CN117203224A

CN117203224A - Artificial protein cage comprising guest cargo encapsulated therein

Info

Publication number: CN117203224A
Application number: CN202280030038.6A
Authority: CN
Inventors: 乔纳森·赫德尔; 阿图尔·比拉; 东佑翼; 安东尼娜·纳斯考斯卡; 金高·博尔泽卡-索拉兹; 扬·罗伊基; 伊扎贝拉·斯图普卡
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: CN117203225A; PL437115A1; CN117157311A

Abstract

The present invention provides an artificial TRAP cage comprising a selected number of TRAP loops and a guest cargo enclosed therein.

Description

Artificial protein cage comprising guest cargo encapsulated therein

Technical Field

The present invention belongs to the field of biochemistry. It relates to an artificial protein cage called "TRAP cage" comprising a selected number of TRAP loops and a guest cargo enclosed therein.

Background

Proteins assembled into monodisperse cage structures are useful molecular containers for diverse applications in biotechnology and medicine. Such protein cages are found in nature, e.g. in viral capsids, but can also be designed and constructed in the laboratory.

Thus, the single cysteine mutant, TRAP-K35C, of tryptophan RNA-binding attenuated protein from Geobacillus stearothermophilus (Geobacillus stearothermophilus) previously described by the present inventors can be assembled into a hollow sphere structure consisting of a plurality of cyclic undecamer subunits by reaction with gold nanoparticles ¹ . The resulting protein cages exhibit extremely high stability under many severe conditions, but are easily disassembled into capsomeric units by addition of a reducing agent.

While these attractive properties of TRAP cages are ideal for developing intracellular delivery vehicles, a key challenge remains in guest packaging.

It is an object of the present invention to provide an easy and robust method for internally loading a protein or therapeutic of interest into a TRAP cage in a stoichiometrically controlled manner.

Disclosure of Invention

The subject of the invention is an artificial TRAP cage comprising a selected number of TRAP loops and at least one guest cargo enclosed therein. Preferably, the artificial TRAP cage comprises a selected number of TRAP loops which are held in place by a cross-linking agent. Preferably, the cross-linking agent is a molecular cross-linking agent or an atomic metal cross-linking agent. Preferably, the TRAP loops are linked by gold or DTME.

Preferably, the guest cargo is no larger than the diameter of the TRAP cage. Preferably, the guest cargo has a diameter of less than 16nm. Preferably, the guest cargo has a diameter between 4nm and 16nm. Guest cargo larger than 4nm will not diffuse into or out of the TRAP cage.

Preferably, the cargo is a protein, preferably selected from the group comprising: enzyme (e.g., protease, nuclease, hydrogenase, dehydrogenase, lipase, lyase, ligase, transferase, reductase, recombinase, nuclease acid-modifying enzyme or other types of enzymes) antigen, antibody. Alternatively, the cargo is other types of protein biomacromolecules (e.g., sterols, steroids, or fatty acids). Alternatively, the cargo is a lipid, a peptide (e.g., a peptide hormone, a cell membrane disrupting peptide, a T cell stimulating peptide, or other type of peptide), a nucleic acid (e.g., DNA, designed DNA nanostructures (including those designed using DNA origami), DNAzymes, RNA, mRNA, miRNA, siRNA, tRNA single-stranded RNA, double-stranded RNA, RNAzymes), a small molecule cargo (such as a drug), a Peptide Nucleic Acid (PNA), a carbon-based structure (e.g., fullerene or buckminsterfullerene, single-walled carbon nanotubes, or multi-walled carbon nanotubes), a metal (e.g., iron, zinc, platinum, copper, sodium, cadmium, lanthanoid, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof), a toxin (e.g., ligand-targeted toxin, protease-activated toxin, bee venom lysopeptide, and a toxin-based suicide therapeutic agent), or a nanoparticle (e.g., a metal nanoparticle such as gold, iron, silver, cobalt cadmium selenide, titanium oxide), or a core-shell metal nanoparticle (such as CDs/ZnS, cde/CDs, and CdSe nanoparticle).

Preferably, the nucleic acid is selected from the group comprising DNA, RNA, mRNA, siRNA, tRNA and micrornas.

Preferably, the therapeutic agent is an enzyme associated with overexpression in a metabolic disorder or disease or with underexpression in a metabolic disorder or disease.

Preferably, the enzyme is selected from the group comprising: hydrogenase, dehydrogenase, lipase, lyase, ligase, protease, transferase, reductase, recombinase, and nuclease acid-modifying enzymes.

Preferably, the therapeutic agent is selected from the group comprising: cancer therapeutic agents, anti-infective therapeutic agents, vascular disease therapeutic agents, immunotherapeutic agents, anti-aging drugs (senolyc) and neurological therapeutic agents.

Preferably, the metal is selected from the group comprising: iron, zinc, platinum, copper, sodium, cadmium, lanthanoid, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof.

Preferably, the toxin is selected from the group comprising: ligand targeting toxins, protease activated toxins, melittin hemolypeptides and toxin-based suicide gene therapeutics.

Preferably, the guest cargo is a protein. Fluorescent proteins are preferred. GFP, mCherry or mOrange are preferred. Interleukin-2 (IL-2) or Neoleukin-2/15 (NL-2) is preferred.

Preferably, the therapeutic agent is selected from the group comprising: cancer therapeutic agents, anti-infective therapeutic agents, vascular disease therapeutic agents, immunotherapeutic agents, anti-aging agents, and neurological therapeutic agents.

Preferably, the cage comprises a plurality of goods, preferably the goods are identical or different from each other.

Preferably, the TRAP cage according to the invention further comprises at least one external ornament.

Preferably, at least one of the external ornaments includes a cell penetrating agent to facilitate intracellular delivery of the cage containing the internal guest cargo.

Preferably, the cell penetrating agent is PTD4.

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 interior surface of the TRAP cage is overcharged. Preferably, the TRAP cage with the super-charged cavity comprises an E48Q or E48K mutation. Preferably, the TRAP cage with the super-charged cavity comprises a K35C/E48Q or K35C/E48K mutation.

Preferably, the guest cargo is genetically fused to the interior surface of the TRAP cage. Preferably, the genetic fusion of the guest cargo of step (ii) with the interior surface of the TRAP cage cavity is by way of the guest cargo with TRAP facing the interior surface of the cavity ^K35C N-terminal fusion of N-terminal of (C). Preferably, the guest cargo is a genetic fusion protein, preferably a fluorescent protein, preferably GFP, mCherry or mOrange. Preferably, the genetic fusion protein is interleukin-2 (IL-2) or Neoleukin-2/15 (NL-2).

Preferably, the guest cargo is conjugated, preferably to the interior surface of the TRAP cage, using SpyCatcher/SpyTag conjugation. Preferably, the guest cargo of step (iii) is conjugated to a SpyCatcher/spyctag of the interior surface of the TRAP cage cavity, wherein the SpyCatcher is introduced into the loop region of the TRAP loop between residues 47 and 48, facing the interior when assembled into the TRAP cage, and the guest cargo comprises a spyctag.

Preferably, the guest cargo is attached to the TRAP cage by a covalent bond, preferably to the interior of the TRAP cage, preferably formed by a chemical or enzymatic bond.

Preferably, the opening of the cage is programmable. Preferably, the specific conditions correspond to specific cleavage characteristics of the crosslinker.

Preferably, the programmable opening of the cage is dependent on the choice of molecular or atomic metal cross-linker that holds the TRAP ring in place in the TRAP cage.

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: 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 photoactivatable 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 crosslinker 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-bromomethyl and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

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

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

Preferably, the TRAP loop is a variant.

Preferably, the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group comprising: K35C, E48Q, E48K, R64S, K C/E48Q, K C/E48K and K35C/R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation or a K35C/E48Q mutation or a K35C/E48K mutation.

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, E48Q, E C/R64Q, E H/R64Q, E33Q, E C/R64Q, E H/R64Q, E C/K35Q, E H/K35Q, E C/K35Q, E H/K35Q, E C/E48Q, E35H/E48Q, E35H/E48Q, E C/E48Q, E C/R64S/E48Q, E H/S33C/R64Q, E H/K35H/R64Q, E C/K35C/R64Q, E C/R64S/E48Q, E H/R64S/E48Q, E C/R64S/E48Q and S33C/E48K. Preferably, the artificial TRAP cage protein is modified to include a K35H/E48Q or K35H/E48 mutation.

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 79 ℃, preferably up to 95 ℃, preferably 95 ℃ 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 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 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 invention also includes the use of an artificial TRAP cage according to the invention as a delivery vehicle for intracellular delivery of its internal guest cargo.

The invention also includes the use of an artificial TRAP cage according to the invention as a vaccine.

The subject of the present invention is also the use of an artificial TRAP cage according to the invention for the treatment of a disease or condition selected from the group comprising: cancer, vascular disease, cardiovascular disease, diabetes, infection, autoimmune disorders, neurodegenerative disease, cell senescence disease, arthritis, and respiratory disease.

The subject of the invention is also a method for preparing an artificial TRAP cage with encapsulated guest cargo, 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 the TRAP ring units via at least one free sulfhydryl bond using a crosslinking agent;

(iii) Modifying the TRAP loop unit to provide a suitable internal surface environment for capturing the guest cargo;

(iv) Forming the TRAP cage by self-assembly to provide a cage cavity in which the guest cargo is encapsulated; and

(v) Purifying and separating the TRAP cage encapsulating the object cargo.

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 modification of step (iii) is selected from the group comprising:

(i) Overcharging the interior surface of the TRAP cage;

(ii) Genetically fusing the guest cargo to an interior surface of the TRAP cage cavity;

(iii) Conjugating the guest cargo SpyCatcher/SpyTag to an interior surface of the TRAP cage cavity; and

(iv) Covalent bond formation is by both chemical and enzymatic methods.

Preferably, the overcharging of step (i) of the inner surface provides a net positive or negative charge on the inner surface of the cage.

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

Preferably, the opening of the cage is programmable. Preferably the specific conditions correspond to specific cleavage characteristics of the crosslinker.

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

Preferably, the interior surfaces of the TRAP cage are overcharged. Preferably, the TRAP cage with the super-charged cavity comprises an E48Q or E48K mutation. Preferably, the TRAP cage with the super-charged cavity comprises a K35C/E48Q or K35C/E48K mutation.

Preferably, part (iii) is used for TRAP ^{K35C E48Q} The cage formation step of (2) is carried out in sodium bicarbonate buffer at pH 9-11.

Preferably, part (iii) is used for TRAP ^{K35C E48k} The cage formation step of (2) is carried out in sodium bicarbonate buffer at pH 10-10.5.

Preferably, the guest cargo may be loaded before or after assembly of the TRAP cage.

Preferably, the genetic fusion of the guest cargo of step (ii) with the interior surface of the TRAP cage cavity is by way of the guest cargo with TRAP facing the interior surface of the cavity ^K35C N-terminal fusion of N-terminal of (C). Preferably, the guest cargo is a genetic fusion protein, preferably a fluorescent protein, preferably GFP, mCherry or mOrange. Preferably, the genetic fusion protein is interleukin-2 (IL-2) or Neoleukin-2/15 (NL-2).

Preferably, the guest cargo of step (iii) is conjugated to a SpyCatcher/spyctag of the interior surface of the TRAP cage cavity, wherein the SpyCatcher is introduced into the loop region of the TRAP loop between residues 47 and 48, facing the interior when assembled into the TRAP cage, and the guest cargo comprises a spyctag.

Preferably, the enzymatic modification is performed by a peptide ligase selected from the group consisting of a localizing enzyme, an asparaginyl, an endoprotease, a trypsin-related enzyme and a subtilisin-derived variant, and the formation of covalent chemical bonds may include strain-promoted alkyne azide cycloaddition and pseudopeptide bonds.

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

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

The invention is also a method of treating a patient comprising administering to the patient a TRAP cage as described herein. The subject of the invention is also a method of treating an individual in need of treatment suffering from a disorder selected from the group comprising: a method of treating cancer, vascular disease, cardiovascular disease, diabetes, infection, autoimmune disorder, neurodegenerative and neurological disease, cell aging disease, arthritis, and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP cage with one or more internal guest cargo selected from the group comprising: nucleic acids, enzymes, therapeutic agents, small molecules, organic or inorganic nanoparticles, peptides, metals, antigens, antibodies and toxins, and therapeutically valuable fragments of all of the foregoing.

The subject of the invention is also a method of vaccinating an individual in need of vaccination suffering from a disorder selected from the group comprising: a method of treating cancer, vascular disease, cardiovascular disease, diabetes, infection, autoimmune disorder, neurodegenerative and neurological disease, cell aging disease, arthritis, and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP cage with one or more internal guest cargo selected from the group comprising: nucleic acids, enzymes, therapeutic agents, small molecules, organic or inorganic nanoparticles, peptides, metals, antigens, antibodies and toxins, and therapeutically valuable fragments of all of the foregoing.

Preferably, the TRAP cage therapeutic agent is administered by intranasal inhalation or injection.

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.

Reference herein to a "TRAP cage cavity" is to the hollow interior of the TRAP cage. It is separated from the external environment by a TRAP ring, which is a wall of the TRAP cage, wherein any hole in the wall is considered to separate the lumen from the external environment by a plane between the edges of the TRAP ring lining the hole.

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 (a 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 is 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 associated 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 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), dithiobismaleimide ethane (DTME), bis-halomethylbenzene and its derivatives, 2-bis-bromomethyl-3-nitrobenzene (BBN), dibromoxylene, and 1, 3-bis-bromomethyl-4, 6-dinitro-benzene (BDNB).

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.

The term "encapsulation" as referred to herein is synonymous with closure, encapsulation, inclusion or confinement with a TRAP cage.

As referred to herein, "guest cargo" refers to a biological product or any item enclosed within a TRAP cage.

Preferably, the guest cargo may be a protein, preferably selected from the group comprising: enzyme (e.g., protease, nuclease, hydrogenase, dehydrogenase, lipase, lyase, ligase, transferase, reductase, recombinase, nuclease acid-modifying enzyme or other types of enzymes) antigen, antibody. Alternatively, the cargo is other types of protein biomacromolecules (e.g., sterols, steroids, or fatty acids). Alternatively, the cargo is a lipid, a peptide (e.g., a peptide hormone, a cell membrane disrupting peptide, a T cell stimulating peptide, or other type of peptide), a nucleic acid (e.g., DNA, designed DNA nanostructures (including those designed using DNA origami), DNAzymes, RNA, mRNA, miRNA, siRNA, tRNA single-stranded RNA, double-stranded RNA, RNAzymes), a small molecule cargo (such as a drug), a Peptide Nucleic Acid (PNA), a carbon-based structure (e.g., fullerene or buckminsterfullerene, single-walled carbon nanotubes, or multi-walled carbon nanotubes), a metal (e.g., iron, zinc, platinum, copper, sodium, cadmium, lanthanoid, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof), a toxin (e.g., ligand-targeted toxin, protease-activated toxin, bee venom lysopeptide, and a toxin-based suicide therapeutic agent), or a nanoparticle (e.g., a metal nanoparticle such as gold, iron, silver, cobalt cadmium selenide, titanium oxide), or a core-shell metal nanoparticle (such as CDs/ZnS, cde/CDs, and CdSe nanoparticle).

