Preferably the molecular crosslinker is photo-labile by exposure to UV light.

Preferably, wherein the number of said TRAP loops in the TRAP cage is between 6 and 60, preferably between 7 and 55, preferably between 8 and 50, preferably between 9 and 45, preferably between 10 and 40, preferably between 11 and 35, preferably between 12 and 34, preferably between 13 and 33, preferably between 14 and 32, preferably between 15 and 31, preferably between 16 and 30, preferably between 17 and 29, preferably between 18 and 28, preferably between 19 and 27, preferably between 20 and 26. Preferably, the number of TRAP loops in the TRAP cage is less than 40, preferably less than 35, more preferably less than 30. Preferably, the number of TRAP loops in the TRAP cage is greater than 6, preferably greater than 10, preferably greater than 15, preferably greater than 20.

Preferably, the number of TRAP loops in the TRAP cage is between 12 and 24.

Preferably, the number of TRAP loops in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP loops in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP loops in the TRAP cage is about 20, preferably 20.

Preferably, the cages according to the invention comprise a mixture of different programmable molecular cross-linking agents.

Preferably, a cage according to the present invention encapsulates cargo, which can be programmed to deliver the cargo at a specific timed and desired location.

Preferably, the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group comprising: K35C, R S and K35C/R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation. Preferably, the cross-linking agent comprises dithiobismaleimide ethane (DTME), and preferably, the artificial TRAP cage protein is modified to include a K35C/R64S mutation.

Preferably, the cage according to the invention is hollow. Preferably, the cage according to the invention is approximately spherical in shape, preferably hollow spherical. The cage herein is a hollow shape that generally approximates a hollow sphere. These approximate the shape obtained when the TRAP ring is placed on the apex or corner of a regular convex polyhedron and then connected together. In any shape, the vertices and edges are imaginary, i.e. there is no actual physical polyhedron on which the TRAP rings are placed, rather the shape of the TRAP cage is placed like a ring on the vertices or corners of a positive convex polyhedron, and then joined together.

Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is raised to normal room temperature or above human/animal body temperature, preferably between 0 and 100 ℃, preferably between 15 and 75 ℃, preferably up to 75 ℃, preferably 75 ℃ and below.

Preferably, the TRAP cage is stable in a non-neutral pH, preferably above pH 7 and below pH 7, preferably between pH 3 and 11, preferably between pH 4 and 10, preferably between pH 5 and 9.

Preferably, the TRAP cage is stable in a chaotropic agent (an agent that breaks down hydrogen bonds in solution, which would break down or denature the structure of proteins or macromolecules) or a surfactant that would otherwise be expected to break down or denature the structure of proteins or macromolecules. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea and urea. Preferably, the TRAP cage is stable in GndHCl up to 4M. Preferably, the TRAP cage is stable in urea up to at least 7M. Preferably, the TRAP cage is stable in up to 15% SDS. These reagents can be used to test the stability of the cages described herein under standard conditions known to those skilled in the art to demonstrate the stability.

The cages described herein exhibit unexpected stability under these conditions, providing a more stable TRAP cage than previously demonstrated.

The subject of the invention is also the use of a cage as defined above according to the invention for delivering goods and reaching a desired location in a controlled time.

The subject of the invention is also the use of any one or more of the group comprising homobifunctional molecule moieties and dihalomethylbenzenes and derivatives thereof as programmable cross-linkers in the construction of programmable TRAP cages.

The subject of the second aspect of the invention is also a method for preparing an artificial TRAP cage, said method comprising:

(i) Obtaining the TRAP loop unit by expressing the TRAP loop unit in a suitable expression system and purifying the unit from the expression system;

(ii) Conjugating said TRAP ring units via at least one free sulfhydryl bond with a programmable molecular cross-linking agent, wherein said cross-linking agent is selected according to its specific characteristics;

(iii) Forming the TRAP cage; and

(iv) Purifying and isolating the TRAP cage.

Preferably, step (ii) comprises first conjugating the TRAP ring units by at least one metal cross-linker, preferably an atomic metal cross-linker. Step (ii) then comprises replacing the metal crosslinker with a molecular crosslinker. Molecular crosslinkers can exchange metal atoms without changing the orientation of the rings in the cage. Preferably, the metal is gold. When the cross-linking agent is a photocleavable linking agent, step (ii) of the modification is preferably applied, preferably wherein the cross-linking agent is bromoxylene or dibromodiphenyl methane (bisbromobimane).

Preferably, the programmable crosslinker may be selected from the group comprising:

(i) A reduction resistant/insensitive linker whereby the cage remains closed under reducing conditions;

(ii) A reduction responsive/sensitive linker whereby the cage opens under reducing conditions; and

(iii) The linker may be photoactivated, whereby the cage opens upon exposure to light.

Preferably the reduction-resistant/insensitive molecular crosslinker may be selected from the group consisting of: a group of Bismaleimidohexanes (BMH) and dibromoxylenes.

Preferably the reduction responsive/sensitive molecular crosslinker may be selected from the group comprising: dihalomethylbenzenes and derivatives thereof, including 1, 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2, 4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB). Preferably, the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group comprising: K35C, R S and K35C/R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation.

Preferably, the cross-linking agent comprises dithiobismaleimide ethane (DTME), and preferably, the artificial TRAP cage protein is modified to include a K35C/R64S mutation.

Preferably, the number of TRAP loops in the TRAP cage is between 12 and 24.

Preferably, step (ii) comprises conjugation with a mixture of different programmable cross-linking agents.

Preferably, the reduction-resistant/insensitive molecular crosslinker may be selected from the group consisting of: a group of Bismaleimidohexanes (BMH) and dibromoxylenes.

Preferably, the reduction responsive/sensitive molecular crosslinker may be selected from the group consisting of: a group of dithiobismaleimide ethanes (DTMEs).

Preferably, the photo-activated molecular cross-linking agent may be selected from the group comprising: dihalomethylbenzenes and derivatives thereof, including 1, 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2, 4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

If cysteine is not present in the biomolecule or is present but not available for reaction, a-SH group, preferably as a cysteine group, may be introduced into the biomolecule.

The introduction of cysteine may be performed by any method known in the art. For example, but not limited to, cysteine introduction is performed by methods known in the art, such as commercial gene synthesis or PCR-based site-directed mutagenesis using modified DNA primers. The above methods are known to those skilled in the art and ready-to-use kits are commercially available.

the-SH moiety may also be introduced into the biomolecules by modifying other amino acids in the biomolecules (i.e., by site-directed mutagenesis or by solid phase peptide synthesis).

The subject of the invention is also the TRAP cage produced by this method. These cages may have any of the features or properties described above with respect to the first aspect of the invention, or any other feature or property described herein.

The subject matter of a third aspect of the invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by at least one cross-linking agent comprising a metal. Preferably the cross-linking agent comprises only metal. Preferably the metal is a metal ion, preferably a single type of metal.

Preferably, the metal cross-linking agent is selected for specific characteristics, whereby the cage can be programmably opened or kept closed as desired under the specific conditions.

Preferably, the metal is selected from the group comprising Ag (I), cd (II), zn (II) and Co (II). The metal may be a derivative of these metals.

Preferably, the metal is a d10 metal having a non-linear coordination geometry or shell. Preferably, the d10 metal with a nonlinear coordination geometry or shell is Zn (II) or Co (II).

Preferably, the metal is a d10 metal having a dual ligand linear coordination geometry or shell. Preferably, the d10 metal with a nonlinear coordination geometry or shell is Ag (I) or Cd (II).

Preferably, the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group comprising: K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S C/R64S, S H/R64S, S C/K35H, S33H/K35H, S C/K35C, S H/K35C.

Preferably, the artificial TRAP cage protein is modified to include a K35C/S33H mutation or a K35H/S33H mutation.

Preferably, the cross-linking agent comprises silver (Ag (I)), and preferably, the artificial TRAP cage protein is modified to include a K35C/R64S mutation.

Preferably, the cross-linking agent comprises cadmium (Cd (II)), and preferably, the artificial TRAP cage protein is modified to include a K35C/R64S mutation.

Preferably, the cross-linking agent comprises cobalt (Co (II)), and preferably, the artificial TRAP cage protein is modified to comprise S33H/K35C or S33H/K35H mutations.

Preferably, the cross-linking agent comprises zinc (Zn (II)), and preferably, the artificial TRAP cage protein is modified to comprise S33H/K35C or S33H/K35H mutations.

