Compositions and methods for the synthesis of vaults

Abstract

Compositions and methods for the in vitro synthesis of vaults are disclosed.

Description

[0001] COMPOSITIONS AND METHODS FOR THE SYNTHESIS OF VAULTS By

[0002] Rodney Burton

[0003] Stephen Thong

[0004] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 779,729, filed March 28, 2025. The foregoing application is incorporated by reference herein.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to the fields of vaults. More specifically, the invention provides compositions and methods for synthesizing vaults in vitro.

[0007] BACKGROUND OF THE INVENTION

[0008] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0009] Vaults, also known as vault cytoplasmic ribonucleoproteins, are very large protein complexes contained within the cytoplasm of eukaryotes. Vaults have a size of approximately 13 MDa (Kedersha, et al. (1991) J. Cell Biol., 112(2):225-35) and can measure about 70-75 nm in length and 40-45 nm in width (Kong, et al. (1999) Structure 7(4):371-9). The outer shell of the vault is composed of 78 copies of the major vault protein (MVP). Vaults also comprise telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VP ARP, also known as PARP4), and small vault RNAs in the interior.

[0010] Notably, vaults are highly conserved among different eukaryotic organisms, including mammals. However, the precise natural function of vaults is not fully understood although it is believed that vaults play a role in several cellular functions. Due to their generally hollow nature and large size (larger than most viruses), vaults have been considered for use as cargo vesicles or drug delivery devices. Moreover, vaults do not elicit damaging immune responses that are often observed with other drug delivery means such as viruses and synthetic carriers. However, one significant drawback to studying and using vaults is the inability to efficiently synthesize vaults in vitro due to their extraordinary size and the complex means of their formation within the cell. While it is possible to synthesize vaults within cells, subsequent purification of the vaults hasproven difficult. Accordingly, there is a strong need for improved in vitro methods for synthesizing vaults.

[0011] SUMMARY OF THE INVENTION

[0012] In accordance with the present invention, major vault protein conjugates are provided. In certain embodiments, the major vault protein conjugate comprises a major vault protein and a scaffold binding protein, wherein the scaffold binding protein is connected (e.g., to the C-terminus) of the major vault protein via a linker such as an amino acid linker. In certain embodiments, the major vault protein is from a mammal, particularly a human. In certain embodiments, the linker of the major vault protein conjugate comprises a protease cleavage site, optionally located near or at the N-terminus of the linker. In certain embodiments, the major vault protein conjugate further comprises at least one affinity tag, detectable agent, or peptide / polypeptide. In certain embodiments, the scaffold binding protein is a DNA binding protein such as Sso7d or protamine. In certain embodiments, the scaffold binding protein is a hyaluronic acid binding protein.

[0013] In accordance with another aspect of the instant invention, nucleic acid molecules encoding a major vault protein conjugate are provided. In certain embodiments, the nucleic acid molecule is contained within a vector, particularly an expression vector.

[0014] In accordance with another aspect of the instant invention, in vitro methods for synthesizing or forming vaults or vault-like nanoparticles are provided. In certain embodiments, the method comprises contacting or incubating a major vault protein conjugate with a scaffold (e.g., linear scaffold), wherein the scaffold binding protein of the major vault protein conjugate binds the scaffold. In certain embodiments, the scaffold is a linear scaffold. In certain embodiments, the method further comprises producing (e.g., recombinantly producing (e.g., in E. coli ) the major vault protein conjugate prior to contacting or incubating with the scaffold. In certain embodiments, the major vault protein conjugate is denatured prior to and / or during the contacting with the scaffold. In certain embodiments, the scaffold is a nucleic acid molecule. In certain embodiments, the scaffold is hyaluronic acid. In certain embodiments, at least one of telomerase-associated protein 1 (TEP1), vault poly(ADP -ribose) polymerase (VP ARP), and small vault RNAs is included in the synthesized vault or vault-like nanoparticle. In certain embodiments, at least one saber protein is included in the synthesized vault or vault-like nanoparticle. In certain embodiments, at least one molecule (e.g., cargo) is encompassed or encapsulatedby the synthesized vault or vault-like nanoparticle. In certain embodiments, more than one unique (e.g., different) major vault protein is contacted with the scaffold, thereby generating a heterogenous vault or vault-like nanoparticle. In certain embodiments, the method further comprises purifying and / or isolating the synthesized vault or vault-like nanoparticle. Vault or vault-like nanoparticles synthesized by the methods of the instant invention and compositions comprising the same are also encompassed by the instant invention.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 provides an image of an SDS-PAGE of purified MVP and tagged MVP variants. From left to right: MARY-MVP elution fraction 1, MARY-MVP elution fraction 2, MVP-HABP35 elution, MVP-CFP elution, MVP-YFP elution, MVP-Sso7d elution fraction 1, MVP-Sso7d elution fraction 2, and MVP with only the 6-His tag.