The enzyme may be a protease selected from the group comprising: bromelain, botulinum toxin a, factor VIIA, protein C, TEV proteases, serine proteases (including SB, SC, SE, SF, SH, SJ, SK, SO, SP, SR, SS, ST, PA, pb, PC and PE superfamily and S48, S62, S68, S71, S72, S79, S81 families, particularly including Lon-a peptidase, CLP protease, lactoferrin, nculeoporin 125), cysteine proteases (including CA, CD, CE, CF, CL, CM, CN, CO, CP, PA, PB, PC, PD and PE superfamily and C7, C8, C21, C23, C27, C36, C42, C53 and C75 families, particularly including papain, cathepsin K, calpain, isolating enzymes, adenine (adenain), localizase a and Hedhehog proteins), aspartic proteases (including AA, AC, AD, AE and AF superfamilies, including the specific examples as follows: BACE1, BACE2, cathepsin D (Cathespin D), cathepsin E chymosin, napsin-AD, nepenthesin, pepsin, presenilin, plasma protease (plasmepsin)), threonine proteases (including PB and PE superfamilies, including in particular ornithine acyl transferase), glutamate proteases (including G1 and G2 superfamilies, metalloproteases including metallopeptidases and metallopeptidases).

The enzyme may be a nuclease selected from the group comprising: endonucleases, such as deoxyribonuclease I; human endonuclease V, CRISPR-associated proteins (including Cas9, cas12, cas 13) with or without associated nucleic acids including guide RNAs; an AP endonuclease; a flap endonuclease.

The protein may be other types of enzymes such as SUMO activating enzyme E1, DNA repair enzymes such as DNA ligase, DNA methyltransferases such as m6A, m C and m5C classes, ten-eleven translocation methylcytosine dioxygenase, early growth reaction protein 1 (EGR 1), oxyguanine glycosylase, caspase enzymes such as E3 ubiquitin ligase (including pVHL, CRBN, mdm2, beta-TrCP 1, DCAF15, DCAF16, RNF114, C-IAP 1) or E1 ligase, E2 ligase, DNA glycosylases, or toxins such as ricin A chain, diphtheria toxin and fragments thereof, pore-forming toxins such as exotoxin a, alpha-hemolysin, gyr-I, myeloid leukemia 1 (Mcl-1), DNA polymerase (including DNA polymerase beta, polymerase delta, and polymerase epsilon) or enzyme replacement therapy enzymes such as arginase beta, arginase alpha, I Mi Gan enzyme (im lucase), taliglucerase alpha, verasidase alpha (velagulosidase alpha), arabinosidase (Alglucerase), sebelipase alpha, laronidase (Laronidase), ai Duliu esterase (Idursulfase), elosulfenase alpha, sulfurase (Galsulfase), and Alglucosidase alpha (Alglucosidase alpha).

The cargo may be a substance that can be recognized as an antigen, e.g., a full length SARS-CoV-2 spike protein, a receptor binding domain, a SARS-CoV-2 spike protein, a peptide thereof, a SARS-CoV-2 spike protein full length, a SARS-CoV-2 spike protein, a receptor binding domain, a SARS-CoV-2 spike protein, a peptide thereof, a AARS-CoV-2 non-spike structural protein, a SARS-CoV-2 non-spike structural protein, a peptide thereof, a SARS-CoV-2 genome encoded protein or a portion thereof, a respiratory syncytial virus spike protein full length, a respiratory syncytial virus spike protein, a receptor binding domain, a respiratory syncytial virus spike protein, a peptide thereof, respiratory syncytial virus non-spike structural protein, a peptide thereof, a protein encoded by the respiratory syncytial virus genome or a portion thereof, a full length of a lassa virus spike protein, a receptor binding domain, a lassa virus spike protein, a peptide thereof, a lassa virus spike protein full length, a lassa virus spike protein, a receptor binding domain, a lassa virus spike protein, a peptide thereof, a lassa virus non-spike structural protein, a peptide thereof, a protein encoded by the lassa virus genome or a portion thereof, an epstein-barr virus spike protein full length, an epstein-barr virus spike protein, a receptor binding domain, an epstein-barr virus spike protein, its peptide, epstein-barr virus spike protein full length, epstein-barr virus spike protein, receptor binding domain, epstein-barr virus spike protein, its peptide, epstein-barr virus non-spike structural protein, its peptide, epstein-barr virus genome encoded protein or part thereof, dengue virus structural protein N, M or E, dengue virus structural protein N, M or E, its peptide, dengue virus structural protein N, M or E, part thereof, cytomegalovirus protein, part thereof and derivative peptides thereof, including capsid protein, cortical protein, polymerase, other proteins encoded by the viral genome, influenza virus HA protein full length, influenza virus HA protein, receptor binding domain, influenza virus HA protein, its peptide, influenza virus non-HA structural protein, its peptide, influenza virus genome encoded protein or part thereof.

The cargo may be antibodies, such as anti-p 53 antibodies, anti-mutated p53 antibodies, anti-JAK mAbs, such as tofacitinib and barbittinib, anti-interleukin inhibitors, such as tobrazumab, kukukuneauzumab and Wu Sinu mAb, anti-CD 20 mAbs, such as rituximab, anti-ofatuzumab and oregroup mAb, anti-TNF mAbs, such as infliximab, adalimumab and golimumab, anti-IgE mAbs, such as omab, hematopoietic growth factors, such as epoetin, anti-PD 1 and PDL-1mAb, such as cocoa-das, anti-CTLA 4 mAb, such as ipilimumab, anti-IL 2 antibodies, anti-IL 12 antibodies, anti-IL 15 antibodies, anti-tgfβ antibodies, anti-angiogenic mAbs, such as avastin, antagonists of A2A and A2B receptors, anti-Her 2 mAb, such as trastuzumab, antibody-EGFR conjugates, anti-fr mAbs, such as anti-EGFR mAb, anti-fr, such as anti-panamab, such as anti-herception, such as anti-CD 52 mAb, such as anti-heruximab, or anti-mAb, such as anti-panamab, such as anti-CD 6, such as anti-mAb, such as anti-panamab, anti-CD-mAb, such as anti-panitumumab, anti-CD-mAb, such as anti-panamab, anti-mAb, or anti-mAb, such as anti-panamab.

The protein may be other types of proteins, for example rapamycin target proteins (TOR), GATA transcription factors Gaf1 (Gaf one), TALE (transcription activator-like effector proteins), zinc finger proteins, tumor suppressor proteins (including proteins involved in controlling gene expression, such as p 16), signal transduction proteins such as (TGF) - β, checkpoint control proteins such as BRCA1, proteins involved in cell adhesion such as CADM1, DNA repair proteins such as p53, transcription factors such as mountain (Yamanaka) factors (Oct 3/4, sox2, klf4, c-Myc), cytochrome c, BCL proteins (including BCL-2 (B-cell lymphoma 2)), transcription control proteins such as NF- κb; cytokines (including chemokines, interferons, interleukins (including interleukin-2 and its artificial versions), lymphokines, and tumor necrosis factors; heat shock proteins (including heat shock beta-1 proteins), growth factors such as GDF11, ubiquitin, DNA double strand break repair proteins such as DNA ligase III alpha, PCSK9 inhibitors such as allo You Shan antibody (evolocumab) and al Mo Luobu mab (alirocumab), brain-derived neurotrophic factor (BDNF) or IL-5 inhibitors such as meplizumab (meplizumab) and rillizumab (relizumab).

The cargo may be other types of biological macromolecules (e.g., sterols, steroids, or fatty acids. Sterols may be cholesterol. Steroids may be progesterone. Fatty acids may be saturated fatty acids such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachic acid, behenic acid, lignoceric acid, cerotic acid, or may be unsaturated fatty acids such as myristoleic acid, palmitoleic acid, soap acid, oleic acid, elaidic acid, linoleic acid, elaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid

The cargo may be a lipid such as a phospholipid, for example phosphatidylcholine (phsophotol choline), phosphatidic acid (phosphatidate ester) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylserine (PS), phosphatidylinositol (PI 0), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP 2) and phosphatidylinositol triphosphate (PIP 3), sphingomyelin (SPH), ceramide phosphatidylethanolamine (sphingomyelin) (Cer-PE).

The cargo may be a peptide, such as a peptide hormone, a cell membrane disrupting peptide, a T cell stimulating peptide, or other type of peptide. Peptide hormones may be adrenocorticotropic hormone (ACTH), amylin, angiotensin, atrial Natriuretic Peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, glucagon, growth hormone, follicle Stimulating Hormone (FSH), insulin, leptin, luteinizing Hormone (LH), melanocyte Stimulating Hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid Stimulating Hormone (TSH), thyroid stimulating hormone releasing hormone (TRH), vasopressin (also known as Arginine Vasopressin (AVP) or antidiuretic hormone (ADH) or Vasoactive Intestinal Peptide (VIP).

The cargo may be molecular cargo such as an antibiotic molecule, e.g., a macrolide antibiotic, nicotinamide adenine dinucleotide (NAD+), nicotinamide mononucleotide, a chlorosterol absorption inhibitor, e.g., ezetimibe (ezetimibe), a Fibrate (Fibrate) such as gemfibrozil (gemfibrozil), bezafibrate (bezafibrate) and cyclopfibrate (cipofibrate), an HMG-CoA reductase inhibitor, ranolazine (Ranolazine), ivabradine (Ivabradine), a nitrate such as glycerol trinitrate, an endothelin antagonist such as Bosentan (Bosentan), hydralazine (Hydroalzine), minoxidil (Minox) calcium channel blockers such as amlodipine (amopine), nifedipine (nifedipine), verapamil (Verapamil) and diltiazem), an antagonist such as chlor (valsalsalan), valsalpran (valsalpran), valsalva (valproic) and an anti-prandial (prandial), an (such as well as an anti-prandial), an active drug such as prandial (prandial), an anti-prandial (prandial) and an anti-prandial drug such as prandial (prandial), an (prandial) such as prandial (prandial), an I), an anti-prandial (prandial) and an (prandial) Cyclizine (cyclizine) and Cetirizine (Cetirizine), glucocorticoids such as Prednisolone (Prednisolone), dexamethasone (dexamethone) and hydrocortisone (hydrocortisone), antiproliferative immunosuppressants, calcineurin inhibitors such as cyclosporin (ciclosporin), diuretics such as Allopurinol (allopurnol) and febuxostat (flibuxostat), DMARDs, cox-2 inhibitors such as Celecoxib (Celecoxib), etoricoxib (etoricoxib) and parecoxib (parecoxib), NSAIDs, DOPA decarboxylase inhibitors such as Carbidopa (Carbidopa) or benserazide (benserazide), selective B3-adrenoceptor agonists, a 1-receptor agonists, B1 receptor agonists such as Dobutamine (Dobutamine), a1 receptor antagonists such as prazosin (prazosin), doxazosin (doxazosin) and tamsulosin (tamsulosin), B2 receptor agonists such as salbutamol (salbutamol) and terbutaline (terbutaline), nicotine partial agonists such as valicarb (Varenicline), peripheral anticholinesterases such as bromhexine (Neostigmine), neuromuscular blockers such as pannu ku-ammonium (panuculonium), vecuronium (vecuronium) and rocuronium (rocuronium), bladder control drugs such as oxybutynin (oxybutynin) and tolterodine (tolbutadine), antimetabolites such as folic acid antagonists, pyrimidine analogues and purine analogues, antimycotics such as grisulmine (griseofluflufen), caspofungin (caspofungin) and ternafine (ternafine), antifungal antibiotics such as Amphotericin (Amphotericin) and nystatin, artemisinin derivatives such as artesunate (artesunate) and artemisinin (artemsinin), folate inhibitors such as proguanil (proguiin), primaquine (primaine), schizophrenic agents in the blood such as chloroquine (chloroquine) and quinine (quine), neuraminidase inhibitors such as Oseltamivir (Oseltamivir) and zanamivir (zanamivir), DNA polymerase inhibitors such as Aciclovir (Aciclovir) and ganciclovir (glaciclovir), protease inhibitors such as Darunavir (Darunavir) and ritonavir (ritavir), reverse transcriptase inhibitors such as nevirapine (primaine) and efavirenz (efenz), antiepileptics such as carbamazepine (carbamazepine), cizol (gabapentin) and triazocine (benzodiazepine), as well as benzodiazepine (benzodiazepine) and benzodiazepine (benzodiazepine), DNA polymerase inhibitors such as acibenzoguanamine (acivir), and benzoguanamine (benzoguanamine), and benzoguanamine (benzoguanamine) as inhibitors such as benzoguanamine, and benzoguanamine (benzoguanamine) and benzoguanamine) inhibitors Isobovazine (isocarboxazine) and mollobemide, norepinephrine reuptake inhibitors such as reboxetine (reboxetine) and maprotiline (maprotiline), SNRI such as venlafaxine (venlafaxine), duloxetine (duloxetine) and norvenlafaxine (desvenlafaxine), SSRI such as fluxetine (fluoxyine), paroxetine (paroxetine), citalopram (citalopram), escitalopram (escitalopram) and sertraline (serraline), tricyclic drugs such as imipramine (imapramine) and clomipramine), antipathogenic drugs such as amisulpride and sulpiride, partial serotonin agonists, receptor antagonists such as NMDA (memantine), cholinesterase inhibitors such as donepezil (donepezil), risstin (rivastigmine) and galantamine (galantamine), monoxidase inhibitors such as selegiline (selegiline) and rasagiline (rasagiline), COMT inhibitors such as entacapone (entacapone) and tolcapone (tolcapone), dopamine agonists such as pramipexole (pramipexole) and rotigotine (rotigotine), type V phosphodiesterase inhibitors such as sildenafil (sildenafil) and tadalafil (tadalafil), uterine agonists such as misoprostal), ergonovel base (ergometrine) and oxytocin (oxymactin), gnRH analogues and inhibitors, alpha-glucosidase inhibitors such as canagliflozin (canagliflozin) and englazin (paglozin), dipeptidyl peptidase inhibitors (Dipeptidyl Petidase Inhibitor) such as sitagliptin, saxagliptin and linagliptin, proton pump inhibitors such as Omeprazole (Omeprazole), lansoprazole (lansoprazole) and pantoprazole (pantoprazole), inhaled glucocorticoids such as beclomethasone (beclomethasone) and budesonide), inhaled muscarinic antagonists such as tiotropium (tiotropium) and glycopyrrolate (glycopyrronium), leukotriene antagonists such as montelukast (montelukast), beta 2-receptor agonists such as amoterol (almet al) and formoterol (formoterol), anticoagulants such as dapagliflozin (dacarbazin), heparin (hepa) and apiban (apiban), STING antagonists, targeted to the tumor cell inhibitors such as tac, tumor cell cycle inhibitors and other drugs, PARP inhibitors such as nilaparib (nirapanib), ALK inhibitors such as aletinib (alectib), HDAC inhibitors such as Bei Linuo he (Belinostat), MEK inhibitors such as cobicitinib (cobimetinib), BRAF inhibitors such as Dabrafenib (Dabrafenib), EGFR inhibitors such as Erlotinib (Erlotinib), mTOR inhibitors such as Everolimus (Everolimus), HER2 inhibitors such as Lapatinib (lapatib), FLT3 kinase inhibitors such as midostaurin (midostaurin), JAK inhibitors such as tofacitinib (tofacitinib) or BCL2 inhibitors such as valnetobax.