Preferably, the number of TRAP loops in the TRAP cage is between 12 and 24.

Preferably, the cage according to the invention comprises a mixture of different cross-linking agents.

Preferably the cage according to the invention is hollow. Preferably, the cage according to the invention is approximately spherical in shape, preferably a hollow sphere. The cage herein is a hollow shape that generally approximates a hollow sphere. These approximate the shape obtained when the TRAP ring is placed on the apex or corner of a regular convex polyhedron and then connected together.

Preferably, the TRAP is stable in chaotropic agents (agents that disrupt or denature the structure of proteins or macromolecules in solution) or surfactants that are otherwise expected to disrupt or denature the structure of proteins or macromolecules. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea and urea. Preferably, the TRAP cage is stable in GndHCl up to 4M. Preferably, the TRAP cage is stable in urea up to at least 7M. Preferably, the TRAP cage is stable in up to 15% SDS. These reagents can be used to test the stability of the cages described herein under standard conditions known to those skilled in the art to demonstrate the stability.

The invention also relates to the use of any one or more of the metals Ag (I), cd (II), zn (II) and Co (II) and derivatives thereof as cross-linking agents in the construction of TRAP cages.

The subject of the fourth aspect of the invention is also a method for preparing an artificial TRAP cage, said method comprising:

(ii) Conjugating said TRAP ring units via at least one free sulfhydryl bond with a metal cross-linking agent, wherein said cross-linking agent is selected according to its specific characteristics;

(iii) Forming the TRAP cage; and

(iv) Purifying and isolating the TRAP cage.

Preferably the metal is a d10 metal having a non-linear coordination geometry or shell. Preferably, the d10 metal with a nonlinear coordination geometry or shell is Zn (II) or Co (II).

Preferably, the number of TRAP loops in the TRAP cage is between 12 and 24.

Preferably, the method is performed partially or wholly in HEPES buffer. Preferably, the process is carried out at about pH 8.

The invention is also directed to the use of any TRAP cage described herein as a medicament.

The invention is also a method of treating a patient comprising administering to the patient a TRAP cage as described herein.

The invention is also directed to the use of any TRAP cage described herein in the treatment of a disease in a patient.

The subject of the invention is also the TRAP cage produced by this method. These cages may have any of the features or properties described above with respect to the earlier aspects of the invention, or any other features or properties described herein.

A further aspect of the invention is directed to an artificial TRAP cage comprising a selected number of TRAP loops held in place by at least one cross-linking agent. These cages may have any of the features or properties described above with respect to the earlier aspects of the invention, or any other features or properties described herein.

Preferably the cross-linking agent is a metal. Preferably the cross-linking agent comprises only metal. Preferably the metal is a metal ion, preferably a single type of metal.

Preferably, the metal is selected from the group comprising Au (I), ag (I), cd (II), zn (II) and Co (II). The metal may be a derivative of these metals.

Preferably, the cross-linking agent comprises gold (Au (I)), and preferably, the artificial TRAP cage protein is modified to comprise S33C/R64S.

Preferably the specific cleavage characteristics of the molecular crosslinker are selected from the group comprising: (i) A reduction-resistant/insensitive molecular crosslinker, whereby the cage remains closed under reducing conditions; (ii) A reduction responsive/sensitive molecular crosslinker whereby the cage opens under reducing conditions; and (iii) a photoactivatable molecular crosslinker, whereby the cage opens upon exposure to light.

Preferably the molecular cross-linking agent is a homobifunctional molecular moiety and derivatives thereof. Preferably the homobifunctional molecular crosslinker is Bismaleimidohexane (BMH).

Preferably the cage is resistant/insensitive to reducing conditions. Preferably the homobifunctional molecular crosslinker is dithiobismaleimide ethane (DTME).

Preferably the cage is responsive/sensitive to reducing conditions. Preferably, the molecular crosslinker is dihalomethylbenzene and its derivatives.

Preferably the molecular crosslinker is selected from the group comprising: 1, 2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

Preferably the molecular crosslinker is photo-labile by exposure to UV light.

Preferably, the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group comprising: K35C, R S and K35C/R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation.

Preferably, the number of TRAP loops in the TRAP cage is between 12 and 24.

Preferably, the cages according to the invention encapsulate the cargo, which can be delivered at specific timed and desired locations.

The present invention is also a TRAP cage comprising a protein modified to comprise any one or more of the following mutations selected from the group comprising: K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S C/R64S, S H/R64S, S C/K35H, S33H/K35H, S C/K35C, S H/K35C.

The present invention is also a TRAP protein modified to include any one or more of the following mutations selected from the group consisting of: K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S C/R64S, S H/R64S, S C/K35H, S33H/K35H, S C/K35C, S H/K35C.

Detailed Description

As used herein, "TRAP protein" refers to tryptophan RNA-binding attenuated protein, a bacterial protein. The protein may be isolated, for example, from wild-type Geobacillus stearothermophilus or other such bacteria. Such proteins may be isolated from a variety of bacteria, but TRAP proteins that function as described herein may be isolated from bacteria such as: bacillus lignin (Alkalihalobacillus ligniniphilus), bacillus isopolyticus (Anaerobacillus isosaccharinicus), bacillus thermolysis protein anaerobic Bacillus (Anoxybacillus caldiproteolyticus), bacillus thermoanaerobacter (Anoxybacillus calidus), bacillus pumilus (Anoxybacillus calidus), bacillus caldovelox (Anoxybacillus calidus), bacillus stearothermophilus (Anoxybacillus calidus), bacillus macerans (Anoxybacillus calidus), bacillus sinusoidal (Anoxybacillus calidus), bacillus FJAT-Anoxybacillus calidus (Bacillus sp. FJAT-Anoxybacillus calidus), bacillus HD4P25 (Bacillus sp. HD4P25) HMF5848 of Bacillus (Bacillus sp.HMF5848), PS06 of Bacillus (Bacillus sp.PS06), REN16 of Bacillus (Bacillus sp.REN 16), SA1-12 of Bacillus (Bacillus sp.SA1-12), V3-13 of Bacillus (Bacillus sp.V3-13), bacillus Anoxybacillus calidus (Anoxybacillus calidus), bacillus strain, and process for producing the same Bacillus validli (Anoxybacillus calidus), sea ditch of Yapu (Anoxybacillus calidus), bacillus el5237 card Anoxybacillus calidus Bacillus natto (Anoxybacillus calidus) Paddy field card Anoxybacillus calidus Bacillus (Anoxybacillus calidus), micro5237 Bacillus (Anoxybacillus calidus), anoxybacillus calidus, bacillus terreus genome sp.3 (Anoxybacillus calidus sp.3), 46C-IIa (Geobacillus sp.46C-IIa), geobacillus stearothermophilus (Geobacillus stearothermophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), geobacillus thermodenitrificans NG80-2 (Geobacillus thermodenitrificans NG-2), geobacillus dactylothermophilus (Halobacillus dabanensis), geobacillus halophilus (Halobacillus halophilus), geobacillus halophilus (Halobacillus halophilus), geobacillus delphinii (Jeotgalibacillus proteolyticus), litchfieldia alkalitelluris, litchfieldia salsa, geobacillus (Mesobacillus harenae), geobacillus parabacillus (Metabacillus), geobacillus (673), geobacillus seashore deposit parabacillus (Oceanobacillus limi), geobacillus, katsurensis (Oceaco-product (Ocalamus) of Geobacillus), organii (Orthosporum kan) and Orthosporium (Ornithinibacillus scapharcae), orthosporium (Orthosporium) and Orthosporium (Orthosporium) co-product (Orthosporium) of Orthosporium, orthosporium (Orthosporium) and Orthosporium (Orthosporium) of co-products, bacillus calantarcticus (Parageobacillus thermantarcticus), bacillus calantarcticus (Parageobacillus thermoglucosidasius), bacillus caldarius (Parageobacillus thermoglucosidasius), bacillus pumilus (Paucisalibacillus globulus), bacillus pumilus EB02 (Pauci alibacillus sp.EB02), brewsteria deep sea (Priestia abyssalis), brewsteria endophyte (Priestia endophytica), lespederia filiformis (Priestia filamentosa), brewsteria koreana (Priestia koreensis), lespederia megateria (Priestia megaterium), bacillus glacialis (Psychrobacillus glaciei), bacillus salicinii (Salinibacillus xinjiangensis), sutcliffiella cohnii, and Thermomyces alpine (Thermolongibacillus altinsuensis).