[0017] Figures 2A-2C provide dynamic light scattering representative histogram plots. Figure 2A: Representative completed refolding reaction of MVP-Sso7d. Figure 2B: Representative completed refolding reaction of MARY-MVP. Figure 2C: Representative completed refolding reaction of MVP-HABP35. All plots from Wyatt DYNAMICS 8 software.

[0018] Figures 3A-3H provide graphs of DLS results used to determine the hydrodynamic radius (nm) of MVP protein during refolding over time (minutes). Fug. 3 A: Untagged MVP (•), MVP-GFP (A), and Sso7d with 1.2 mg / mL copurified DNA (0). Fig. 3B: Sso7d-MVP vault formation with the addition of universal nuclease (+UN A) vs. control without universal nuclease (-UN A), both with 1.2 mg / ml copurified DNA. Fig. 3C: Time-resolved refolding of MARY-MVP in absence of nuclease (o). Fig. 3D: Refolding of MARY-MVP across a pH gradient. Fig. 3E: HABP35-MVP vault formation: 8 nm HABP35-MVP in the presence of 1 nm HA (o) or absence of HA (A).

[0019] Fig. 3F: Summary of DLS radii of vesicles formed from negative controls, untagged MVP and GFP-MVP, and from MVP with various scaffold-binding tags. The untagged MVP value was from n = 2 and lacks a standard deviation. Figure 3G provides a graph of the radius of vaults over time as determined by dynamic light scattering. Sso7d-MVP was incubated with the indicated amounts of DNA. Figure 3H provides a graph of the radius of vaults over time formed by placing Sso7d-MVP into refolding buffer (without preincubation and in the absence of added DNA) with universal nuclease (circle), without universal nuclease (square), or inactivated DNase II (triangle).Figure 4A provides TEM of VLPs formed with Sso7d-MVP scaffold-binding tag. Figure 4B provides TEM of VLPs formed with MARY-MVP. Scale bars are 40 nm and 75 nm in length. Figure 4C shows large irregular vesicle-like disordered complexes formed with untagged MVP.

[0020] Figures 5A-5E show FRET and DLS analysis of heterogeneous VLPs. Fig. 5 A: Rendering of heterogeneous vault particles with CFP-MVP and YFP-MVP co-inserted with MARY-MVP. Fig. 5B: Fluorescence intensity of FRET on VLPs produced by refolding denatured MVP variants. Solution MCY (top line), control

[0021] solution MY, and control solution CY. lex= 433 nm. Fig. 5C:DLS hydrodynamic radii of solutions MCY, MY, and CY. Fig. 5D: DLS hydrodynamic radii of varying concentrations of MARY-MVP at a fixed 8.3 nM concentration of CFP-INT. Fig. 5E: Peak fluorescence emission intensity at 480 nm of CFP-INT of varying concentrations of MARY-MVP at a fixed 8.3 nM concentration of CFP-INT. lex= 433 nm. Dashed line is linear trendline fit.

[0022] Figures 6A and 6B provide images of the Alphafold 3 simulations of fifteen Sso7d-MVP monomers in the absence and presence of DNA scaffold, respectively.

[0023] Figure 6C provides images of the Alphafold 3 simulation of in vitro folding of untagged MVP monomers, illustrating the untagged C-terminal tail folding into the interior.