"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.

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).

Reference herein to the cavity "supercharge" of a TRAP cage means that the cavity-facing surface of the cage undergoes a net change in charge equivalent to at least +1 or-1 per TRAP loop, as compared to the unmutated (wild-type) loop. Thus, when overcharged, the 24-ring TRAP cage will carry a minimal charge change of-24 or +24 compared to the non-overcharged variant.

In addition, the following abbreviations are also used: TRAP (trp RNA binding attenuated protein), GFP (green fluorescent protein), PTD4 (protein transduction domain), CPP (cell penetrating peptide), SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), TEM (transmission electron microscope), DMEM (Dulbecco's modified Eagle's medium), FBS (fetal bovine serum).

The transport of molecular cargo to cells is ideal for a range of applications including the delivery of drugs, genetic material or enzymes. A number of nanoparticles have been used to achieve this, including liposomes, virus-like particles, non-viral protein cages, DNA paper folding cages, and inorganic nanoparticles, each with its own advantages and disadvantages. Protein cages are a promising approach, as demonstrated by viruses in nature, which are capable of delivering genetic material to cells, often with high efficiency and specificity.

Artificial cages are constructed from proteins that do not naturally form a cage structure, and wherein interactions between constituent proteins may be modified to facilitate their assembly. The advantage of using this approach is that the resulting cage may be given properties and capabilities that may not be available or viable in naturally occurring forms. To date, many artificial protein cages have been created, including tandem fusions of proteins with 2 and 3 fold rotational symmetry capable of forming 12-subunit tetrahedral cages, nanocube structures of 24 subunits with octahedral symmetry, constructed from trimeric proteinsA block self-assembled 60-subunit icosahedral cage structure, and a co-assembled two-component 120-subunit icosahedral protein complex that can be comparable to a small viral capsid complex, and a designed peptide capable of forming a network that closes to form a cage. There are several examples in which artificial protein cages have been filled with various cargo, including siRNA, mRNA ² And fluorescent dyes. However, only a few cases have demonstrated the delivery of cargo to cells through artificial cages. To our knowledge, delivery of protein/therapeutic cargo to cells mediated by artificial protein cages (as opposed to natural cages) has not been previously demonstrated.

We have previously produced artificial protein cages using building blocks consisting of naturally occurring circular proteins, TRAP (trp RNA binding attenuation protein) termed TRAP cages (FIG. 1 a) ¹ . In nature, TRAP is involved in controlling tryptophan synthesis and is well characterized structurally and biochemically. It is also used as a multifunctional building block in biological nanoscience. The TRAP cage consisted of 24 TRAP rings forming hollow spheres of approximately 22nm diameter and 2.2MDa with a cavity diameter of approximately 16nm. Each TRAP ring in the cage is associated with 5 adjacent TRAP rings and the structure contains 6 square holes of about 4nm diameter. Surprisingly, in contrast to other natural and most artificial cages, the cyclic subunits in the cage are not held together by a network of protein-protein interactions. Instead, a single gold (I) ion bridges the opposing sulphur of the cysteine residues between the loops in the protein, with the lysine at naturally occurring position 35 being replaced by cysteine. The cysteines of ten out of the 11 monomers of each ring in the cage bridge with the eleventh remaining unbridged monomer in this way and can be used for example for reaction with maleimide-labeled dyes.

TRAP cages are very stable, capable of surviving at temperatures of 95 ℃ for at least 3 hours, and capable of surviving in high levels of denaturants such as urea. Despite this high stability, TRAP cages are prone to rupture in the presence of low concentrations of reducing agents, including the cytoreductive agent glutathione. This feature holds the prospect that the TRAP cage can be used as a system for delivering cargo to cells, as it can be expected to maintain its structure, protecting the cargo until it enters the cell where intracellular reducing agents will cause decomposition and subsequent release of the cargo.

We have demonstrated that TRAP cages can be deliberately filled with protein cargo, and we use the super negative variant GFP (-21) of green fluorescent protein as an example molecule. We have also demonstrated that TRAP cages can be used to deliver these cargo to the interior of human cells. This cell penetration is itself controllable, as it only occurs when the surface of the TRAP cage is modified, for example by cell penetrating peptides. These results are the first step in developing TRAP cages as a potentially useful tool for delivering medically relevant cargo to cells, and more generally demonstrate the potential of artificial protein cage systems as therapeutic agents.

Here we demonstrate that TRAP cages can be used to deliberately encapsulate protein cargo and transport it into the cell interior. TRAP cages used in unmodified or externally modified form had no significant effect on cell viability.

In the first case, use was made of the TRAP cage we previously developed with a positively charged patch inside ¹ Cargo filling is achieved to electrostatically capture the negatively charged GFP by diffusion into the cages. Attempts to deliver filled cages to cells showed that if TRAP cages were not modified, there was no evidence that they penetrated into the cells. In contrast, attachment of Cell Penetrating Peptide (CPP) PTD4 to the TRAP cage will result in significant penetration into the cell interior.

The previous few efforts on artificial protein cage mediated delivery of cargo to cells have demonstrated the success of non-protein cargo. Notably, it has been shown that artificial protein cages loaded with siRNA can be taken up and released from their cargo by different mammalian cells to induce RNAi and knockdown target gene expression ³ . In this case, high gene silencing efficiency combined with low toxic effects suggests that protein cage vectors have potential as therapeutic delivery systems. Packaging of protein cargo in artificial protein cages has been previously demonstrated. However, these cages are not shown to be able to deliver their cargo directly to the cells, rather, multiple copies of the cage themselves are used As cargo within lipid envelopes prepared in cells, derived from host cell membranes, and purified as envelope protein nanocages' (EPNs). EPN capable of delivering the cage means that access to the cells is achieved by the envelope, the host-derived membrane, and not the protein cage.

Considering the overall high stability of the TRAP cage, its ability to decompose in the presence of a cell reducing agent has been demonstrated ¹ It is interesting to know if the cages are easily broken down inside the cell. Once in the cells, changes in the relative signal intensities of Alexa-647 and GFP associated with TRAP cages suggested intracellular rupture of the cages and release of cargo. One possible explanation is that when Alexa647 and GFP are very close to each other due to association with TRAP cages, GFP fluorescence may be reduced due to the quenching effect of the dye. Once GFP is released by TRAP cage breakdown, the average distance of GFP to Alexa-647 becomes greater, resulting in an increase in GFP fluorescence detected. This observation supports this possibility when delivered using TRAP cages lacking Alexa-647, with significantly brighter signals from intracellular GFP (figure 10).

In summary, the work presented herein provides the first demonstration of protein delivery to cells mediated by artificial protein cages. The proven cargo filling efficiency is quite low and this can be solved by further modifying the TRAP cage such that it carries a higher density of positive charges inside the cage.

Furthermore, it was demonstrated that functional protein cargo was loaded into TRAP cages using genetic fusion of cargo to the lumen-oriented N-terminus of TRAP monomers. The splice expression method (patchwork expression approach) allows mixed expression of TRAP monomers with fusion and TRAP monomers without fusion, thereby allowing control of the amount of cargo so that it does not exceed the capacity of the cage. In this way, the TRAP cage carrying mCherry cargo and carrying mixed mCherry and mOrange cargo was demonstrated, and the presence of cargo was confirmed.

Furthermore, the genetic fusion approach was adapted to provide multiple copies of the cavity-oriented SpyCatcher protein and demonstrated that this was able to capture SpyTag-bearing functional proteins, green Fluorescent Protein (GFP) and computationally designed interleukin-2 mimetics (Neoleukin-2/15，NL-2)(Silva DA, et al, nature,2019,565,186-191). We further demonstrate that by encapsulation in TRAP cages significantly inhibits the interaction of NL-2 with its target cell receptor, when the TRAP cage is opened by an externally applied trigger (trigger), the cargo becomes functionally similar to free NL-2.

Alternatively, different cargo capture methods (such as covalent attachment) may be explored, as described for other protein cages. ^4,5 Furthermore, we contemplate further modification of TRAP cages to increase targeting specificity and expand the scope and utility of their packaged cargo. Finally, future studies will require accurate localization and tracking of the exact intracellular location of the TRAP cage and its quaternary states. 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 damage 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 that are linked together by a reducible linker will spontaneously open and release cargo when the cytoplasm of the cells is highly reduced. The cages may also be used in combination with or as vaccines, where anti-T cell responses are expected to be stimulated The pro (i.e., peptide) is captured within the TRAP cage and then targets T cells, which then trigger opening.

Drawings

FIG. 1TRAP cage protein (a) TRAP cage (PDB: 6 RVV) structure, each TRAP loop showing a different color. The gold atoms appear as yellow spheres. (b) Surface illustrations of the exterior (left) and interior (right) of TRAP cages stained by charge distribution. (c) A surface view of a single TRAP ring, showing the faces directed to the internal cavity, is colored according to charge. (d) The negatively charged GFP (-21) is shown in the cartoon representation (left) and in the surface view according to charge coloring (right). (e) Scheme for encapsulation of TRAP cage with GFP (-21) and external modification with Alexa-647 dye and PTD4 peptide.

Filling and decoration of the TRAP cage of fig. 2. (a) Non-denaturing PAGE gels, showing purified TRAP cages incubated with His-tagged GFP (-21) after passage through Ni-NTA columns in the absence (-TCEP) or in the presence of TCEP (+TCEP). Lane 1: GFP (-21) positive control; 2: marker molecular weight for non-denaturing PAGE; 3: an empty TRAP cage; 4: input (TRAP cage with GFP (-21)); 5 and 8: circulating; 6 and 9: washing; 7 and 10: eluting. The fractions collected were subjected to protein staining (left) or analysis by fluorescence detection (right, extract.48nm). (b) The collected fractions were subjected to SDS-PAGE and then Western blotted with anti-GFP detection. Lane 1: GFP (-21) positive control; 2: marker molecular weight for SDS-PAGE; 3: an empty TRAP cage; 4: input (TRAP cage with GFP); 5 and 8: circulating; 6 and 9: washing; 7 and 10: eluting. (c) The non-denaturing PAGE gel shows encapsulation of GFP (-21) by either unmodified TRAP cages or TRAP cages modified externally by Alexa-647 and PTD 4. Lane 1: TRAP cages with GFP (-21); 2: TRAP cage with GFP (-21) decorated with Alexa-647; 3: TRAP cage with GFP (-21) decorated with Alexa-647 and PTD 4; 4: molecular weight markers for non-denaturing PAGE. The gel was protein stained (upper panel) and analyzed by fluorescence detection GFP (middle panel, ecto.488 nm) and Alexa-647 (lower panel, ecto.647).

(d) Negative staining transmission electron microscopy of TRAP cages with GFP (-21) (left panel); TRAP cage with GFP (-21) decorated with Alexa-647 (middle panels); TRAP cage with GFP (-21) decorated with Alexa-647 and PTD4 (right panel).

FIG. 3 delivers TRAP cages carrying GFP (-21) to MCF-7 cells. (a) Representative flow cytometry plots of MCF-7 cells at 15min, 2h and 4h after 4h treatment with Alexa-647 labeled TRAP cages carrying GFP (-21) (denoted (TC+GFP) +Alexa-647) and with GFP (-21) labeled with Alexa-647 and PTD4 peptides (denoted (TC+GFP) +Alexa-647+PTD4). The X-axis and Y-axis represent fluorescence intensities of GFP and Alexa-647, respectively. Untreated cells served as negative controls. (b) Representative red and green fluorescence overlap histograms from MCF-7 cells from the same experiment. (c) Median fluorescence intensity after 15min, 2h and 4h incubation with Alexa-647 and GFP positive cells treated with either a TRAP carrying GFP and decorated with Alexa-647 or a TRAP carrying GFP and decorated with both Alexa-647 and PTD 4. Data were normalized to untreated cells and based on three independent experiments. Control: 1: untreated cells; 2: cells incubated with (TC+GFP) +Alexa-647. (d) confocal microscopy image: untreated cells (control cells, upper row), cells incubated with TRAP cages filled with GFP (-21) and labeled with Alexa-647 only (middle row): cells incubated with TRAP cages filled with GFP (-21) and labeled with Alexa-647 and PTD4 (bottom row). Actin filaments were stained with Alexa-568 conjugated phalloidin and nuclei were stained with DAPI. Green channel-GFP; red channel-Alexa-647; blue channel-DAPI; gray channel-Alexa-568; (scale: 10. Mu.M).

FIG. 4 tracking of TRAP cage and GFP (-21) in MCF-7 cells. Cells were incubated with a GFP (-21) -carrying TRAP cage modified with Alexa-647 and PTD4, and pooled images were obtained from confocal microscopy fixed at different time points. Actin was stained with phalloidin conjugated to Alexa-568, while DAPI was used for nuclear staining; (Scale: 10 μm). The rectangular image below each main image is a representative orthogonal view on the yz axis. (a) -an image with a maximum projection of the red channel; (b) -a maximum projection image with a green channel.

FIG. 5 estimates the number of His-tagged GFP (-21) molecules in TRAP cages. (a) Standard curves were obtained from fluorescence measurements of GFP (-21) protein at concentrations ranging from 0-100nM. The fitting equation is: y=0.0258x+4.4；R ² = 0.9786. (b) Western blot for band densitometry analysis. Lanes 1-4: GFP (21); lane 5: GFP (-21) -loaded TRAP cage (denoted (tc+gfp)).