The Trp RNA-binding attenuated protein is a bacterial circular homo11-mer (see A.A.Antson, J.Otridge, A.M.Brzozowski, E.J.Dodson, G.G.Dodson, K.S.Wilson, T.Smith, M.Yang, T.Kurecki, P.Gollnick, incorporated herein by reference), and the structure of the Trp RNA-binding attenuated protein can be found in the literature (Nature 374,693-700 (1995), incorporated herein by reference).

Suitably, the protein used herein is a modified form of wild-type TRAP isolated from bacillus stearothermophilus. As shown in table 1:

TABLE 1

The gene sequences of wild-type TRAP bacillus stearothermophilus are shown in table 2:

TABLE 2

Preferably, the preparation of the protein is performed by expressing the biomolecules in a suitable expression system and purifying the expression product. Preferably a modified form having the above-described wild-type TRAP Bacillus stearothermophilus gene sequence.

TRAP proteins form loops, referred to herein as "TRAP loops," and loops are the natural state of TRAP proteins. Typically, as demonstrated herein for the case of the geobacillus stearothermophilus protein, the TRAP monomer protein spontaneously assembles into a loop (toroid) or ring (ring) prepared from the monomer.

TRAP cages are formed only under specific conditions, e.g., as demonstrated herein, in the presence of cysteine, cross-linking can result in the assembly of the loops into a cage. For example, as demonstrated herein, in the presence of cysteine at position 35 (the result of the K35C mutation), these will form.

Reference herein to a "TRAP loop" is synonymous with a TRAP member, subunit of a TRAP cage complex, or TRAP monomer assembly. Reference herein to a "analog" of a particular protein or nucleotide sequence refers to a protein or nucleic acid sequence that has sufficient identity or homology to the protein or nucleotide sequence to be able to perform a particular function (e.g., forming a TRAP cage under the conditions described herein) or to encode a protein able to perform a particular function (e.g., forming a TRAP cage under the conditions described herein).

To determine the percent identity/homology of two sequences, the sequences in question and the reference sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment purposes, and non-homologous sequences may be omitted for comparison purposes). A sequence may be identified as an analog of a particular sequence when the sequence has amino acids or nucleotides of preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% of the reference sequence related length. When comparing amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions, a molecule is identical at that position if that position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap.

Suitably, the TRAP protein comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% identity or homology to the amino acid sequence of SEQ ID NO. 1. Preferably, the TRAP protein comprises an amino acid sequence having at least 85% identity or homology with the amino acid sequence of SEQ ID NO. 1.

References herein to "TRAP cage" refer to an assembled protein complex formed from multiple biomolecules where multiple TRAP protein loops form the complex. The TRAP protein loops may be linked together by a cross-linking agent, in this case a molecular cross-linking agent. "complex", "assembly", "aggregate" are used interchangeably in the specification and refer to a superstructural structure constructed by reactions between biomolecules. The amount of units included in the complex depends on the nature of the biomolecule. More particularly, it depends on the amount of biomolecules and the amount of-SH groups present in the biomolecules. "TRAP cage" and "artificial TRAP cage" are used interchangeably herein.

TRAP proteins are suitable biomolecular models for use in the methods of the invention. This may be due to its high inherent stability, ring shape, lack of natural cysteine residues (easier control of the conjugation process) and available residues which can be changed to cysteines and the resulting cysteines are suitable for forming appropriate bonds in suitable chemical and steric environments.

References herein to "programmable" are intended to express that the TRAP cages of the present invention have properties that are imparted or designed such that they tend or are susceptible or susceptible to being exhibited in a particular and selected manner upon exposure to particular environmental conditions or stimuli.

Reference herein to "opening" is synonymous with TRAP cage breaking, leaking, crushing, fracturing or generally allowing cargo to escape from the interior of the cage.

Reference herein to "closing" is synonymous with the TRAP cage remaining intact, non-destructible, impermeable, or generally remaining the entire cage.

Reference herein to "bifunctional" refers to a molecular crosslinker having two functional groups, e.g., a molecule having two functional groups herein, wherein each cysteine thiol group has one functional group crosslinked to join the TRAP rings in the TRAP cage. Reference herein to "homobifunctional" refers to a bifunctional linker in which both groups are identical.

Preferably homobifunctional linkers include Bismaleimidohexane (BMH), dithiobismaleimidoethane (DTME), bis-halomethylbenzene and its derivatives, 2-bis-bromomethyl-3-nitrobenzene (BBN), dibromoxylene, and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

"Unit", "subunit", "molecule", "biomolecule" and "monomer" are used interchangeably throughout the specification to refer to one molecule linked to another molecule to form a complex.

A "molecular crosslinker" is a molecule that connects a unit, subunit, molecule, biomolecule, or monomer to the same other instance by forming one or more chemical bonds. The molecular crosslinker is not a monoatomic crosslinker, which is a different entity.

Reference herein to a "reduction-resistant/insensitive molecular crosslinker" refers to a crosslinker that is not cleaved by a reduction reaction, such as is typically seen when disulfide bonds are cleaved by a reducing agent. These cross-linking agents are stable under conditions that would result in cleavage of the reduction-sensitive bonds. These Bismaleimidohexanes (BMH) and dibromoxylenes.

Reference herein to a "reduction reactive/sensitive molecular crosslinker" refers to a crosslinker that is cleaved by a reduction reaction, such as is typically seen when disulfide bonds are cleaved by a reducing agent. These crosslinking agents are unstable under conditions that would result in cleavage of the reduction-sensitive bonds. These include dithiobismaleimide ethane (DTME).

Reference herein to a "photoactivatable molecular crosslinker" refers to a crosslinker that is photoreactive or sensitive to light, i.e., a crosslinker that will be cleaved upon exposure to light. The light may be UV or other light having a specific range of wavelengths. These include 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2, 4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

In order to achieve a programmable disassembly, rather than a simple triggerable disassembly, a protein cage has been proposed in which the building blocks are held together by simple cross-linking agents by easy chemistry, allowing easy interchangeability (Fig 1 a). The removal of such cages will depend on the cleavage characteristics of the crosslinker used. To test this possibility, TRAP (K35C/R64S) has been used and attempts have been made to link the TRAP ring with simple bifunctional molecular crosslinkers (dithiobismaleimidoethane (DTME) or Bismaleimidohexane (BMH)). Due to their thiol-specific reaction at neutral pH, they are assembled in gold-mediated (TRAP cage ^Au(I) ) Acts as Au (I) -like linkers. Since DTME contains cleavable disulfide bonds and BMH does not, when exposed to reducing agents, the resulting two cages (TRAP cages ^DTME And TRAP cage ^BMH ) With very different disassembly characteristics (fig. 1 a).

To obtain covalently crosslinked cages, TRAP (K35C/R64S) was mixed with DTME or BMH in aqueous buffer. Size Exclusion Chromatography (SEC) of the resulting reaction mixture in the presence of TRAP cage ^Au(I) A significant peak was shown at a similar elution volume, indicating successful cage formation with both crosslinkers (fig. 1). The separated fractions were further analyzed by SEC (fig. 1 c), dynamic Light Scattering (DLS) and negative-stained Transmission Electron Microscopy (TEM) (fig. 1d, fig. 2), yielding the result as expected of a monodisperse gabion structure with a diameter of about 25 nm. The assembly appears to be substantially complete within 60min (fig. 1 b), with a typical yield of about 20% for both cross-linked TRAP cages. No free cysteines were detected after the reaction. Further analysis of the obtained TRAP cage using size exclusion chromatography in combination with right angle and small angle light scattering (SEC-RALS/LALS) showed two particles The apparent average molecular weight of (2.2 MDa) indicates a 24-ring arrangement.

Determination of TRAP cage using frozen EM single particle reconstruction ^DTME And TRAP cage ^BMH Is a detailed structure of (a). Two types of cages have been obtainedAnd->Electron density map at resolution. These reveal that each structure consists of 24 TRAP loops arranged in two chiral forms, similar to the TRAP cage seen ^Au(I) (FIG. 1 e). The TRAP loop model was improved according to the figure, yielding a good fit. A more careful examination at the ring-ring interface found two significant electron densities bridging two adjacent subunits, which may correspond to bismaleimide crosslinkers (fig. 1 e). The cross-linker appears to bend into a horseshoe shape between the cysteine residues of the opposing subunits.

TRAP cage ^DTME And TRAP cage ^BMH Exhibit similarly high stability in response to elevated temperatures, chaotropic agents and surfactants. In particular, they did not show significant morphological changes after incubation for 10 minutes at 75 ℃ at pH ranging from 2-11 up to 4M gnd hcl, up to at least 7M urea and 7% SDS. However, TRAP cage ^DTME Is easily removed when the reducing agent tris (2-carboxyethyl) phosphine (TCEP) or Dithiothreitol (DTT) is added (fig. 2a, c). In contrast, TRAP cage ^BMH Unaffected (FIG. 2b, c). TRAP cages were also observed at high temperature, over a large pH range and in the presence of high concentrations of the cahortopic agent and surfactant ^DTME (FIGS. 2 d-g) and TRAP cage ^BMH Is high in stability. Further real-time studies of DTT-dependent disassembly were performed at the single cage level using a high-speed atomic force microscope (HSAFM, fig. 2 l). This indicates a TRAP cage ^BMH Has resistance to disassembly in the presence of DTT. In contrast, TRAP cage ^DTME Easy to disassemble under the same conditions, discrete patches of TRAP subunits appear to "peel" from the cage surface, ultimately resulting in a complete about 3min after the first loop separationOpening of the individual structures (fig. 2 d). TRAP cage ^DTME Is gradually disassembled and TRAP cage ^Au(I) Unlike, it shows a more coordinated disassembly on a shorter time scale.

In order to effectively track the stability of the cages to various thiol-containing reagents, a spectroscopic method was employed to monitor the removal of most of the cages from the solution in real time. In this system, two fluorescent proteins, mOrange2 and mCherry, are encapsulated as the donor and acceptor of the Forster Resonance Energy Transfer (FRET), respectively. These fluorescent proteins are genetically fused to the N-terminus of TRAP, which in the assembled configuration faces the interior of the cage. To avoid steric hindrance during cage formation, they are co-produced with unmodified TRAP to form a "splice" loop.

The resulting TRAP rings were then assembled into a cage structure using Au (I) or DTME (fig. 3 a). After purification using size exclusion chromatography, the isolated particles were analyzed by non-denaturing PAGE (FIG. 3 b) and TEM imaging (FIG. 3 c), confirming encapsulation of the guest in the monodisperse cavities of the ball cages. The presence of these two proteins in the restricted volume of the TRAP cage should be able to achieve efficient FRET (22, 23). Indeed, the TRAP cage co-packaging both guests, compared to the corresponding control sample comprising a cage encapsulating only mOrange2 or mCherry and mixed in solution ^Au(I) And TRAP cage ^DTME Shows a signal about 1.5 times higher in mCherry emission at 610nm (fig. 4a, b). However, for both types of cages, the addition of DTT to induce disassembly resulted in FRET subtraction, as observed in the resulting spectra, which was very similar to the control samples (fig. 4a, b). These results indicate that there is efficient energy transfer between the encapsulated objects and is eliminated by release, indicating that the FRET cage is suitable for monitoring the removal process of the TRAP cage.

Next, we measured TRAP cage after DTT addition ^Au(I) And TRAP cage ^DTME Detachment kinetics of both. The change in fluorescence intensity ratio at 568/610nm was used as an indicator of the time-dependent disassembly process (FIG. 4 c). For both cages, the increase in fluorescence reached the plateau about 5min after DTT addition, indicating complete cage removal and object release. However, the mechanism of this process It appears that the difference is different depending on the cage type. With TRAP cage ^Au(I) In contrast, TRAP cage ^DTME Exhibit S-shaped detachment behavior, indicating the multiple steps involved in the object release process. These results are in conjunction with the TRAP cage observed by HSAFM ^Au(I) Or TRAP cage ^DTME The respective co-disassembly is consistent with the progressive disassembly.

Protein cages that are capable of carrying cargo and that are removable in the presence of reducing agents have the potential for intracellular delivery. Ideal nanocarriers for this purpose should remain assembled under extracellular conditions, but disassemble to release their cargo when exposed to intracellular conditions. We assessed this possibility by monitoring the kinetics of cage disassembly in the presence of cysteine and glutathione as model thiols. For TRAP cage ^Au(I) FRET depletion was observed to increase with time after addition of cysteine, and tended to smooth at about 15min, indicating complete detachment (fig. 4d, black circles). In contrast, TRAP cages containing identical FRET pairs ^DTME Shows little change in fluorescence, indicating that the cage was not disassembled under these conditions (fig. 4d, red circle). The stability of this cysteine may be due to the slow kinetics of this free thiol-catalyzed disulfide cleavage, conversely, for the TRAP cage ^Au(I) The ligand exchange of (c) may be much faster. However, by using higher concentrations of free sulfhydryl compounds, TRAP cages ^DTME Still can dismantle: TRAP cage was observed when 50mM reduced glutathione was used ^DTME Although with TRAP cage ^Au(I) The dissociation rate was significantly slower compared (fig. 4e, red and black circles).

According to one aspect, the cages described herein can be used as medicaments. This may be used to treat a patient, such as including administering a cage as described herein to a patient, or a cage as described herein for treating a disease in a patient. In particular, this may be a cage designed to load cargo and to disintegrate in the presence of a reducing agent for intracellular delivery. These cages may be administered together with or in the presence of pharmaceutically acceptable carriers, adjuvants or excipients. The use of the cage as a medicament or as a cargo for treating a patient would be beneficial to said patient. For example, as a Drug Delivery System (DDS) -for active molecules (in particular biological macromolecules such as RNA, DNA, peptides and proteins). They offer advantages because biological macromolecules are often susceptible to destruction or digestion by conditions such as those found in vivo. The biomacromolecules are too large to diffuse out of the pores in TRAP, and as large proteins, TRAP cages can undergo significant changes without disrupting the overall structure. This means that it can be modified to capture therapeutic cargo and at the same time be externally modified to target therapeutic targets. Programmable linkers can be used that cleave under the desired conditions associated with reaching the site of action. For example, light may be irradiated on the target site to cleave open the photo-cleavable TRAP cage. If the TRAP penetrates the cells, those cells held together by the reducible linker will spontaneously open and release cargo when the cytoplasm of the cells is highly reduced. The cage may also be used in conjunction with or as a vaccine, wherein an antigen (i.e., peptide) intended to stimulate a T cell response is captured within the TRAP cage and then targets the T cells, followed by triggering opening.

Drawings

FIG. 1 molecular cross-linking mediated TRAP cage formation. a, schematic representation of the crosslinking reaction with dithiobismaleimide ethane (DTME) or bismaleimide hexane (BMH). The TRAP (K35C/R64S) ring (shown in the left figure) and cysteine (shown in the outside as circles) are covalently linked to each other by a reaction between cysteine and a bismaleimide compound (line above the first arrow, detailed chemical structure below) to form a cage-like structure. Addition of Dithiothreitol (DTT) results in a DTME-mediated cage (TRAP cage) ^DTME Roof) is disassembled, but for cages assembled with BMH (TRAP cages) ^BMH Bottom) has no effect. b, TRAP cage ^DTME And TRAP cage ^BMH The non-denaturing PAGE gel formed and the black arrows mark the height of the cages formed. c, purified TRAP cage ^DTME (light grey line) and TRAP cage ^BMH Size exclusion chromatography (grey line). Providing TRAP cage ^Au(I) As a control. mAu, milliabsorbance unit d, TRAP cage ^DTME (right) and TRAP cage ^BMH Transmission Electron Microscope (TEM) image (left). Scale bar, 50nm. e, TRAP cage ^DTME The density maps of the frozen electron microscopes in the left-hand (top) and right-hand (bottom) form, respectively, are accurate toAnd->Is a single-layer structure. The inset shows an enlarged image of the ring-ring interface with the fitted crosslinker models of DTME (middle) and BMH (right) highlighted in side view (top) and top view (bottom).