FIG. 6 shows an external-decoration (a) RP-HPLC chromatogram of a TRAP cage with GFP (-21) showing purified PTD4 peptide for decorating a TRAP cage filled with GFP (-21). (b) After titration of Alexa-647 in the conjugation reaction, a non-denaturing PAGE gel of TRAP cages carrying GFP (-21) was shown. Gels, alexa647 (left panel, extract.647) and GFP (middle and small panels, extract.48nm) and protein staining (right panel) were analyzed by fluorescence detection. The arrow indicates the optimal decoration conditions for further experiments. (c) SDS-PAGE gels, comparing TRAP cages carrying GFP (-21) with no decoration, alexa-647 or both Alexa-647 and PTD 4. Left: detection at 488 nm; intermediate: detection at 647 nm; right: western blot of the same samples detected with anti-GFP antibody. Lanes: 1: molecular weight markers for SDSPAGE electrophoresis; 2: TRAP cages with GFP (-21); 3: TRAP cage with GFP (-21) decorated with Alexa-647; 4: TRAP cage with GFP (-21) decorated with Alexa-647 and PTD 4; 5: GFP (-21) -positive control.

FIG. 7 stability and cell viability test of TRAP cage in culture medium. (a) Non-denaturing PAGE gels, showing the stability of TRAP cages in DMEM medium in the absence and presence of FBS during 18h incubation. (b) Cell viability of MCF-7 and HeLa cells after 4h exposure to empty TRAP cages, GFP (-21) -loaded TRAP cages, and TRAP cages with GFP (-21) decorated with Alexa-647 and PTD 4. M = molecular weight marker for non-denaturing electrophoresis; TC: an empty TRAP cage; (TC+GFP): TRAP cages filled with GFP (-21); (TC+GFP) +Alexa-647+PTD4: TRAP cage with GFP (-21) decorated with Alexa-647 and PTD4 was used.

FIG. 8 delivers TRAP cages with GFP (-21) to HeLa cells. (a) Representative flow cytometry plots of HeLa cells after 4h treatment with Alexa-647 labeled TRAP cage with GFP (-21) (expressed as (TC+GFP) +Alexa-647) and 15min, 2h and 4h treatment with Alexa-647 labeled TRAP cage with GFP (-21) and PTD4 peptide (expressed as (TC+GFP) +Alexa-647+PTD4). The x-axis and y-axis represent fluorescence intensities of GFP and Alexa-647, respectively. Untreated cells served as negative controls. (b) Representative red and green fluorescence overlap histograms from HeLa cells from the same experiment. (c) Median fluorescence intensities after incubation of GFP positive cells with Alexa-647 treated with (TC+GFP) +Alexa-647 and (TC+GFP) +Alexa-647+PTD4 for 15min, 2h and 4 h. Data were normalized to untreated cells and based on three independent experiments. Control: 1: untreated cells; 2: confocal microscopy images of cells (d) incubated with (TC+GFP) +Alexa-647: untreated cells (control cells, upper row), cells incubated with Alexa-647-labeled (tc+gfp) alone (middle row), cells incubated with TRAP cages filled with GFP (-21) and labeled with Alexa-647 and PTD4 (lower row). Actin filaments were stained with Alexa-568 conjugated phalloidin and nuclei were stained with DAPI. Green channel-GFP; red channel-Alexa-647; blue channel-DAPI; gray channel-Alexa-568; (scale: 10. Mu.M).

FIG. 9 tracking of TRAP cage and GFP in HeLa cells. Cells were incubated with TRAP cages with GFP (-21) labeled with Alexa-647 and PTD4, and pooled images from confocal microscopy fixed at different time points. Actin was stained with phalloidin conjugated to Alexa-568, while DAPI was used for nuclear staining; (Scale: 10 μm). The rectangular image is a representative orthogonal view on the yz axis. (a) -an image with a maximum projection of the red channel; (b) -a maximum projection image with a green channel.

FIG. 10Alexa-647 effect on GFP (-21) fluorescence. (a) Cells were exposed to (TC+GFP) labeled with Alexa-647 and PTD4 (upper panel) or (TC+GFP) labeled with PTD4 alone (lower panel). Actin filaments were stained with Alexa-568 conjugated phalloidin and nuclei were stained with DAPI. Green channel-GFP; red channel-Alexa-647; blue channel-DAPI; gray channel-Alexa-568; (scale: 10. Mu.M). (b) Average GFP fluorescence intensities recorded from three different fields of view of the samples, where cells were exposed to either (tc+gfp) labeled with Alexa-647 and PTD4 or (tc+gfp) labeled with PTD4 alone. Fluorescence intensity was quantified with ImageJ and background intensity subtraction was considered. (c) Average fluorescence of GFP (21) encapsulated in unmodified TRAP and fully modified TRAP measured in solution.

FIG. 11 uses genetic fusion and splice formation to encapsulate a single type of guest of a guest protein. (a) Schematic of mCherry encapsulation into TRAP cages using genetic fusion and splicing strategies. Ptet/tetO, tetracycline promoter/operator; pt7/lacO, T7 promoter/lac operator system. (b) Negative staining transmission electron microscopy images of TRAP cages containing different numbers of mCherry in the cavity.

FIG. 12 uses genetic fusion and splice formation to encapsulate guests of two different types of guest proteins. (a) schematic representation of a TRAP cage loaded with fluorescent protein. The spliced TRAP ring fused at the N-terminus with mCherry (dark cylinder) or mOrange2 (light cylinder) is mixed together with DTME or triphenylphosphine monosulfate (TPPMS) -Au (I) -Cl. (b) Non-denaturing PAGE shows the fluorescent properties of purified TRAP cages associated with fluorescent cargo. Gels were visualized using an fast blue (InstantBuue) protein stain (left) and using fluorescence excited at 532nm and emitted at 610nm (right). (c) TEM images: empty (left) TRAP and fluorescent protein filled TRAP (right) were assembled using Au (I) (top) or DTME (bottom). Scale bar, 50nm.

FIG. 13 confirmation of double protein loading in TRAP cages. 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 568nm and that of mCherry was 610 nm. 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.

FIG. 14 uses the concept of the SpyTag/SpyCatcher system to encapsulate objects. (a) SpyTag/Spycatcher mediated guest loading. (b) constructs of TRAP and GFP variants. SpyT, spyTag; spyC, spyCatcher; spycatchers are included at positions between residues 47 and 48. The construct was generated with His-tagged SUMO and cleaved with SUMO protease after Ni-NTA affinity chromatography to yield TRAP-K35C-ring SpyC.

FIG. 15 produces a TRAP cage containing Spycatchers in the cavity. (a) SDS-PAGE analysis of TRAP 11-mer consisting of TRAP-K35C and SPYC-TRAP or TRAP-cycloSPYC was performed. SN, supernatant after cell lysis and centrifugation; ni is Ni-NTA affinity chromatography; SU, SUMO protease cleavage. (b) non-denaturing-PAGE analysis using Au (I) to form cages. The reaction was performed in 50mM sodium phosphate buffer (pH 8.0) containing 100. Mu.M TRAP, 0 or 100. Mu.M TPPMS-Au (I) -Cl (Au (I) (-) or (+)) and 0 or 600mM NaCl (NaCl (-) or (+).

FIG. 16 SDS- (a) and non-denaturing- (b) PAGE analysis of mixtures of GFP encapsulated in TRAP cages using the SpyTag-SpyCatcher system (a, b) containing the SpyCatcher moiety in the cavity, spyC-TRAP cage or TRAP-Ring SpyC cage and SpyTag-GFP. For non-denaturing PAGE, the same gels were visualized by fluorescent and quick Blue (Instant Blue) staining.

FIG. 17 isolation and imaging of GFP-filled TRAP cages. (a, b) size exclusion chromatography (a) and negatively stained TEM images (b) of TRAP cages containing SpyCatcher parts in the cavity mixed with Spy-labeled GFP induced with 30ng/mL tetracycline. Cages consisting of the TRAP-K35C variants and not filled with any cargo were also provided for comparison. (c) negative staining TEM image of SpyT-TRAP variants.

Fig. 18 strategy for encapsulating Neoleukin-2/15 in a photo-openable TRAP. The spliced TRAP loop consists of a TRAP variant that contains a K35C, R S mutation and lacks lysines at positions 73 and 74, and a TRAP variant that contains a K35C mutation, with His-tag and SUMO at the N-terminus, and SpyCatcher in the luminal loop. BBN,1, 2-dibromomethyl-3-nitrobenzene; beta-ME, beta-mercaptoethanol.

Fig. 19 Neoleukin-2, triggered release from TRAP cages, stimulated target cells. a, a graph reflecting SEAP activity, indicated by absorbance measured 24h after stimulation of HEK-Blue cells with NL-2, hIL-2, spy-Tag-NL-2 conjugated to SPYCatcher-TRAP-loop; b SEAP activity after 24h stimulation with NL-2, empty TRAP before and after UV irradiation and SpyCatcher-TRAP filled with spycag-NL-2 before and after UV irradiation.

Examples

Techniques for implementing the invention

Electron microscope

TRAP cages filled with GFP (-21), TRAP cages filled with GFP (-21) and labeled with Alexa-647, and TRAP cages filled with GFP (-21) and fully decorated were imaged using transmission electron microscopy. The sample is typically diluted to a final protein concentration of 0.025mg/ml, centrifuged at 10000g for 5min at room temperature, and the supernatant applied to a hydrophilized carbon coated copper mesh (STEM Co). The samples were then negatively stained with 3% phosphotungstic acid (pH 8) and observed using a JEOL JEM-2100 instrument operating at 80 kV.

Flow cytometry

For TRAP cage internalization experiments, MCF-7 and HeLa cells were plated at 2.5X10 per well ⁵ Is inoculated into 12-well plates (VWR) in 800 μl of DMEM medium containing 10% fbs and cultured for 16h before the experiment. Cells were then incubated with 50 μg (6 nM) of cargo-filled TRAP cage labeled with Alexa-647 alone or decorated with Alexa-647 and PTD4 peptides in 50mM HEPES, pH 7.5, containing 150mM NaCl, supplemented with 10% FBS for 15min, 2h and 4 h. After incubation, cells were washed three times with Phosphate Buffered Saline (PBS) (EURx), 5min each, harvested with trypsin (1 mg/ml) and centrifuged at 150g for 5min. Subsequently, the cells were washed three times in PBS by centrifugation (150 g,3 min) and resuspended in PBS. Cells were run in a Navios flow cytometer (Beckman Coulter) and 12000 cell fluorescence per sample was collected. Untreated cells and cells treated with TRAP cages filled with cargo and labeled with Alexa-647 alone were used as negative controls. The data obtained from three independent experiments were analyzed using Kaluza software (Beckman Coulter). The percentage of Alexa-647/GFP positive cells and the median fluorescence intensity were determined for each sample.

Confocal microscope for laser scanning

For fluorescent laser scanning confocal microscopy, cells grown on 15-mm glass coverslips were transplanted into 12-well plates (2.5x10 per well) ⁵ In 800 μl DMEM medium with 10% fbs) and further stimulated as described above to conduct flow cytometry experiments. Next, the cells were washed with PBS (3 times, 5 min), fixed with 4% paraformaldehyde solution (15 min, room temperature), and permeabilized with 0.5% Triton-X100 in PBS (7 min, room temperature). Actin filaments were conjugated with Alexa-568The combined phalloidin was stained in PBS (1:300,Thermo Fisher Scientific,1.5h, room temperature). The coverslip was then mounted onto the slide using Prolong Diamond medium with DAPI (Thermo Fisher Scientific). Fluorescence images were obtained under an Axio observer.z1 inverted microscope (Carl Zeiss, jena, germany) equipped with an LSM 880 confocal module and a 63x oil immersion objective. Images were processed using ImageJ 1.47v (national institutes of health).

Example 1 filling of trap cage.

To fill the TRAP cage we exploit the fact that the only significant positive charge on the surface of the TRAP ring is located on the surface lining (lining) inside the cage fig. 1a-c 1b, c. In principle, this may allow capturing negatively charged cargo by electrostatic interactions, as with other protein cages (e.g. ⁶ ) As demonstrated. In fact, in the addition of gold (I) ¹ Previously, the constituted TRAP loops did not assemble into the TRAP cage, which means that protein cargo below about 4nm had two possible encapsulation routes-they could be combined with the TRAP loops prior to assembly, or they could be added after TRAP cage formation and allowed to diffuse through four times the pores into the cage. We selected the negatively charged GFP (-21) as model cargo (fig. 1 d). The cylindrical protein has a diameter of about 2.4nm and is therefore expected to be able to diffuse into the assembled TRAP cage (fig. 1 e). His-tagged GFP (-21) was mixed with TRAP cages and incubated overnight, then purified by size exclusion chromatography to remove the remaining free GFP (-21). As shown by co-migration of fluorescent signals on non-denaturing gels, two proteins were found to associate (FIG. 2 a). To verify if His-tagged GFP (-21) is within the TRAP cage and not bound to its outside, we performed a pullout assay using Ni-NTA affinity chromatography, followed by Western blot analysis. Observations that GFP (-21) associates with TRAP cages but not with Ni-NTA columns indicate that successful encapsulation renders His-tags inaccessible. This is further supported by a pull-out assay which shows that the associated GFP (21) is available only for interaction with the Ni-NTA column after cage decomposition by addition of a reducing agent (fig. 2 b). These results strongly suggest that encapsulation of GFP in TRAP cages is in a full partial mode (partial encapsulation is GFP "blocking") The plug "hole in TRAP cage and His tag directed to the inside). The amount of GFP (-21) per cage was about 0.3, comparable to that found in many other filled protein cages, although some have shown considerable amounts of cargo.

Preparation and purification of GFP (-21) -filled TRAP cage

The preparation and purification of the TRAP cage was performed as described previously. ¹ The relevant plasmid and amino acid sequence information is shown in Table 1. The super-charged (21) His-tagged GFP protein was expressed with pET28a encoding the GFP gene and produced in BL21 (DE 3) cells. The protein was purified using Ni-NTA. Briefly, in the presence of protease inhibitor (Thermo Fisher Scientific), the protease inhibitor is prepared by washing in 50mM Tris-HCl, pH7.9, 150mM NaCl,5mM MgCl ₂ ，5mM MgCl ₂ The cells were lysed at 4℃by sonication and the lysate was centrifuged at 20000g for 0.5h at 4 ℃. Coupling the supernatant with Ni ²⁺ Agarose beads of bound nitrilotriacetic acid (His-Pur Ni-NTA, thermo Fisher Scientific) were incubated together and the agarose beads pre-equilibrated in 50mM Tris,pH7.9, 150mM NaCl,20mM imidazole (buffer A). After washing the resin three times (with buffer a), the protein was eluted with 50mM Tris,pH7.9, 150mM NaCl,300mM imidazole (buffer B). Fractions containing His-tagged GFP (-21) were pooled and size exclusion chromatography was performed on a HiLoad 26/600Superdex 200pg column (GE Healthcare) in 50mM Tris-HCl, pH7.9, 150mM NaCl at room temperature. Protein concentration was measured using a Nanodrop spectrophotometer with a wavelength of 280 nm.