FIG. 2 stability redox response of crosslinked TRAP cage a, b TRAP cage in the presence of DTT and tris (2-carboxyethyl) phosphine (TCEP) ^DTME (a) And TRAP cage ^BMH (b) Non-denaturing PAGE analysis of (C). TRAP cages exhibited a distinct band running between 1048 and 1236 kDa. C=trap cage ^DTME (a) And TRAP cage ^BMH (b) A. The invention relates to a method for producing a fibre-reinforced plastic composite M = molecular weight marker. c, TEM image shows TRAP cage treated with 0.1mM (left) and 1mM (middle) TCEP ^DTME And TRAP cage after treatment with 10mM TCEP (right) ^BMH . Scale bar, 50nm. d-g TRAP cage ^DTME Is stable. d, non-denaturing PAGE shows TRAP cage ^DTME Thermal stability at the indicated incubation times and temperatures. The images below the gel show that the TRAP cage retains its structure after incubation for 10min at 95 ℃, scale bar, 100nm, e, non-denaturing PAGE shows the effect of pH on stability: the cages were stabilized at pH 3-11 using non-denaturing PAGE. The image below the gel is a TEM image of the sample after incubation at the pH indicated. Scale bar, 100nm. f, evaluation of TRAP cage Using non-denaturing PAGE ^DTME Shows that in the presence of guanidine hydrochloride (GdnHCl), urea and g, SDS, the structure is unaffected within the test range. TEM images (bottom), scale bar, 100nm were obtained after incubation of the cages with 4M GndHCl. h-kTRAP cage ^BMH Stability to TRAP-cage ^DTME The results (panels d-g) were identical (except for the use of TRAP cages ^BMH Outside of (c). Black arrows indicate the location of the complete TRAP cage on the gel. l, frames from HSAFM films showed addition of 4mM DTT to TRAP cage ^DTME Is effective in (1). The time after DTT addition is shown.

FIG. 3 is a schematic diagram of TRAP cage a loaded with fluorescent protein using FRET. The spliced TRAP ring fused at the N-terminus with mCherry (black cylinder) or mOrange2 (gray cylinder) is mixed together with DTME or triphenylphosphine monosulfate (TPPMS) -Au (I) -Cl. b, non-denaturing PAGE shows the fluorescence properties of the purified TRAP cages associated with fluorescent cargo. Gels were observed using an fast blue (InstantBuue) protein stain (right) and using fluorescence excited at 532nm and emitted at 610nm (left). Note that the exact location of the prominent band corresponds to the TRAP cage operating at approximately 1028-1236kDa, with slight variation depending on the presence/absence of cargo and the nature of the cross-linker used. c, TEM image: empty (left) TRAP and fluorescent protein filled TRAP (right), assembled using Au (I) (top) or DTME (bottom). Scale bar, 50nm.

Fig. 4 object release. a. b, TRAP cage loaded with mOrange2 and mCherry before and after addition of 10mM DTT ^Au(I) (a) And TRAP cage ^DTME (b) Normalized emission spectrum at 510nm excitation. The emission peak of mOrange2 was 618 nm and the emission peak of mCherry was 610nm. The additional lines represent spectra of cages loaded with only the mOrange2 or mCherry proteins, respectively, mixed together immediately prior to measurement in the absence or presence of DTT, respectively. c, d, e, TRAP cage after addition of 10mM DTT (c), 2.5mM cysteine (Cys) (d) or 50mM Glutathione (GSH) (e) ^Au(I) (black circles) and TRAP cage ^DTME (grey circles) disassembly over time. 100% leakage represents the highest donor intensity after 10mM DTT treatment for 10min after each experiment.

Figure 5 TRAP cages with different metal linkers. a. Non-denaturing PAGE analysis of TRAP cages assembled using Ag (I) or Cd (II); TRAP cage ^Ag(I) A TEM image of (a); TRAP cage ^Cd(I) A TEM image of (a); d. non-denaturing PAGE analysis of TRAP cages assembled using Co (II) or Zn (II); e.TRAP cage ^Co(II) A TEM image of (a); TRAP cage ^Zn(II) A TEM image of (a); samples were run on 3-12% non-denaturing Bis-Tris acrylamide gels for a.and d.samples. These gels were visualized by coomassie blue staining. For all TEM images-scale bar, 100nm.

FIG. 6 non-denaturing PAGE of template reactions; m-labelling; an R-TRAP ring; C-TRAP cage control; C+T-TRAP cage+10 mM TCEP showed cage disassembly due to the presence of Au (I); d = TRAP ring + DBX-cageless assembly; DC-TRAP cage+DBX; DCT-TRAP cage+DBX+10 mM TCEP-cage was still present, indicating successful exchange reaction; b.1, 3-dibromoxylene structure; dls shows about the size-24 nm of DBX TRAP cage; d. TEM image of purified DBX TRAP cage after treatment with 10mM DTT, scale bar, 100nm; frozen EM structures of left-handed (left) and right-handed (right) structures of dbx cross-linked cages; au (I) -induced wire frame models of cage (left) and DBX cage (right), amplifying ring-to-ring connectivity properties g.1, 2-dibromomethyl-3-nitrobenzene (BBN) structure; h. SDS PAGE before and after reaction with BBN template; m-tag, R-TRAP ring, C-Au (I) -induced TRAP cage, BBN-TRAP cage after mixing with BBN-additional bands appear, indicating the presence of covalently bound TRAP dimer; i. TEM images of purified BBN TRAP cages, scale bar, 100nm (left), 50nm (right), j. Native PAGE shows j. Dependency of photocleavage on concentration of different quenchers (DTT) present after 10min UV irradiation, k. Different time points after starting UV irradiation in the presence of 10mM DTT; for both gels: m-tag, C-TRAP cage, C+ or CDTT-crosslinked TRAP cage, reducing agent is added to show its resistance to reducing conditions.

FIG. 7 shows a frozen EM density chart showing the structure of TRAP cages made using TRAP S33C/R64S, resulting in 20-ring cages. From left to right, with 4-fold hole as the center; a view centered on a bow hole (bowtie hole); a perspective view; scale bar-5 nm.

Examples

Techniques for implementing the invention

Transmission Electron Microscope (TEM)

The samples were typically diluted to a final protein concentration of 0.025mg/ml, briefly centrifuged in a bench top centrifuge, and the supernatant applied to a hydrophilized carbon coated copper mesh (STEM co.) negatively stained with 4% phosphotungstic acid (pH 8) and observed using a JEOL JEM-1230 80kv instrument.

Non-denaturing PAGE

Samples were run on 3-12% non-denaturing Bis-Tris gels according to the manufacturerIs operated by proposal (Life Technologies). Samples were mixed with 4x native PAGE sample buffer (200mM BisTris,pH7.2, 40% w/v glycerol, 0.015% w/v bromophenol blue). As a qualitative guide for the molecular weight of the migrating bands, native mark undyed protein standards (Life Technologies) were used. Protein bands were observed according to the manufacturer's protocol (Life Technologies) when performing blue native PAGE, otherwise using fast blue ^TM Protein staining (Expedeon).

Protein expression and purification

In a typical purification, E.coli (E.coli) BL21 (DE 3) cells (Novagen) transformed with pET21b plasmid carrying TRAP (K35C/R64S) gene were grown by shaking at 37℃in 3L LB medium with 100. Mu.g/ml ampicillin until OD ₆₀₀ =0.6, induced with 0.5mM IPTG, followed by shaking for 4h. Cells were collected by centrifugation and the pellet was kept at-80 ℃ until use. Cells were lysed by sonication in 50ml of 50mM Tris-HCl, pH7.9, 50mM NaCl (in the presence of protease inhibitor (thermo scientific) and in the presence or absence of 2mM DTT) at 4℃and the lysate was centrifuged at 66,063g for 0.5h at 4 ℃. The supernatant fraction was heated at 70 ℃ for 10min, cooled to 4 ℃ and centrifuged again at 66,063g for 0.5h at 4 ℃. By at least one ofThe supernatant fractions were purified by ion exchange chromatography on a purifier (GE Healthcare Life Sciences) using a 4X 5ml HiTrap QFF column, combined in 50mM Tris-HCl, pH7.9,0.05M NaCl, +/-2mM DTT buffer and eluted with a 0.05-1M NaCl gradient. Fractions containing TRAP protein were pooled and concentrated using an Amicon Ultra 10kDa MWCO centrifugal filter unit (Millipore) and samples were size exclusion chromatographed on a HiLoad 16/60Superdex 200 column at room temperature in 50mM Tris-HCl, pH7.9, 0.15M NaCl. Protein concentration was calculated using BCA protein assay kit (Pierce Biotechnology).

Stability of cage

Use and the method described above ¹ Similar method testTRAP cage stability to chemicals and heat. All reagents used for the assay (DTT, TCEP, SDS, gdn-HCl and urea) were reconstituted in PBS pH 7.4 and mixed with TRAP cage samples overnight at room temperature. Thermal stability checks were performed by heating the samples at different temperatures for 10 min. The samples were then subjected to non-denaturing PAGE. These experiments were repeated twice, each with consistent results.

Frozen EM

Vitreous ice was prepared in PBS using 4. Mu.L of 1mg/mL protein sample. After blotting the samples on an EM grid (Quantifoil 1.2/1.3, cu,300 wood), they were frozen in liquid ethane using a FEI Vitrobot; FEI Vitrobots have parameters; imprinting force=0, imprinting time=4 sec, waiting time=0 sec, discharge time=0 sec. Photomicrographs were collected using a FEI TitanKrios freeze microscope with 300kV operation and a Falcon III camera at 75k magnification. 4942 and 10169 TRAP cages were collected respectively ^DTME And TRAP cage ^BMH Is a microscopic photograph of (a). Using MotionCorr2 ⁶ Motion correction was performed on all micrographs and CTFFIND4 was used ⁷ CTF estimation is performed. Use of frozen SPARC v2.12.4 ⁸ The particles were selected and extracted first in manual mode (about 4000 particles) and then in automatic mode, the initial class 2D was used as template. The extracted particles are again subjected to a 2D classification to select the best particles for the subsequent reconstruction step. Using EMD-4443 and EMD-4444 (TRAP cage) ^Au(I) ) As a search model, 3D reconstruction is performed by a heterogeneous refinement scheme (Heterogenous Refinement protocol).

EXAMPLE 1 preparation of TRAP cage ^DTME And TRAP cage ^BMH

Molecular cloning

For all cloning steps, E.coli NEB 5. Alpha. Strain was used. Plasmid sequences were confirmed by the sanger sequencing method by Eurofins. A tetracycline-inducible protein expression vector was constructed by subcloning the gene fragment encoding TRAP (K35C) into pACTet_H-mCherry or pACTet_H-mOrange. The gene of TRAP (K35C) was amplified by PCR using pET21b_TRAP-K35C as template, and the oligonucleotides FW_XhoI_TRAP and RV_MluI_TRAP (see Table 3) as primers. Amplified (amplified)The PCR product was used directly as template for a second PCR using FW_BsrGI_tev and RV_MluI_TRAP oligonucleotides as primers to introduce the adaptor gene fragments. The PCR products were cloned into pACTet_H-mCherry or pACTet_H-mOrange via BsrGI and MluI sites to give pACTet_H-mCherry-TRAP-K35C and pACTet_H-mOrange-tev-TRAP-K35C. Here, only the single point mutation K35C critical for subunit ligation was introduced into the TRAP sequence, whereas the previously used R64S was only important for avoiding gold nanoparticle binding ¹ 。

Table 3.Oligonucleotide sequences

Protein expression and purification

TRAP protein was produced using the same method previously described in A.D. Malay et al 'An ultra-stable gold-coordinated protein cage displaying reversible assembly' Nature 569,438-442 (2019), which is incorporated herein by reference, but 2mM DTT was kept in buffer during the initial purification step to avoid undesired cysteine oxidation. To generate the spliced TRAP loop, E.coli strain BL21 (DE 3) cells were co-transformed with pACTet_H-mOrange-TRAPK35C or pACTet_H-mCherry-TRAP-K35C with pET21_TRAP-K35C (see Table 4 in materials and methods). Cells were grown at 37℃in 100ml LB medium supplemented with ampicillin and chloramphenicol until OD ₆₀₀ =0.5-0.7. At this time, protein expression was induced by adding 0.2mM IPTG and 10ng/ml tetracycline in the case of pACTet_H-mCherry-TRAP-K35C, or by adding 30ng/ml tetracycline in the case of pACTet_H-mOrange-TRAP-K35C, and then the cells were cultured at 25℃for 20 hours. Cells were then harvested by centrifugation at 5,000Xg for 10 min. The cell pellet was stored at-80 ℃ until purification. They were then resuspended in 40ml lysis buffer (50 mM sodium phosphate buffer, 600mM NaCl,10mM imidazole, pH 7.4) supplemented with spatula DNase I end and lysozyme, 1-plate protease inhibitor cocktail and 2mM DTT and stirred at room temperature for 30min. The samples were then sonicated and clarified by centrifugation at 10,000Xg for 20min at 4 ℃. The supernatant was then incubated with 4ml of Ni-NTA resin previously equilibrated in lysis buffer in a gravity flow column for 20min. The resin was then washed in lysis buffer containing 20 and 40mM imidazole to more than 10 column volumes. His-tagged protein was eluted using 5ml of 50mM sodium phosphate buffer (pH 7.4) containing 500mM imidazole. Protein sample buffer was then exchanged into 2 Xphosphate buffered saline (PBS) supplemented with 5mM ethylenediamine tetraacetic acid (EDTA), hereinafter referred to as 2 XPBS-E) using an Amicon Ultra-15 centrifugal filtration device (50 k Molecular Weight Cut Off (MWCO), merck Millipore). The proteins were then size exclusion chromatographed using a Superdex 200Incure 10/300GL column (GE Healthcare) at a flow rate of 0.8 ml/min. Amicon Ultra-15 (50 k MWCO) was used to combine and concentrate the main peaks showing absorbance at 548nm or 587 nm. Protein purity was checked by SDS-PAGE, using extinction coefficient by using UV-1900UV-Vis spectrophotometer (Shimadzu): epsilon _mCherry ₅₈₇ ＝72000M ^-1 cm ^-1 、ε _mOrange ₅₄₈ ＝58000M ^-1 cm ^-1 ² 、ε _TRAP ₂₈₀ ＝8250M ^-1 cm ^-1 (http:// expasy.org/tools/protparam.html) Absorbance was measured to determine protein concentration. The protein was stored at 4℃until use.

Table 4.Plasmid and amino acid sequence

Free thiol concentration measurement

TRAP cage was evaluated using 5,5 '-dithiobis- (2-nitrobenzoic acid) (DTNB) reagent according to the manufacturer's protocol ^DTME And TRAP cage ^BMH The free thiol concentration of either. Centrifugal filtration device using Amicon Ultra-4The two samples were concentrated to 0.3mM by standing (100 k MWCO). Absorbance at 412nm was measured using a Spectramax190UV/VIS plate reader (Molecular Devices). According to the molar extinction coefficient (14150M) of 2-nitro-5-thiobenzoic acid ^-1 cm ^-1 ) The concentration of free thiol in the sample was calculated and for TRAP cage ^DTME And TRAP cage ^BMH Is undetectable.

Cage assembly and purification

For cross-linker induced cage assembly, TRAP (K35C/R64S) (100-500. Mu.M) in 2 XPBS-E was mixed with 5-fold molar excess of DTME or BMH and stirred at room temperature for 1 hour. The final DMSO concentration in the solution was maintained at no greater than 12.5%. After the reaction, insoluble portions that may be caused by low solubility of the crosslinking agent in the aqueous solution were removed by centrifugation at 12,000Xg for 5 min. Then using Superose 6 Increate 10/300GL column (GE Healthcare) at a flow rate of 0.5ml/minThe supernatant was purified by size exclusion chromatography on a purifier (GE Healthcare). Fractions containing cross-linked TRAP cages were pooled and concentrated using an Amicon Ultra-4 (100 k MWCO) centrifugal filtration device. Typical yields of crosslinked TRAP cages were obtained at about 20%. Formation and purification of gold (I) -induced TRAP-cage as previously described ¹ . Cage formation of fusion proteins was performed using the same protocol as described for cross-linking and gold (I) -induced cages, with an additional Ni-NTA purification step prior to size exclusion chromatography to purify the samples away from only partially assembled cages (unprotected His-tagged mCherry and mcarange 2 bound to Ni-NTA columns within the cages). The ratio of protein concentration and encapsulated guest was estimated using absorbance at 280/548nm or 280/587 nm. The extinction coefficient used for calculation is epsilon _mCherry ₅₈₇ ＝72000M ^-1 cm ^-1 ，ε _mOrange2 ₅₄₈ ＝58000M ^-1 cm ^-1 ，ε _TRAP ₂₈₀ ＝8250M ^-1 cm ^-1 . Due to the spectral overlap between mCherry and mOrange2, the concentration of both encapsulated objects was calculated correctly, also at 548nm (ε _mCherry ₅₄₈ ＝42538M ^-1 cm ^-1 ) The absorbance ratio of mCherry at 548/587nm without fusion to TRAP was used to estimate mCherry extinction coefficient. Similarly, the extinction coefficients of mCherry and mOrange2 at 280nm were experimentally determined to be ε, respectively _mCherry ₂₈₀ ＝56744M ^-1 cm ^-1 And epsilon _mOrange2 ₂₈₀ ＝52200M ^- ¹ cm ^-1 。

EXAMPLE 2 confirmation of TRAP cage Structure Using frozen EM

Determination of TRAP cage using frozen EM single particle reconstruction ^DTME And TRAP cage ^BMH Is a detailed structure of (a). Two types of cages have been obtainedAnd->Electron density map at resolution. These reveal that each structure consists of 24 TRAP loops arranged in two chiral forms, similar to the TRAP cage seen ^Au(I) . The TRAP loop model was improved according to the figure, yielding a good fit. A more careful examination at the ring-ring interface found two significant electron densities bridging two adjacent subunits, which may correspond to bismaleimide crosslinkers. The cross-linker appears to bend into a horseshoe shape between the cysteine residues of the opposite subunits (fig. 1).