GFP encapsulation was performed by mixing an equal volume of 100. Mu.M of the negatively charged (-21) His-tagged GFP with 1. Mu.M preformed TRAP cage, and incubating overnight in 50mM Tris,150mM NaCl (pH 7.9). Purification of GFP-loaded TRAP was performed by size exclusion chromatography in 50mM HEPES,pH 7.5, 150mM NaCl using a Superose 6increase 10/300 column (GE Healthcare). Fractions containing TRAP cages were collected and analyzed by non-denaturing PAGE using 3-12% non-denaturing Bis-Tris gel (Life Technologies) followed by fluorescence detection at 488nm using Chemidoc detector (BioRad).

Estimation of the number of His-tagged GFP (-21) molecules in TRAP cage

Two methods were used to estimate GFP (-21) loading:

1. based on detection of GFP fluorescence in cargo-filled TRAP cages. GFP (21) standard curves were prepared at concentrations ranging from 0-100 nM. Using RF-6000The Spectro fluorescence photometer obtains fluorescence spectrum at 26deg.C, fixed excitation wavelength of 488nm, and emission wavelength of 495-550nm, lambda _em The interval is 1.0nm, the scanning speed is 6000nm min, lambda _ex Bandwidth of 5nm lambda _em The bandwidth was 5nm. Using an emission maximum lambda _em Fluorescence at 510nm was used for calculation. TRAP protein concentration was determined from absorbance at 280 nm. TRAP cage-GFP (-21) stoichiometry of 1:0.28.+ -. 0.07 was obtained (FIG. 5 a).

2. Densitometry analysis briefly, a series of His-tagged GFP (-21) dilutions (0.4 ng;0.8ng;4ng;8ng; measured at wavelength 280nm by NanoDrop) and loaded TRAP cages, samples (2. Mu.g, measured at wavelength 280nm by NanoDrop) were separated by SDS-PAGE and Western blotted (FIG. 5 b). Signals from His-tagged GFP (-21) protein were detected with anti-GFP antibody and HRP conjugated secondary antibody in a chemiluminescent detector (Chemidoc, bioRad). Densitometric analysis of the resulting blots using ImageLab (BioRad) software showed that 0.6ng of His-tagged GFP (-21) was present in 2 μg of cargo-filled TRAP cages. Densitometry analysis resulted in approximately 1:0.4 TRAP cage: GFP (-21) stoichiometry.

Ni-NTA "pull out"

Samples of purified TRAP cages filled with His-tagged GFP (-21) protein were split into two parts and incubated under either reducing (1 mM TCEP) or non-reducing (no TCEP) conditions. Next, the samples were passed under gravity flow through Ni-NTA resin (Thermo Fisher Scientific), with 100. Mu.l of each sample being introduced onto 50. Mu.l of the resin equilibrated with buffer A. Three samples were collected:

(i) flow-through, (ii) washing with buffer a, and (iii) eluting with buffer B. Samples were analyzed by non-denaturing PAGE and then subjected to fluorescence detection (excitation at 488nm, chemidoc, bioRad) and Western blotting. For SDS-PAGE and Western blotting, samples collected from Ni-NTA pull experiments were denatured by adding TCEP (final concentration 0.1 mM) and boiling for 15min, and then separated by Tris/glycine gel electrophoresis. The gel was electrotransferred (2 h,90 v) to activated PVDF membranes in 25mM Tris, 192mM glycine, 20% methanol buffer. Membranes were blocked with 5% skim milk in Tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T) and then incubated with mouse monoclonal anti-GFP antibody (1: 2500;St.John's Laboratories,UK) and horseradish peroxidase-conjugated anti-mouse (1:5000,Thermo Fisher Scientific) secondary antibody for 1.5h. Signals were generated using Pierce ECL blotting substrate (Thermo Fisher Scientific) and visualized in a BioRad Chemidoc detector.

TABLE 1 plasmid information and amino acid sequence

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The TRAP cages herein may have a super-charge cavity. To achieve this, the TRAP cage may include an E48Q or E48K mutation. Preferably, TRAP cages with a super-charged cavity will include K35C/E48Q or K35C/E48K mutations. This provides and additional.

Example 2. Decoration of TRAP cage with fluorescent dye and cell penetrating peptide label.

Our aim was to modify the TRAP cage to facilitate its cell entry. We selected PTD4 (YARAAARQARA, SEQ.ID No. 8) -optimized TAT-based cell penetrating peptides with significantly improved ability to penetrate cell membranes, higher amphiphilicity, reduced arginine numbers, and increased α -helix content. ⁷ Many work have shown that coating nanoparticles with PTD4 or the like promotes cell penetration (e.g ⁸ ). We attached a PTD4 derivative, ac-YARAAARQARAG (SEQ. ID No. 9), to the amino group on the exposed lysine on the TRAP cage surface. In TRAPCages had three such surface-exposed lysines per monomer, potentially allowing 792 peptides per cage to be linked. Acetylation of the N-terminal amino groups eliminates the possibility of cross-reacting those amino groups with activated carboxyl moieties that are intended to react with available amino groups of the TRAP protein. In addition, the extended C-terminal glycine residue acts as a flexible linker and, since it is not a chiral amino acid, the opportunity for racemization during the carboxyl activation process is eliminated. Peptides were synthesized using a solid phase method and purified by reverse phase high performance liquid chromatography (fig. 6 a). In the optimized reaction, we observed an increase in apparent molecular weight of the TRAP cage after reaction with PTD4 (fig. 6 c), as observed by non-denaturing PAGE.

To be able to track the TRAP cage independently of its cargo we labeled it with Alexa-647 fluorescent dye. To this end, we crosslinked the maleimide groups on the dye with 24 available cysteines in six 4 nm-pore liners of the TRAP cage that did not participate in the ring-ring interaction. By titration we determined the optimal number of Alexa-647 to be added (which is equal to the number of TRAP cysteine groups), where the TRAP cage was easily labelled and no free dye was present in the sample. This was assessed by non-denaturing PAGE in combination with a fluorometry to detect both GFP (-21) and Alexa-647 (FIG. 6 b). Although the cargo GFP contained 3 cysteine residues, the control reaction showed no detectable label for GFP with Alexa647 (fig. 6 c). Negative staining Transmission Electron Microscopy (TEM) confirmed that the modified TRAP cages retained their characteristic shape (fig. 6 d).

PTD4 peptide synthesis

PTD4 peptide derivatives (Ac-YARAAARQARAG, (SEQ. ID No. 10), referred to herein as PTD4 for simplicity), were synthesized on a 0.1mmol scale using a Liberty Blue automated microwave synthesizer (CEM, USA) according to the Fmoc-based solid phase peptide synthesis method. Fmoc-Gly-Wang resin (100-200 mesh, degree of substitution 0.70mmol/g, novabiochem, germany) was swelled overnight with Dichloromethane (DCM)/Dimethylformamide (DMF) (1:1). Fmoc deprotection was performed with 25% morpholine in DMF at 85℃for 5 min. The coupling reaction was carried out at 85℃for 5min using DIC/oxyma activator and five times the excess of Fmoc-protected amino acid derivative according to the manufacturer's recommended protocol. All Fmoc-Arg (Pbf) couplings were double. N-terminal acetylation was performed on the resin with 10% acetic anhydride in DMF at 60 ℃. Cleavage of the resin and deprotection of the side chains was achieved by vigorous shaking with TFA/Triisopropylsilane (TIS)/water (94:3:3) at 30℃for 4 h. The resin was filtered and TFA evaporated under a gentle stream of nitrogen. The crude peptide was precipitated by addition of cold diethyl ether and then centrifuged (3000 rpm,10 min). The residue was washed with cold diethyl ether (2 x) and ethyl acetate (2 x). The precipitated crude peptide was dried in vacuo overnight. The crude peptide was dissolved in 8M urea and purified on an Agilent 1260RP-HPLC using a semi-preparative C18 (10X 150 mm) column (Cosmosil, nacalai tesque). The collected peptide-containing fractions were lyophilized. The purified peptide was analyzed on an analytical C18 column (Zorbax SBC18 5mm 4.6x150 mm,Agilent) with a linear gradient of 0-20% acetonitrile and 0.1% TFA for 30min at a flow rate of 1.0ml/min. Peak signals were detected at 220 and 280nm (fig. 6 a).

TRAP cage labeled with Alexa-647 and decorated with cell penetrating peptides

TRAP cages carrying GFP labeled with Alexa-647 and decorated with the cell-penetrating peptide Alexa Fluor-647 C2 maleimide fluorescent dye (Alexa-647, thermo Fisher Scientific) and the cell-penetrating peptide PTD4 were conjugated to GFP-filled TRAP cages.

To achieve fluorescent labeling, GFP-carrying TRAP cage (300. Mu.l, 16 nM) was mixed with Alexa-647C2 maleimide dye ((50. Mu.l, 1. Mu.M)) and reacted in 50mM HEPES containing 150mM NaCl,pH 7.5 with continuous stirring at 450rpm for 2.5h at room temperature. The optimal interaction ratio of maleimide conjugated Alexa-647 to TRAP cage was assessed by titration (fig. 6 b). Briefly, aliquots of TRAP cages loaded with GFP (-21) (11.36 nM) were mixed with 0.1. Mu.M to 100. Mu.M maleimide conjugated Alexa-647. The samples were then separated by non-denaturing gel electrophoresis and visualized by fluorescence detection of excitation at 647nm in Chemidoc. The absence of free Alexa-647 in the sample and the absence of GFP reaction interfering with Alexa-647 signal was observed was considered the best decorative condition and used for further experiments.

Furthermore, to exclude the possibility of directly labeling GFP by Alexa-647, the TRAP cages loaded with and without Alexa-647 labeled GFP (-21) were subjected to denaturing gel separation and Western blotting, followed by detection with anti-GFP antibodies. No band shift was observed from the potential interaction of GFP with Alexa-647 dye (fig. 6 c).

For cell-penetrating peptide decoration, PTD4 peptide (50 μl,0.5 mM) was mixed with 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 10 μl,83 mM) and N-hydroxysuccinimide (NHS, 10 μl,435 mM), all reagents were dissolved in ddH ₂ O. Subsequently, excess activated PTD4 peptide was added to TRAP cage filled with GFP (-21) and labeled with Alexa-647, and incubated for 2.5h at room temperature while continuously stirring at 450 rpm. The reaction was stopped by adding 5. Mu.L of 200mM Tris-HCl pH 7.5. Conjugation efficiency was verified by non-denaturing PAGE and fluorescent gel imaging. The change in molar weight of the decorated TRAP cage resulted in a band shift observed in non-denaturing PAGE (fig. 6 c).

Example 3 stability of trap cage and effect on cell viability.

Before starting the cell delivery test we first assessed whether the TRAP cage is structurally stable, i.e. not disassembled under cell culture conditions. At 37℃with 5% CO ₂ Stability was checked under atmosphere in Dulbecco's Modified Eagle Medium (DMEM) without or with varying concentrations of Fetal Bovine Serum (FBS). The results showed that 5% CO was used at 37℃in DMEM medium ₂ After 18h of lower culture, the cage structure was stable (FIG. 7 a).

To determine the effect of TRAP cages on cell viability, alamarBlue experiments were performed. This test is based on the natural ability of living cells to convert resazurin (a blue non-fluorescent compound) into resorufin (red fluorescent molecule) under the action of mitochondria and other reductases. ⁹ Human cancer cell lines MCF-7 and HeLa were incubated in the presence of TRAP cages, filled with GFP (21) and decorated with Alexa-647 and PTD4 peptides. The number of cells, TRAP cage dose and stimulation time used in the cell viability test correspond to the conditions under which the TRAP cage internalization assay was performed. Untreated cells were used as controls. DataIt was shown that unmodified TRAP cages and TRAP cages filled with GFP (-21) and decorated with Alexa-647 and PTD4 did not significantly affect viability of MCF-7 and HeLa cells during at least 4h incubation (FIG. 7 b).

Cell culture and cytotoxicity assessment of TRAP cages:

HeLa and MCF-7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, sigma) supplemented with 10% FBS (EURX), 100. Mu.g/ml streptomycin, 100IU/ml penicillin (Gibco). Culturing at 37deg.C and 5% CO ₂ The following is performed. To test the stability of the TRAP cage in the medium, purified samples were added to DMEM medium containing 0, 2 and 10% Fetal Bovine Serum (FBS) and incubated at 37 ℃ with 5% CO ₂ Incubate for 2h, 6h and 18h under incubation. Samples were then analyzed by non-denaturing PAGE and then subjected to fast blue gel staining (FIG. 7 a).

Cell viability after TRAP cage treatment was determined using alamarBlue test (VWR). 2.5X10 per well in 96 well plates ⁴ Cell density of individual cells were cultured. Cells were then treated with 5 μg (0.6 nM) TRAP cage, filled with GFP (21) and decorated with Alexa-647 and PTD4 in 50mM HEPES pH 7.5 containing 150mM NaCl supplemented with 10% FBS for 4h. After treatment, 10. Mu.l alamarBlue diluted in 90. Mu.l DMEM medium was added to each well and incubated at 37℃with 5% CO ₂ Cells were incubated for an additional 3h. The active ingredient resazurin of alamarBlue was reduced to the highly fluorescent compound resorufin only in living cells and the absorbance of the dye was recorded (excitation 570nm, emission 630 nm). Untreated cells were used as negative controls (fig. 7 b). Three measurements were made on all samples in three independent experiments.

Example 4. Delivery of protein cargo to cells.

The delivery of TRAP cages to cells was studied using the human cancer cell lines MCF-7 and HeLa. Cells were incubated with purified TRAP cages containing encapsulated GFP (-21) and labeled with Alexa-647 alone or with Alexa-647 and PTD4 for various periods of time and analyzed by flow cytometry. In both cell lines treated with TRAP cages with GFP labeled with Alexa-647 and PTD4 peptides, as the fluorescence signal of Alexa647 and GFP increased with the extension of incubation time (FIGS. 3a, b, c). These results indicate that external modification of TRAP cages with cell penetrating peptides promotes their cellular entry and efficient cargo delivery. Interestingly, this effect was more pronounced in the MCF-7 cell line compared to the HeLa cell line (FIGS. 8a, b, c).