EXAMPLE 3 specific cleavage Properties of the molecular Cross-linker used in TRAP cage (FIGS. 2-4)

TRAP cage ^DTME And TRAP cage ^BMH Exhibit similarly high stability in response to elevated temperatures, chaotropic agents and surfactants. In particular, they did not show significant morphological changes after incubation for 10 minutes at 75 ℃ at pH ranging from 2-11 up to 4M gnd hcl, up to at least 7M urea and 7% SDS. However, TRAP cage ^DTME Is easily removed when the reducing agent tris (2-carboxyethyl) phosphine (TCEP) or Dithiothreitol (DTT) is added. In contrast, TRAP cage ^BMH Unaffected. DTT-dependent disassembly on single cage level using High Speed Atomic Force Microscopy (HSAFM)Further real-time studies were performed. This indicates a TRAP cage ^BMH Has resistance to disassembly in the presence of DTT. In contrast, TRAP cage ^DTME Easy to disassemble under the same conditions, discrete patches of TRAP subunits appear to "peel" from the cage surface, ultimately resulting in an opening of the entire structure about 3 minutes after the first loop separation. TRAP cage ^DTME Is gradually disassembled and TRAP cage ^Au(I) Unlike, it shows a more coordinated disassembly on a shorter time scale. TRAP cage carrying FRET protein pair crog ^DTME Also allowing disassembly in the presence of a reducing agent to be kinetically characterized (fig. 4).

Example 4 assembled cage Using dissimilar metals

Protein expression and purification

TRAP (K35C/R64S) protein was expressed and purified as described previously.

TRAP (S33H/K35C) and TRAP (S33H/K35H) proteins were expressed and purified according to the same protocol as TRAP (K35C/R64S), but the pH of all buffers was 8.5.

The protein concentration was determined from absorbance measurements at 280 nm.

Assembled cage using different metals

TRAP cages were formed by mixing purified TRAP variants (final concentration of 0.1mM monomer subunits) with salts of the relevant metals in a suitable buffer at a ratio of TRAP monomer to metal ion between 4:1 and 2:1: agNO in 50mM Tris ₃ ，pH 7.9，0.15M NaNO ₃ The method comprises the steps of carrying out a first treatment on the surface of the Cd (NO) in 50mM Tris ₃ ) ₂ pH 7.9,0.15M NaCl; coCl in 50mM HEPES ₂ Or ZnCl ₂ pH 7.9,0.15M NaCl. The reaction is typically incubated for 3 days at room temperature. TRAP cage formation was confirmed using non-denaturing PAGE and TEM. Any precipitated material was removed by centrifugation at 12,045g for 5 min.

Jin Qudong TRAP cage [ TRAP (K35C/R64S) +Au (I) ]

The double mutant TRAP (K35C/R64S) of tryptophan RNA binding attenuated protein can be assembled into a hollow globular structure by reaction with monovalent gold ions. Frozen EM single particle reconstitution showed that by linear thio-Au (I) -thio cross-linking between the opposite cysteines, a 22nm cage consisting of 24 cyclic undecolymer subunits was obtained.

Silver and cadmium driven TRAP cage [ TRAP (K35C/R64S) +Ag (I) or Cd (II) ]

Metals other than Au (I), i.e., hg (II), ag (I), cd (II), can promote cage formation, indicating that metal driven cage formation requires a water stable d10 metal ion with a preferred dual ligand linear geometry.

Ag (I) -TRAP cages were formed and remained stable only in the absence of chloride ions.

TRAP (K35C/R64S) cages made by addition of Ag (I) or Cd (II) showed bands on native PAGE with similar mobility to Au (I) -mediated TRAP cages (fig. 5 a). Cage formation was further confirmed by negative-working Transmission Electron Microscopy (TEM), showing a spherical hollow structure with a diameter of 22-24nm (fig. 5b and 5 c). These results indicate that silver and cadmium driven cages may form structures with morphology similar to Au (I) -TRAP cages.

Cobalt and zinc driven TRAP cage [ TRAP (S33H/K35C) or TRAP (S33H/K35H) +Co (II) or Zn (II) ]

TRAP metal binding sites have been redesigned to target metal ions with tetrahedral coordination preference. Based on the crystal structure, a pair of histidines or cysteines and histidines are introduced at the i and i+2 positions of the β -sheet motif around the edges of the TRAP loop, resulting in TRAP (S33H/K35C) and TRAP (S33H/K35H) such that a single monomer unit provides two ligands to coordinate the divalent metal. When Zn (II) and Co (II) are added, these variants assemble into a cage structure.

Non-denaturing electrophoresis showed that the migration of TRAP (S33H/K35H) using both Zn (II) and Co (II) was similar to Au (I) -mediated TRAP cages (FIG. 5 d). Cage formation was confirmed by negative staining Transmission Electron Microscopy (TEM) with a diameter of about 22nm, indicating that the structure was similar to Au (I) -TRAP cages (fig. 5e and 5 f).

EXAMPLE 5 TRAP cage assembled Using a photocleavable crosslinker

Materials and methods:

the preparation of gold-induced TRAP cages was performed as previously described (Malay et al, nature,2019, which is incorporated herein by reference). 1, 3-dibromoxylene and 1, 3-dibromomethyl-4-nitrobenzene were purchased from commercial suppliers and dissolved in N, N-Dimethylformamide (DMF). While stirring for 1 hour at room temperature, 2 molar excess (relative to TRAP monomer) of either cross-linking agent was mixed with freshly purified gold-induced TRAP cage in 50mM sodium phosphate buffer (pH 7.4) containing 5mM EDTA. 10mM Dithiothreitol (DTT) was then added to the reaction to capture Au (I). The samples were then purified by size exclusion chromatography using a Superose6 Increate 10/300GL column (GE Healthcare).

Photo-induced disassembly of 1, 3-dibromomethyl-4-nitrobenzene TRAP cages was tested by exposing the samples to light at 365nm wavelength for different times in the presence of 10mM Dithiothreitol (DTT) for quenching free radical species. Cage morphology and cross-linker cleavage process were monitored using Dynamic Light Scattering (DLS), SDS, native PAGE, and TEM of Zetasizer (Malvern).

Results

In the first attempt, we tried a cage assembly method similar to that we did for BMH/DTME cross-linker (simple mixing of TRAP ring with excess cross-linker), but was unsuccessful, probably due to the difficulty in controlling the correct orientation of the ring in the structure.

We have found a new method to overcome this problem, using a previously assembled Au (I) -induced TRAP cage instead of just a ring, so that the dibromo-crosslinker can displace the gold atoms without changing the orientation of the ring in the cage, we call the "template reaction". We further used 1, 3-Dibromoxylene (DBX) crosslinker (fig. 6 b) as the basis for optimizing the process. The gold-induced TRAP cage has gold (I) as a linker between the loops, which is easily removed under reducing conditions (fig. 6a lane CT). We used this property to check if DBX was built into the structure of TRAP cages as a result of the "template reaction" because there was no disassembly property under the same conditions. Indeed, after mixing DBX with Au (I) -induced TRAP and further purification, the TRAP became unable to disassemble under reducing conditions, demonstrating the different types of linkages between the rings. We further characterized the structure of the obtained DBX TRAP cage by DLS method (fig. 6 c)), indicating that the size of the DBX TRAP cage is about 24nm with high monodispersity. The DBX TRAP cages were 2nm larger than the Au (I) -induced TRAP cages, also indicating the presence of cross-linking agents in the structure, which become larger in size as the cage structure is maintained, which can be observed on TEM (FIG. 6 d). Finally, we resolved the structure of the DBX TRAP cage, demonstrating the presence of DBX cross-linker between the rings. The resulting frozen EM structure of the cage (fig. 6 e) reveals other interesting features of the assembly. Indeed, as in the case of Au (I) -induced cages, we were able to distinguish between the two chiral forms of the cage (left-handed and right-handed), which is not surprising, since gold-induced cages have chiral properties. A significant difference between the template cage (gold induced) and the final template cage (containing DBX) is the number of junctions between TRAP loops. In the case of Au (I) induced cages, 120 linkages were recognized (-S-Au-S-bridge), but in the case of DBX cages the number of linkages was reduced to half of this number. 60 linker molecules and the same overall geometry (based on snub cube) forced slightly different orientations of the TRAP loops. Wire frame models of Au (I) -induced cages and DBX cages showed the difference between TRAP ring orientation and transition from edge-to-edge (Au (I)) to vertex-to-vertex (DBX) (fig. 6 f).