In order to distinguish between fluorescent signals from internalized TRAP cages in cells and fluorescent signals from externally adsorbed TRAP cages of cell membranes, confocal microscopy was used. TRAP cages containing GFP (-21) and labeled with Alexa-647 but lacking PTD4 were not observed in the cells. In contrast, TRAP cages containing GFP (-21) and modified with PTD4 showed clear signals inside the cells at 4h after stimulation (FIG. 3d and FIG. 8 d).

Example 5 intracellular kinetics of trap cages.

The high stability of the TRAP cage combined with its ability to divide in the presence of moderate concentrations of cell reducing agents suggests that the TRAP cage in the cytoplasm should be easily disassembled, releasing GFP (-21) cargo. Since TRAP cages and GFP have discrete and trackable signals, we hypothesize that if Alexa-647 and GFP signals become non-co-localized after cell entry, removal of the cage and release of GFP (-21) can be strongly inferred. To assess this possibility, we followed the change over time of both signals after addition of MCF-7 and HeLa cancer cells. Notably, in both cell lines tested, TRAP cages were present mainly at the cell boundary during the first 90 minutes of incubation, as shown by the strong localization of Alexa-647 signal (fig. 4a, 9 a), and little GFP signal was detected (fig. 4b, 9 b). However, after 3h incubation, the TRAP cage signal (Alexa-647) became weaker and appeared to be more evenly distributed in the cells, whereas the GFP signal was clearly detectable, probably due to its release from the TRAP cage (fig. 4a, b and fig. 9a, b).

Example 6 Effect of Alexa-647 on GFP (-21) fluorescence

To assess the potential effect of Alexa-647 on GFP (-21) fluorescence (as shown in fig. 6a, middle panel), we compared cargo-filled TRAP cages by confocal microscopy imaging, where the compared cages were either decorated with PTD4 peptide alone or fully modified (PTD 4 and Alexa-647) (fig. 10 a). Briefly, cells were treated with the corresponding samples as described in materials and methods. Next, the cells were fixed and stained according to the protocol described above. Fluorescence intensity in the green channel was quantified with ImageJ. The calculation of the average fluorescence intensity (fig. 10 b) takes into account the background signal from each field of view.

In addition, RF-6000 is usedSpectro fluorometer, the in-solution fluorescence of GFP (-21) encapsulated in fully decorated TRAP cages was compared to the fluorescence of cargo in TRAP cages without Alexa-647. As shown in fig. 10c, the presence of Alexa-647 dye on the TRAP cage resulted in a decrease in fluorescence of its cargo of approximately 30%.

EXAMPLE 7 filling TRAP cage with protein cargo by genetic fusion

By genetically fusing the guest to the caged TRAP, efficient protein packaging is achieved. As an initial model we used the far red fluorescent protein mCherry (fig. 11 a). Genetic fusion of N-terminal His-tagged fluorescent proteins to TRAP that faces inward upon Assembly ^K35C The N-terminus. Since excessive modification of an 11-mer TRAP unit with these fluorescent proteins may interfere with cage assembly due to steric hindrance, it is highly desirable to use these fluorescent proteins in E.coli (Escherichia coli) host cells with unmodified TRAP ^K35C Co-production of fusion proteins is possible by different inducers (tetracycline and isopropyl-beta- _D Thiogalactoside (IPTG)) to control individual transcript levels. Using this co-production system, the mCherry number per TRAP loop can be well regulated by varying the tetracycline concentration. The spliced TRAP loops fused to mCherry were then assembled into cages using Au (I) (fig. 11 b). The same method can be used to fill the TRAP cage with more than one cargo protein.

Protein yield:

co-transformation was performed using pACTet_H-mCherry-TRAP-K35C and pET21_TRAP-K35C to produce a spliced TRAP loop. Protein expression was induced by the addition of 0.2mM isopropyl- β -d-thiogalactoside and tetracycline (8 ng/ml). After sonicating the cells, the spliced TRAP rings were then separated using Ni-nitrilotriacetic acid (NTA) affinity chromatography, followed by SEC analysis using Superdex 200Increase 10/300GL chromatography columns.

Cage assembly and characterization:

TRAP cage formation was performed by mixing equimolar amounts of purified TRAP loops comprising mCherry and chloro (triphenylphosphine monosulfoxide) gold (I) - (TPPMS-Au (I) -Cl) in 50mM sodium phosphate buffer (pH 8.0) comprising 600mM NaCl and holding overnight at room temperature. The stoichiometry of the guest protein was determined using the absorbance ratio 280/587 nm. The morphology of the isolated cages was checked using negatively stained TEM according to the protocol described above.

TRAP cage with mCherry ornament using cell penetrating peptide:

the maleimide moiety was introduced at the N-terminus of the peptide on the resin using 6-maleimidocaprooic acid and DIC/Oxyma coupling protocol. 0.5mM 6-maleimidocaprooic acid-PTD 4 or HA/E2 (25. Mu.l, 0.5 mM) peptide was mixed with TRAP cage filled with mCherry (75. Mu.l, 0.3 mg/mL) and incubated overnight at room temperature while stirring continuously at 450 rpm. Conjugation efficiency was verified by non-denaturing PAGE and fluorescent gel imaging. Variation in the molar weight of decorated TRAP cages resulted in band shifts observed in non-denaturing PAGE.

Example 9 filling TRAP cages with two different protein cargo by genetic fusion. More than one type of protein may be used to fill the TRAP cage. Two fluorescent proteins, mOrange2 and mCherry, act as a donor and acceptor, respectively, of the Forster Resonance Energy Transfer (FRET), and are encapsulated by genetic fusion of the respective cargo proteins to the N-terminus of the TRAP monomer. The fusion protein is co-expressed with unmodified TRAPK35C in E.coli host cells, and the individual transcription levels can be controlled by different inducers, tetracycline and isopropyl-beta-D-thiogalactoside (IPTG). The amount of expression inducer added was optimized to obtain 0.3mOrange2 and 1mCherry proteins per TRAP loop, which could avoid steric hindrance during the cage formation process.

Cargo modified TRAP rings were then mixed in a 1:1 molar ratio and Au (I) -or DTME was added to promote cage assembly (fig. 12 a).

The resulting cages were then purified by size exclusion chromatography and analyzed by non-denaturing PAGE in combination with fluorescence detection and TEM imaging (fig. 12b, c). Non-denaturing PAGE demonstrates the successful encapsulation of TRAP cages with two fluorescent cargo, which can be seen by fluorescence excitation of the cage at 532nm (emission 610 nm) (FIG. 12 b).

TEM images show a monodisperse population of TRAP cages, which are obviously packed with cargo after assembly with a mixture of cargo-modified TRAP-rings. The resulting products retained their morphology compared to empty Au (I) or DTME induced cages (fig. 12 c).

The presence of the mOrange2 and mCherry proteins in the vicinity of the TRAP cage should allow for Frst Resonance Energy Transfer (FRET), a physical process of energy transfer from the excited fluorophore to other molecules. Energy transfer between fluorescent proteins encapsulated within protein cages has been described, but has never been applied to monitor the removal kinetics of artificial protein cages.

To evaluate TRAP cage ^DTME And TRAP cage ^Au(I) FRET efficiency between the mOrange2 and mCherry proteins, spectral data were collected using excitation values of mOrange 2. All spectral data were normalized at the mOrange2 fluorescence peak and co-localization with mCherry was judged by the relative value of the fluorescence intensity ratio. Not only fluorescence spectra of FRET versus (pair) -packaged TRAP cages but also of control samples, which were TRAP cages packaged with mCherry or mOrange2 protein alone and then mixed in solution, were measured ^Au(I)/DTME . Such control samples cannot exhibit FRET because the fluorophores are remote from each other, encapsulated in a distant cage. Indeed, the TRAP cage packaged with FRET pairs compared to the corresponding control samples ^Au(I) And TRAP cage ^DTME The spectrum of (c) shows that mCherry at 610nm emits a signal about 1.5 times higher (fig. 13a, b), indicating that there is efficient energy transfer between mCherry 2 and mCherry in DTME and Au (I) -induced TRAP cages.

Protein yield:

coli strain BL21 (DE 3) cells were co-transformed with pACTet_H-mOrange-TRAPK35C or pACTet_H-mCherry-TRAP-K35C and pET21_TRAP-K35C. 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, followed by incubation 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. The pellet was resuspended in 40ml lysis buffer (50 mM sodium phosphate buffer, 600mM NaCl,10mM imidazole, pH 7.4) supplemented with DNase I 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 over 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 XPBS saline (PBS) plus 5mM EDTA, hereinafter referred to as 2 XPBS-E, using an Amicon Ultra-15 centrifugal filtration device (50K 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. Ab Amicon Ultra-15 (50 k MWCO) was used to combine and concentrate the main peaks showing absorption at 548nm or 587 nm. Protein purity was checked by SDS-PAGE and by using UV-1900UV-Vis spectrophotometer (Shimadzu), extinction coefficient was used: epsilon _mCherry ₅₈₇ ＝72,000M ^-1 cm ^-1 、ε _{mOrange 548} ＝58,000M ^-1 cm ^-1 、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.

Cage assembly and purification:

for cross-linker induced cage assembly, TRAP (K35C/R64S) (100-500. Mu.M) in 2 XPBS-E was combined with 5-fold molar excess of DTME orBMH was mixed 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 purifier FPLC (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 cross-linked TRAP cages obtained were about: 20%. Formation and purification of the gold (I) -induced TRAP cage was performed 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 the partially assembled cages (His-tagged mCherry and mcarange 2 binding to Ni-NTA columns that were not fully protected within the cages). Using absorbance at 280/548nm or 280/587nm, protein concentration and ratio of encapsulated guest were estimated using a method similar to that previously reported (4). 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 . Morphological fidelity of the assembled cages was confirmed by negative staining TEM and non-denaturing PAGE analysis.

Fluorescence measurement: fluorescence spectra were obtained at room temperature on an RF-6000 fluorescence spectrophotometer (Shimadzu) in polystyrene cuvettes of 1cm path length using 70nM mOrange2 in 2 XPBS-E. The protein is excited at 510nm and the emission is scanned over a wavelength range of 530 to 700 nm. The obtained spectra were normalized to the mOrange2 fluorescence peak. After each measurement, 10mM DTT was added to the sample to trigger complete cage disassembly.

TABLE 2 plasmid information and amino acid sequence

EXAMPLE 10 filling TRAP cage with protein cargo by formation of isopeptide bond

Although systems using genetic fusion are robust and versatile, this strategy still suffers from the disadvantage of requiring exposure of the guest protein to Au (I) or maleimide cross-linkers. This process can be particularly problematic if the guest protein contains free cysteine residues important for activity, such as cysteine proteases. To overcome this problem, we designed an post-assembly loading system using the SpyTag/SpyCatcher system, the 13-amino acid peptide SpyTag interacted with the protein SpyCatcher to spontaneously form isopeptidic linkages (zakri B, et al, proc.Natl. Acad.Sci.U.S. A.2012,109,690-697, which is incorporated herein by reference). In the case of filling protein cages, two strategies can be used (fig. 14 a). In strategy 1, the inner wall of the TRAP cage carries SpyCatcher, while the cargo carries SpyTag. In the second strategy, the inner wall of the TRAP cage carries SpyTag, while the cargo carries SpyCatcher, we designed various constructs based on this approach, first to capture GFP in the TRAP cage (fig. 14 b). For strategy 2, spyCatcher was introduced into the N-terminal or loop-facing cavity of TRAP (particularly between residues 47 and 48). All host TRAPs are equipped with hexahistidine tags to facilitate purification, whereas the tags can be cleaved off using carboxypeptidase or SUMO protease to produce TRAP-K35C variants with SpyTag or SpyCatche at the N-terminus, referred to as SpyT-TRAP or SpyC-TRAP, respectively, and variants with SpyCatche in the luminal loop, referred to as TRAP loop SpyC.

TRAP variants with SpyCatcher were co-produced with unlabeled TRAP-K35C in host bacteria to produce splice loops as described in example 9. To avoid failure to form sufficient cages due to excessive SpyCatcher moieties, we tested two concentrations (10 or 30 ng/mL) of tetracycline that regulated the gene expression level of SpyCatcher fusion variants. The generation of splice loops with different fusion content was confirmed by SDS-PAGE analysis, cleavage of His-tag by SUMO protease (fig. 15 a). When Au (I) was added to the phosphate buffer, all variants showed cage formation, while further enhancing the assembly efficiency by increasing the ionic strength of the buffer (fig. 15 b). After separation of the assembled cages, they were mixed with different concentrations of SpyTag-equipped GFP (SpyTag-GFP) in Phosphate Buffered Saline (PBS). Isopeptides formation and host-guest association of all variants were observed, as judged by analysis using SDS-PAGE (fig. 16 a) and non-denaturing PAGE (fig. 16 b), indicating successful guest packing. However, further analysis of SpyC-TRAP cage samples obtained from 30ng/mL tetracycline using SEC and TEM showed slight changes in elution time compared to some objects outside the empty TRAP cage and cage assembly, indicating that guest GFP may leak partially out of the cage (fig. 17). Meanwhile, for Trap-ring SPYC cages, no such leakage was observed. UV-Vis absorption spectra of the isolated filled cages showed that these TRAP cages could be loaded with up to 28 GFP molecules, while the packing density could be controlled by varying the tetracycline concentration during bacterial production or the mixing ratio during packing. Strategy 1 was found to be challenging compared to TRAP-SpyCatcher fusion, as SpyT-TRAP variants showed an aggregation trend, and therefore no efficient Au (I) -mediated cage formation was observed (fig. 17 c). In summary, it can be concluded that the TRAP loop SpyC variant is the most robust and efficient variant for guest packaging.

The second guest demonstrated in this way was Spy-labeled Neoleukin-2/15 (Silva DA, et al, nature,2019,565,186-191, incorporated herein by reference), which was also successfully loaded into TRAP cages with Spycatchers in the cavity. Photo-openable TRAP consisting of a variant comprising two mutations K35C and R64S and lacking the C-terminal two lysine residues d73K and d74K and a TRAP ring SpyC, wherein the TRAP rings are interconnected by the photo-cleavable crosslinker 1, 2-dibromomethyl-3-nitrobenzene (BBN) (fig. 18). Similar to the case of GFP, spy-labeled NL-2 was mixed with TRAP cages and the filled cages were separated by size exclusion chromatography (FIG. 19 a). Subsequent analysis using negatively stained TEM and SDS-PAGE showed successful packing of the NL-2 protein into TRAP cages by isopeptide bond formation (FIG. 19 b).