After successful results of basic dibromo-crosslinking of the TRAP cage, we decided to change DBX to the photolabile crosslinker-1, 2-dibromo-3-nitrobenzene, which is very similar in structure but which can break after UV (365 nm) light irradiation due to the presence of nitro groups (fig. 6 g). We optimized the conditions of the "template reaction" using BBN crosslinker, with the same results as the DBX used previously. After the "template reaction", SDS PAGE showed a clear appearance of TRAP dimer, demonstrating the presence of covalent bonds (fig. 6 h), indicating that BBN cross-linker built into TRAP cage structure in a similar manner as DBX used previously. TEM confirmed the presence of monodisperse TRAP cages, which were about 24nm in diameter (FIG. 6 i).

To further investigate the potential of the obtained photolabile TRAP cages, we tested their removal ability under UV light. Such reactions depend on the presence of a quencher, which does not allow the reaction to be reversible. We tested different concentrations of quencher (DTT) and also showed this dependence in the case of BBN TRAP cages. BBN TRAP cages were successfully disassembled under UV light and in the presence of 10mM DTT (FIG. 6 j). We also tested the time required for a fully detachable BBN TRAP cage. Non-denaturing PAGE shows that the bands of the cages fade over time, and it is estimated that complete disassembly occurs approximately 2min from UV irradiation (FIG. 6 k).

Throughout the description and claims of this specification the words "comprise" and "comprising" and variations thereof mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

EXAMPLE 6 TRAP cage made of twenty rings

Materials and methods

The TRAP protein was expressed as described above, except for the expression plasmid encoding the TRAP protein with the mutation S33C instead of K35C (the mutation R64S is also). In addition, incubation with the Au (I) source was similar to that described above. Subsequent purification of the TRAP cage formed was similar to that described above. The structure of the resulting TRAP cage was determined using frozen EM similar to that described above.

Results

Structural analysis of the assembled cage (fig. 7) shows that it consists of 20 linked TRAP S33C/R64S loops and bridging density suggests the case of the cage seen when using TRAP K35C mutants. This is believed to be the same as the gold staple structure seen in previous TRAP cages, although in this case the resolution is poor, the Au (I) ions act as a bridge between two opposing Cys residues.

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Claims

1. An artificial TRAP cage comprising a selected number of TRAP loops held in place by a molecular cross-linking agent, wherein the cross-linking agent is selected according to its particular characteristics, whereby the cage is programmably opened or kept closed as required under particular conditions.

2. Cage according to claim 1, wherein the specific cleavage characteristics of the molecular cross-linker are selected from the group comprising:

(iii) A photoactivatable molecular cross-linking agent, whereby the cage opens upon exposure to light.

3. Cage according to claim 1 or 2, wherein the cross-linking agent is a homobifunctional molecule moiety and derivatives thereof.

4. A cage according to any one of claims 1 to 3, wherein the cage is resistant or insensitive to reducing conditions.

5. The cage of claim 4, wherein the cross-linking agent is bismaleimide hexane (BMH) or dibromoxylene.

6. A cage according to any one of claims 1 to 3, wherein the cage is responsive or sensitive to reducing conditions.

7. The cage of claim 6, wherein the cross-linking agent is dithiobismaleimide ethane (DTME).

8. A cage according to any one of claims 1 to 3, wherein the cage is photoactivatable.

9. The cage of claim 8, wherein the cross-linking agent is dihalomethylbenzene and derivatives thereof, comprising: 1, 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2, 4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

10. Cage according to claim 8 or 9, wherein the cross-linking agent is photo-labile by exposure to UV light.

11. Cage according to any of the previous claims, wherein the number of TRAP loops in the TRAP cage is between 6 and 60.

12. Cage according to claim 11, wherein the number of TRAP loops in the TRAP cage is 12, 20 or 24, preferably 24.

13. Cage according to any of the preceding claims, comprising a mixture of different programmable cross-linking agents.

14. Cage according to any of the preceding claims, which encloses a cargo, which can be programmed to deliver the cargo at a specific timed and desired location.

15. Cage according to any of the previous claims, wherein the artificial TRAP cage protein is modified to comprise any one or more of the following mutations selected from the group comprising: K35C, R S and K35C/R64S.

16. Cage according to any of the preceding claims, wherein the cage is stable at elevated temperatures, stable in non-neutral pH and/or stable in chaotropic agents.

17. An artificial TRAP cage comprising a selected number of TRAP rings held in place by at least one metal cross-linker, wherein the metal is selected from the group comprising Ag (I), cd (II), zn (II) and Co (II).

18. The cage of claim 17, wherein the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group consisting of: K35C, K35H, R64S, K C/R64S, K35H/R64S, S33C, S33H, S C/R64S, S H/R64S, S C/K35H, S H/K35H, S C/K35C and S33H/K35C.

19. A cage according to claim 17 or claim 18, wherein the cross-linking agent comprises cadmium and preferably the artificial TRAP cage protein is modified to include a K35C/R64S mutation.

20. A cage according to claim 17 or claim 18, wherein the cross-linking agent comprises silver and the artificial TRAP cage protein is modified to include a K35C/R64S mutation.

21. A cage according to claim 17 or claim 18, wherein the cross-linking agent comprises cobalt and the artificial TRAP cage protein is modified to include S33H/K35C or S33H/K35H mutations.

22. A cage according to claim 17 or claim 18, wherein the cross-linking agent comprises zinc and the artificial TRAP cage protein is modified to include S33H/K35C or S33H/K35H mutations.

23. A cage according to any one of claims 17 to 22, wherein the number of TRAP loops in the TRAP cage is between 6 and 60.

24. Cage according to claim 23, wherein the number of TRAP loops in the TRAP cage is 12, 20 or 24, preferably 24.

25. A cage according to any one of claims 17 to 24, comprising a mixture of different programmable cross-linking agents.

26. A cage according to any one of claims 17 to 25, which encloses cargo, which can be programmed to deliver the cargo at a specific timed and desired location.

27. Cage according to any of the previous claims, which is approximately spherical in shape.

28. Use of a cage according to any of the preceding claims to deliver cargo and to a desired location within a controlled period of time.

29. Use of a cage according to any of the preceding claims as a medicament.

30. A method of treating a patient comprising administering to the patient a cage according to any one of claims 1 to 27.

31. Cage according to any one of claims 1 to 27, for use in the treatment of a disease in a patient.

32. Use of any one or more of the group comprising homobifunctional molecule moieties, dihalomethylbenzenes and derivatives thereof as programmable cross-linking agents in the construction of programmable TRAP cages.

33. Use of any one or more of the metals Ag (I), cd (II), zn (II) and Co (II) and derivatives thereof as cross-linking agents in the construction of TRAP cages.

34. A method of making an artificial TRAP cage, the method comprising:

(iii) Forming the TRAP cage; and

(iv) Purifying and isolating the TRAP cage.

35. The method of claim 34, wherein the programmable crosslinker is selected from the group consisting of:

36. The method of claim 34 or 35, wherein step (ii) comprises conjugation with a mixture of different programmable cross-linking agents.

37. A TRAP cage produced by the method of any one of claims 34 to 36.

38. A method of making an artificial TRAP cage, the method comprising:

(iii) Forming the TRAP cage; and

(iv) Purifying and isolating the TRAP cage;

wherein the metal is selected from the group comprising Ag (I), cd (II), zn (II) and Co (II).

39. A TRAP cage produced by the method of claim 38.

40. An artificial TRAP cage comprising a protein modified to include any one or more of the following mutations selected from the group consisting of: K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S C/R64S, S H/R64S, S C/K35H, S33H/K35H, S C/K35C, S H/K35C.