HEK-Blue IL-2 cell assay was used to evaluate the properties of encapsulated SpyTag-NL-2 in TRAP cages. HEK-Blue is a type of HEK 293T cells that is engineered to co-express stably the human IL-2 receptor and its signaling pathway as well as additional Secreted Embryonic Alkaline Phosphatase (SEAP) reporter genes. Binding of IL-2 or NL-2 to the IL-2 receptor (IL-2R) results in the initiation of a signaling cascade that results in transcriptional activation and secretion of SEAP, allowing its monitoring by colorimetry.

HEK-Blue cells were seeded on 96-well plates. The next day, empty UV-cleavable SpyCatcher-TRAP cage samples were encapsulated with NL-2/15 and added with 10mM cysteine (quencher) and treated with UV light for 10min. The treated samples and controls were diluted in cell culture medium (DMEM) to various concentrations in the pM range. Control samples included unconjugated SpyTag-NL-2 and purchased human IL-2, spyTag-NL-2 conjugated to TRAP ring and empty TRAP cage before and after UV treatment. Cells were stimulated for 24 hours, then Quanti Blue assay was performed, which was able to assess the amount of SEAP secreted.

HEK-Blue cells treated with SpyTag-NL-2, hIL-2 and TRAP-NL-2 control samples showed very similar SEAP production levels after stimulation, indicating that IL-2R binding was not affected by NL-2/15 conjugation to the TRAP loop and its modification with SpyTag (FIG. 20 a). Treatment of cells with empty TRAP cages did not lead to any signal transduction either before or after UV irradiation of the samples (fig. 20 b).

The SEAP yield was significant after treatment with NL-2 filled TRAP cage after UV irradiation (FIG. 20 b). Samples not treated with UV light were also able to signal transduction by IL-2R, but at much lower levels.

Protein yield:

using essentially the same protocol as mCherry fusion, a splice structure consisting of TRAP variants containing the K35C mutation, the N-terminal His6-SUMO, and SpyPatcher downstream of SUMO or between TRAP-K35C residues 47 and 48 (or TRAP-K35C, R S, d73K, d K for NL-2 encapsulation) was generated. Tetracycline (10 or 30 ng/ml) and ITPG (0.2 mM) were used to induce protein expression. After lysing the cells by sonication, the fusion proteins were purified from the soluble fraction using Ni-NTA affinity chromatography. Then, his6-SUMO units were cleaved from the full-length fusion by treatment with SUMO protease 1 (25 units/mg total protein) overnight at 4deg.C, followed by treatment with Ni-NTA agarose resin to remove unreacted material and His-tagged protease. The desired splice assembly was further purified by size exclusion chromatography. In SDS-PAGE analysis, the band intensity ratio was used to estimate the fidelity of the protein and the number of Spycatchers per 11 mer TRAP loop.

N-terminal His6 and Spy tagged GFP and Neoleukin-2/15, referred to as H-SpyT-GFP or H-SpyT-NL-2, were produced using E.coli BL21 (DE 3) strain transformed with pET 28-H-SpyT-GFP or pET 28-H-SpyT-NL-2, a pET 28-based plasmid with ColE origin of replication, kanamycin resistance gene, lac repressor and H-SpyT-GFP or H-SpyT-NL-2 under the control of the T7 promoter and lac operator system. Proteins were expressed using 0.2mM IPTG at 25℃for 20 hours and purified using Ni-NTA affinity chromatography and size exclusion chromatography.

Cage assembly and characterization: spliced TRAP loops containing SpyCatcher (400. Mu.M relative to TRAP monomer) were mixed with TPPMS-Au (I) -Cl (200. Mu.M) in 50mM sodium phosphate buffer (pH 8.0) containing 600mM NaCl (2M NaCl for spliced loops containing TRAP-K35C, R64S, d K, d K) and kept overnight at room temperature. The assembled cages were then separated using size exclusion chromatography. For the photo-openable cages, au (I) -mediated cages (200 μm relative to TRAP monomer) were added to DMF (final 5%) along with 1, 2-bromomethyl-3-nitrobenzene (300 μm,3 euiv.) and stirred at room temperature for 1 hour. Beta-mercaptoethanol (4. Mu.L) was then added to the reaction and stirred at room temperature for a further 30 minutes to quench unreacted benzyl bromide and remove Au (I). These small molecule reactants were removed by ultrafiltration using an Amicon ultra-4 centrifuge (30,000 molecular weight cut-off) and the resulting cages were used for packaging without further purification. In SDS-PAGE analysis, the band intensity ratio was used to estimate the number of Spycatchers per cage. The morphology of the isolated cages was checked using negatively stained TEM according to the protocol described above.

Guest loading (small scale): the spliced TRAP loop containing Spycatchers (20. Mu.M relative to Spycatchers) was mixed with H-SpyT-GFP (0-20. Mu.M) in PBS and kept overnight at room temperature. The reaction mixture was then analyzed by SDS-PAGE and non-denaturing-PAGE. For non-denaturing-PAGE analysis, blue excitation and emission filters (530/28 nm) on a Biorad ChemiDoc MP imager were used, and fast blue staining and fluorescence were used to visualize the bands.

Guest loading (large scale): the spliced TRAP loop containing SpyCatcher (20. Mu.M relative to SpyCatcher) was mixed with H-SpyT-GFP (20. Mu.M) or H-SpyT-NL-2 (40. Mu.M) in PBS and kept overnight at room temperature. The cages were then separated by size exclusion chromatography using a Superose 6 in create 10/300 column followed by TEM and spectroscopic analysis. TEM imaging was performed as described above. By using a UV-1900UV-Vis spectrophotometer (Shimadzu), an extinction coefficient was used: epsilon _{GFP 488} ＝52,700M ^-1 cm ^-1 、ε _{GFP 280} ＝26,850M ^-1 cm ^-1 、ε _{TRAP 280} ＝8250M ^-1 cm ^-1 (http://expasy.org/ tools/protparam.html) Absorbance was measured to estimate the number of objects per cage.

TABLE 3 plasmid information and amino acid sequences

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HEK-Blue-IL-2 reporter cell assay

HEK-Blue-IL-2 cells were cultured in DMEM-high glucose medium containing 10% FBS, 100U/mL penicillin, 100ug/mL streptomycin and 50ug/mL calcitonin. HEK-Blue supplementation in cells after 2 passages ^TM CLR selection and puromycin to ensure sustained transgene expression in cells. Before inoculation, the CLR selection medium was replaced with DMEM-high glucose medium containing 10% FBS, 100U/mL penicillin, 100ug/mL streptomycin (P/S) and 50ug/mL Normocin. Cells were separated from the surface of a culture flask (VWR) by air flow, centrifuged at 70xg for 8min, and resuspended in 2ml fresh medium without addition of Normocin. To assess the number, 10 μl of cell suspension was transferred to a counting plate (BioRad) and placed in a TC20 automated cell counter (BioRad). Cells were grown at 1X10 ⁴ Is inoculated on 96-well plates (VWR) in 180ul of medium at 37℃and 5% CO ₂ For 20 hours.

The proteins tested were prepared as 10x stock dilutions in DMEM-high glucose with 10% FBS. The next day HEK-Blue-IL-2 cells were stimulated by adding 20. Mu.l of protein at different concentrations and at 37℃and 5% CO ₂ And incubated for 24 hours. By combining QB-buffer and QB-reagent in sterile H ₂ Diluting 100x in O, and incubating for 10min with mild shaking in dark place to prepare Quanti-Blue ^TM A solution. 180ul of Quanti-Blue ^TM The solution was transferred to each well of a fresh 96-well plate and 20. Mu.l of HEK-Blue-IL-2 cell supernatant was added. Plates were incubated for 1 hour at 37 ℃. Secreted Embryonic Alkaline Phosphatase (SEAP) activity was assessed by absorbance measurement at 630 nm.

Reference to the literature

1 Malay,A.D.et al.An ultra-stable gold-coordinated protein cage displaying reversible assembly.Nature 569,438-442(2019).

2 Butterfield,G.L.et al.Evolution of a designed protein assembly encapsulating its own RNA genome.Nature 552,415-420(2017).

3 Edwardson,T.G.,Mori,T.&Hilvert,D.Rational Engineering of aDesigned Protein Cage for siRNA Delivery.J.Am.Chem.Soc.(2018).

4 Azuma,Y.,Zschoche,R.,Tinzl,M.&Hilvert,D.Quantitative packaging of active enzymes into a protein cage.Angew.Chem.Int.Ed.55,1531-1534(2016).

5 Dashti,N.H.,Abidin,R.S.&Sainsbury,F.Programmable in vitro coencapsidation of guest proteins for intracellular delivery by virus-like particles.ACS nano 12,4615-4623(2018).

6 B.,Pianowski,Z.&Hilvert,D.Efficient in vitro encapsulation of protein cargo by an engineered protein container.Journal of the American Chemical Society 134,909-911(2012).

7 Ho,A.,Schwarze,S.R.,Mermelstein,S.J.,Waksman,G.&Dowdy,S.F.Synthetic protein transduction domains:enhanced transduction potential in vitro and in vivo.Cancer research 61,474-477(2001).

8 Berry,C.C.Intracellular delivery of nanoparticles via the HIV-1 tat protein.Nanomedicine 3,357-365(2008).

9 Rampersad,S.N.Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays.Sensors 12,12347-12360(2012)。

Sequence listing

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Ser Glu Cys Leu Asp Lys Gly Glu Val Leu Ile Ala Gln Phe Thr Glu

35 40 45

His Thr Ser Ala Ile Lys Val Arg Gly Lys Ala Tyr Ile Gln Thr Ser

50 55 60

His Gly Val Ile Glu Ser Glu Gly Lys Lys

65 70

<210> 7

<211> 249

<212> PRT

<213> Artificial work

<220>

<223> GFP(-21)

<220>

<221> misc_feature

<222> (16)..(16)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (19)..(20)

<223> Xaa can be any naturally occurring amino acid

<400> 7

His His His His Gly Ser Ala Cys Glu Leu Met Val Ser Lys Gly Xaa

1 5 10 15

Glu Leu Xaa Xaa Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp

20 25 30

Val Asn Gly His Glu Phe Ser Val Arg Gly Glu Gly Glu Gly Asp Ala

35 40 45

Thr Glu Gly Glu Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu

50 55 60

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln

65 70 75 80

Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys

85 90 95

Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys

100 105 110

Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp

115 120 125

Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp

130 135 140

Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn Ser His Asp

145 150 155 160

Val Tyr Ile Thr Ala Asp Lys Gln Glu Asn Gly Ile Lys Ala Glu Phe

165 170 175

Glu Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His

180 185 190

Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp

195 200 205

Asp His Tyr Leu Ser Thr Glu Ser Ala Leu Ser Lys Asp Pro Asn Glu

210 215 220

Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile

225 230 235 240

Thr His Gly Met Asp Glu Leu Tyr Lys

245

<210> 8

<211> 12

<212> PRT

<213> Artificial work

<220>

<223> PTD4 derivatives

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> acetylation of N-terminal amino group

<400> 8

Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala Gly

1 5 10

<210> 9

<211> 11

<212> PRT

<213> Artificial work

<220>

<223> PTD4

<400> 9

Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala

1 5 10

<210> 10

<211> 335

<212> PRT

<213> Artificial work

<220>

<223> His6-mCherry-TRAP-K35C

<400> 10

Met His His His His His His Gly Gly Ser Ser Met Val Ser Lys Gly

1 5 10 15

Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys Val

20 25 30

His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu

35 40 45

Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val

50 55 60

Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln

65 70 75 80

Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro

85 90 95

Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val

100 105 110

Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser

115 120 125

Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn

130 135 140

Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu

145 150 155 160

Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu

165 170 175

Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu

180 185 190

Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala

195 200 205

Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr

210 215 220

Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly

225 230 235 240

Gly Met Asp Glu Leu Tyr Lys Leu Ser Glu Asn Leu Tyr Phe Gln Ser

245 250 255

Gly Gly Ser Gly Ser Ser Tyr Thr Asn Ser Asp Phe Val Val Ile Lys

260 265 270

Ala Leu Glu Asp Gly Val Asn Val Ile Gly Leu Thr Arg Gly Ala Asp

275 280 285

Thr Arg Phe His His Ser Glu Cys Leu Asp Lys Gly Glu Val Leu Ile

290 295 300

Ala Gln Phe Thr Glu His Thr Ser Ala Ile Lys Val Arg Gly Lys Ala

305 310 315 320

Tyr Ile Gln Thr Arg His Gly Val Ile Glu Ser Glu Gly Lys Lys

325 330 335

<210> 11

<211> 335

<212> PRT

<213> Artificial work

<220>

<223> His6-mOrange-TRAP-K35C

<400> 11

Met His His His His His His Gly Gly Ser Ser Met Val Ser Lys Gly

1 5 10 15

Glu Glu Asn Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys Val

20 25 30

Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu

35 40 45

Gly Glu Gly Arg Pro Tyr Glu Gly Phe Gln Thr Ala Lys Leu Lys Val

50 55 60

Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro His

65 70 75 80

Phe Thr Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro

85 90 95

Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val

100 105 110

Met Asn Tyr Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser

115 120 125

Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn

130 135 140

Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu

145 150 155 160

Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Lys

165 170 175

Ile Lys Met Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Thr Ser Glu

180 185 190

Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala

195 200 205

Tyr Ile Val Asp Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr

210 215 220

Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly

225 230 235 240

Gly Met Asp Glu Leu Tyr Lys Leu Ser Glu Asn Leu Tyr Phe Gln Ser

245 250 255

Gly Gly Ser Gly Ser Ser Tyr Thr Asn Ser Asp Phe Val Val Ile Lys

260 265 270

Ala Leu Glu Asp Gly Val Asn Val Ile Gly Leu Thr Arg Gly Ala Asp

275 280 285

Thr Arg Phe His His Ser Glu Cys Leu Asp Lys Gly Glu Val Leu Ile

290 295 300

Ala Gln Phe Thr Glu His Thr Ser Ala Ile Lys Val Arg Gly Lys Ala

305 310 315 320

Tyr Ile Gln Thr Arg His Gly Val Ile Glu Ser Glu Gly Lys Lys

325 330 335

<210> 12

<211> 300

<212> PRT

<213> Artificial work

<220>

<223> His6-SUMO-SpyCatcher-TRAP-K35C

<400> 12

Met His His His His His His Gly Ser Ser Met Ala Ser Met Lys Asp

1 5 10 15

His Leu Ile His Asn His His Lys His Glu His Ala His Ala Glu His

20 25 30

Leu Gly Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys

35 40 45

Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly

50 55 60

Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg

65 70 75 80

Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu

85 90 95

Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu

100 105 110

Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln

115 120 125

Ile Gly Gly Ser Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp

130 135 140

Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser

145 150 155 160

Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp

165 170 175

Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro

180 185 190

Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln

195 200 205

Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile

210 215 220

Gly Ser Ser Tyr Thr Asn Ser Asp Phe Val Val Ile Lys Ala Leu Glu

225 230 235 240

Asp Gly Val Asn Val Ile Gly Leu Thr Arg Gly Ala Asp Thr Arg Phe

245 250 255

His His Ser Glu Cys Leu Asp Lys Gly Glu Val Leu Ile Ala Gln Phe

260 265 270

Thr Glu His Thr Ser Ala Ile Lys Val Arg Gly Lys Ala Tyr Ile Gln

275 280 285

Thr Arg His Gly Val Ile Glu Ser Glu Gly Lys Lys

290 295 300

<210> 13

<211> 309

<212> PRT

<213> Artificial work

<220>

<223> His6-SUMO-TRAP-K35C

<400> 13

Met His His His His His His Gly Ser Ser Met Ala Ser Met Lys Asp

1 5 10 15

His Leu Ile His Asn His His Lys His Glu His Ala His Ala Glu His

20 25 30

Leu Gly Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys

35 40 45

Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly

50 55 60

Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg

65 70 75 80

Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu

85 90 95

Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu

100 105 110

Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln

115 120 125

Ile Gly Gly Ser Gly Ser Gly Gly Ser Ser Tyr Thr Asn Ser Asp Phe

130 135 140

Val Val Ile Lys Ala Leu Glu Asp Gly Val Asn Val Ile Gly Leu Thr

145 150 155 160

Arg Gly Ala Asp Thr Arg Phe His His Ser Glu Cys Leu Asp Lys Gly

165 170 175

Glu Val Leu Ile Ala Gln Phe Thr Gly Ser Ser Asp Ser Ala Thr His

180 185 190

Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala

195 200 205

Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile

210 215 220

Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr

225 230 235 240

Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile

245 250 255

Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys Ala

260 265 270

Thr Lys Gly Asp Ala His Ile Pro Gly Thr Glu His Thr Ser Ala Ile

275 280 285

Lys Val Arg Gly Lys Ala Tyr Ile Gln Thr Arg His Gly Val Ile Glu

290 295 300

Ser Glu Gly Lys Lys

305

<210> 14

<211> 117

<212> PRT

<213> Artificial work

<220>

<223> SpyTag-TRAP-K35C-srt-His6

<400> 14

Met Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gln Gly

1 5 10 15

Ser Gly Gly Ser Gly Ser Ser Tyr Thr Asn Ser Asp Phe Val Val Ile

20 25 30

Lys Ala Leu Glu Asp Gly Val Asn Val Ile Gly Leu Thr Arg Gly Ala

35 40 45

Asp Thr Arg Phe His His Ser Glu Cys Leu Asp Lys Gly Glu Val Leu

50 55 60

Ile Ala Gln Phe Thr Glu His Thr Ser Ala Ile Lys Val Arg Gly Lys

65 70 75 80

Ala Tyr Ile Gln Thr Arg His Gly Val Ile Glu Ser Glu Gly Lys Lys

85 90 95

Gly Thr Gly Gly Ser Leu Pro Ser Thr Gly Gly Ala Pro Val Glu His

100 105 110

His His His His His

115

<210> 15

<211> 268

<212> PRT

<213> Artificial work

<220>

<223> His6-SpyTag-GFP

<400> 15

Met Gly Ser Ser His His His His His His Gly Gly Ser Ala His Ile

1 5 10 15

Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Ser Gly Thr Ala Ser

20 25 30

Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu

35 40 45

Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu

50 55 60

Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr

65 70 75 80

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr

85 90 95

Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp

100 105 110

Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

115 120 125

Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe

130 135 140

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

145 150 155 160

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn

165 170 175

Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys

180 185 190

Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu

195 200 205

Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu

210 215 220

Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp

225 230 235 240

Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala

245 250 255

Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys

260 265

<210> 16

<211> 130

<212> PRT

<213> Artificial work

<220>

<223> His6-SpyTag-NL-2

<400> 16

Met Gly Ser Ser His His His His His His Gly Gly Ser Ala His Ile

1 5 10 15

Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Ser Gly Thr Pro Lys

20 25 30

Lys Lys Ile Gln Leu His Ala Glu His Ala Leu Tyr Asp Ala Leu Met

35 40 45

Ile Leu Asn Ile Val Lys Thr Asn Ser Pro Pro Ala Glu Glu Lys Leu

50 55 60

Glu Asp Tyr Ala Phe Asn Phe Glu Leu Ile Leu Glu Glu Ile Ala Arg

65 70 75 80

Leu Phe Glu Ser Gly Asp Gln Lys Asp Glu Ala Glu Lys Ala Lys Arg

85 90 95

Met Lys Glu Trp Met Lys Arg Ile Lys Thr Thr Ala Ser Glu Asp Glu

100 105 110

Gln Glu Glu Met Ala Asn Ala Ile Ile Thr Ile Leu Gln Ser Trp Ile

115 120 125

Phe Ser

130

<210> 17

<211> 72

<212> PRT

<213> Artificial work

<220>

<223> TRAP mutation K35C, R64S, dK73, dK74K

<400> 17

Met Tyr Thr Asn Ser Asp Phe Val Val Ile Lys Ala Leu Glu Asp Gly

1 5 10 15

Val Asn Val Ile Gly Leu Thr Arg Gly Ala Asp Thr Arg Phe His His

20 25 30

Ser Glu Cys Leu Asp Lys Gly Glu Val Leu Ile Ala Gln Phe Thr Glu

35 40 45

His Thr Ser Ala Ile Lys Val Arg Gly Lys Ala Tyr Ile Gln Thr Ser

50 55 60

His Gly Val Ile Glu Ser Glu Gly

65 70

Claims

1. An artificial TRAP cage comprising a selected number of TRAP loops and at least one guest cargo enclosed therein.

2. The cage of claim 1, wherein the guest cargo is selected from the group consisting of: proteins, enzymes, antigens, antibodies, protein macromolecules, lipids, peptides, nucleic acids, small molecule cargo, peptide nucleic acids, carbon-based structures, metals, toxins, or nanoparticles.

3. The cage of claim 2, wherein the nucleic acid is selected from the group consisting of DNA, RNA, mRNA, siRNA, tRNA and micrornas.

4. Cage according to claim 2, wherein the enzyme is an enzyme associated with overexpression in a metabolic disorder or disease or with underexpression in a metabolic disorder or disease.

5. The cage of claim 4, wherein the enzyme is selected from the group consisting of: hydrogenase, dehydrogenase, lipase, lyase, ligase, protease, transferase, reductase, recombinase, and nuclease acid-modifying enzymes.

6. The cage of claim 2, wherein the therapeutic agent is selected from the group consisting of: cancer therapeutic agents, anti-infective therapeutic agents, vascular disease therapeutic agents, immunotherapeutic agents, anti-aging agents, and neurological therapeutic agents.

7. Cage according to claim 2, wherein the metal is selected from the group comprising: iron, zinc, platinum, copper, sodium, cadmium, lanthanoid, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof.

8. The cage of claim 2, wherein the toxin is selected from the group consisting of: ligand targeting toxins, protease activated toxins, melittin hemolypeptides and toxin-based suicide gene therapeutics.

9. Cage according to any of the preceding claims, wherein the guest cargo is a protein, and preferably the protein is a fluorescent protein, interleukin-2 (IL-2) or Neoleukin-2/15 (NL-2).

10. Cage according to any of the previous claims, wherein the cage comprises a plurality of object cargo, and wherein the object cargo are identical or different from each other and are any combination of the cargo from claims 2 to 9.

11. The cage of any one of the preceding claims, further comprising at least one external ornament.

12. The cage of claim 11, wherein at least one of the external ornaments comprises a cell penetrating agent to facilitate intracellular delivery of the cage comprising an internal guest cargo.

13. The cage of claim 12, wherein the cell penetrating agent is PTD4.

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

15. Cage according to claim 14, wherein the number of TRAP loops in the TRAP cage is 12, 20 or 24, preferably 24.

16. A cage according to any one of the preceding claims, wherein the interior surface of the TRAP cage cavity is super charged and the TRAP cage protein comprises an E48Q or E48K mutation.

17. A cage according to any one of the preceding claims, wherein the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group comprising: including K35C, K35 3834 64S, E48Q, E48 4635C/R64S, K H/R64S, S33C, S33H, S C/R64S, S H/R64S, S C/K35H, S33H/K35H, S C/K35C, S H/K35C, K C/E48Q, K35C/E48K, K H/E48Q, K H/E48K, S C/E48Q, S C/E48K, S C/E48Q and S33C/E48K.

18. A TRAP cage according to any preceding claim, wherein the opening of the cage is programmable.

19. The TRAP cage of claim 18, wherein the programmable opening of the cage is dependent on the selection of molecular or atomic cross-linking agents that fix the TRAP ring in place in the TRAP cage.

20. The TRAP cage of claim 19, wherein the cross-linking agent is (i) a reduction-responsive/sensitive cross-linking agent, whereby the cage opens under reducing conditions; or (ii) a photoactivatable crosslinking agent, whereby the cage opens upon exposure to light.

21. Use of an artificial TRAP cage according to any of the preceding claims as a delivery vehicle for intracellular delivery of its internal guest cargo.

22. Use of an artificial TRAP cage according to any one of claims 1 to 20 as a vaccine.

23. Use of an artificial TRAP cage according to any one of claims 1 to 20 for the treatment of a disease or disorder selected from the group comprising: cancer, vascular disease, cardiovascular disease, diabetes, infection, autoimmune disorders, neurodegenerative disease, cell senescence disease, arthritis, and respiratory disease.

24. A method of making an artificial TRAP cage with encapsulated guest cargo, the method comprising:

(v) Purifying and separating the TRAP cage encapsulating the object cargo.

25. The method of claim 24, wherein the modification of step (iii) is selected from the group consisting of:

(i) Overcharging the interior surface of the TRAP cage;

(iv) Covalent bond formation is by both chemical and enzymatic methods.

26. A method according to claim 24 or 25, wherein step (ii) comprises first conjugating the TRAP ring units by means of at least one metal cross-linker, preferably an atomic metal cross-linker, followed by substitution of the metal cross-linker with a molecular cross-linker.

27. A method according to any one of claims 24 to 26, wherein the overcharging of the interior surface of step (i) provides a net positive or negative charge on the interior surface of the cage.

28. The method of any one of claims 24 to 27, wherein the artificial TRAP cage protein is modified to include any one or more of the following mutations selected from the group consisting of: including K35C, K35 3834 64S, E48Q, E48 4635C/R64S, K H/R64S, S33C, S33H, S C/R64S, S H/R64S, S C/K35H, S33H/K35H, S C/K35C, S H/K35C, K C/E48Q, K35C/E48K, K H/E48Q, K H/E48K, S C/E48Q, S C/E48K, S C/E48Q and S33C/E48K.

29. A method according to any one of claims 28, wherein the portion (iii) is for TRAP ^K35CE48Q The cage formation step of (2) is carried out in sodium bicarbonate buffer at pH 9-11.

30. A method according to any one of claims 28, wherein the portion (iii) is for TRAP ^K35CE48k The cage formation step of (2) is carried out in sodium bicarbonate buffer at pH 10-10.5.

31. The method of any one of claims 24 to 30, wherein the guest cargo may be loaded before or after assembly of the TRAP cage.

32. The method of any one of claims 24 to 31, wherein the genetic fusion of the guest cargo of step (ii) with the interior surface of the TRAP cage cavity is by way of TRAP of the guest cargo with the interior surface facing the cavity ^K35C N-terminal fusion at the N-terminus of (C).

33. The method of claim 32, wherein the guest cargo of step (iii) is conjugated to SpyCatcher/spyctag of an interior surface of the TRAP cage cavity, wherein the SpyCatcher is introduced into the annular region of the TRAP ring between residues 47 and 48, faces inward when assembled into the TRAP cage, and the guest cargo comprises spyctag.

34. The method of any one of claims 24 to 33, wherein the enzymatic modification is performed by a peptide ligase selected from the group consisting of a localizing enzyme, an asparaginase, a trypsin-related enzyme, and a subtilisin-derived variant, and the formation of covalent chemical bonds may include strain-promoted alkyne-azide cycloadditions and pseudopeptide bonds.

35. A TRAP cage produced by the method of any one of claims 24 to 34.

36. Use of a cage according to any one of claims 1 to 20 as a medicament.

37. A method of treating a patient comprising administering to the patient a cage according to any one of claims 1 to 20.

38. Cage according to any one of claims 1 to 20, for use in the treatment of a disease in a patient or as a vaccine.

39. An artificial TRAP cage protein modified to include any one or more of the following mutations selected from the group consisting of: K35C, K35 3834 35S, S4S, E48Q, E48 4635C/R64S, K H/R64S, S33C, S33H, S C/R64 3833H/R64S, S C/K35H, S33H/K35H, S C/K35C, S H/K35C, K C/E48Q, K35C/E48K, K H/E48Q, K H/E48K, S C/E48Q, S C/E48K, S C/E48Q and S33C/E48K.

40. A method of treating an individual in need of treatment suffering from a disorder selected from the group consisting of: a method of treating cancer, vascular disease, cardiovascular disease, diabetes, infection, autoimmune disorder, neurodegenerative and neurological disease, cell aging disease, arthritis, and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP cage with one or more internal guest cargo selected from the group comprising: nucleic acids, enzymes, therapeutic agents, small molecules, organic or inorganic nanoparticles, peptides, metals, antigens, antibodies and toxins, and therapeutically valuable fragments of all of the foregoing.

41. A method of vaccinating an individual in need of vaccination suffering from a disorder selected from the group consisting of: a method of treating cancer, vascular disease, cardiovascular disease, diabetes, infection, autoimmune disorder, neurodegenerative and neurological disease, cell aging disease, arthritis, and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP cage with one or more internal guest cargo selected from the group comprising: nucleic acids, enzymes, therapeutic agents, small molecules, organic or inorganic nanoparticles, peptides, metals, antigens, antibodies and toxins, and therapeutically valuable fragments of all of the foregoing.

42. The method of claim 40 or 41, wherein the TRAP cage therapeutic agent is administered by intranasal inhalation or injection.