[0024] DETAILED DESCRIPTION OF THE INVENTION Physiological vault formation in the cell involves the translation of multiple vault protein monomers simultaneously on a polyribosome (Michiels, et al. (2020) Cell Rep., 30(8):2834-2845). This allows vault proteins to assemble into the vault complex co-translationally while aligned along their long axis; properly aligning them positionally and rotationally to catalyze assembly. This mechanism also supports the exposure of the C-terminal tails (a flexible region comprising about 68 amino acids) of the major vault proteins on the exterior of the vault. Indeed, the C-terminus of the major vault protein is translated only after vault assembly has already begun. Therefore, the C-terminus of the major vault protein is sterically less likely to be incorporated into the interior of the vault. While the flexible nature of the C-terminus of the major vault protein makes visualization by X-ray crystallography very difficult, the exterior location of the C-terminus of the major vault protein has been further confirmed by the attachment of C-terminal tags such as binding tags and subsequent localization of the C-terminal tags outside of the vault.Mature vaults comprise the assembly of two half vaults into a singular mature vault. Cargo may be encapsulated with the vault during the assembly of the two half vaults into a mature vault. Moreover, fully assembled vaults may uncouple / couple the two half vaults to allow for cargo encapsulation and / or delivery. Conformation changes within the major vault protein may lead to disassociation of the two vault halves (Guerra, et al. (2022) Sci. Adv.,8:eabj7795).

[0025] This model of physiological vault formation and the major vault protein C-terminal exterior positioning contrasts sharply with in vitro refolding models from denatured major vault protein monomers. The lack of the polyribosome complex exponentially increases the degrees of freedom available to each major vault protein monomer, thereby making vault assembly far less favorable entropically. During in vitro refolding, C-terminal orientation can also be random. However, AlphaFold simulations (an artificial intelligence program which performs predictions of protein structures) of native multimers of eight major vault proteins predict that the C-terminal tail folds towards the interior (Fig. 6C). Accordingly, spontaneous in vitro vault self-assembly is either very inefficient or not possible.

[0026] Herein, in vitro scaffold-coordinated refolding of denatured major vault protein monomers into assembled vault-like nanoparticles was performed. DNA or hyaluronic acid-binding tags were added to the MVP monomers, allowing MVP to align rotationally and translationally along these linear molecules. This mimics the polyribosome assembly in vivo. Tagged MVP variants were expressed in E. coli and purified under denaturing conditions. Dynamic light scattering showed the formation of nanoparticles with a hydrodynamic radius of ~26 nm, consistent with the formation of vault-like nanoparticles. This was confirmed by transmission electron microscopy, FRET analysis, and cargo loading of CFP-INT fusion. CFP- and YFP -tagged MVP showed FRET only in the presence of MVP with a DNA-binding tag. This is the first successful instance of bioengineering of homogenous and heterogeneous vault-like nanoparticles, and at a much larger scale than current protocols.

[0027] In a particular embodiment, the present invention has attached the Sso7d DNA binding protein to the C-terminus of the major vault protein via a flexible linker. Sso7d is DNA binding protein 7d from the thermophilic archaea Saccharolobus solfataricus and can revert protein aggregation and mediate disassembly of protein aggregates (Guagliardi, et al. (2004) Biochem. J., 381(Pt 1) :249-55). Herein, it is demonstrated that the addition of a stable DNA binding domain such as Sso7d to the C-terminus of the major vaultprotein allows for in vitro reproduction of the physiological polyribosome assembly mechanism by attaching the major vault protein to a DNA strand. As Sso7d binds, the DNA acts as a molecular scaffold and provides for alignment of the tagged major vault protein monomers positionally and rotationally. The linker between major vault protein and the DNA binding domain prevents significant steric hindrance during assembly.

[0028] In accordance with the instant invention, major vault protein conjugates are provided. In certain embodiments, the major vault protein conjugates comprise a major vault protein and a scaffold binding protein (e.g., a linear scaffold binding protein). In certain embodiments, the major vault protein and scaffold binding protein are directly connected (e.g., by a bond such as a peptide bond). In certain embodiments, the major vault protein and scaffold binding protein are connected by a linker. In certain embodiments, the scaffold binding protein is connected, e.g., via the linker, to the N-terminus of the major vault protein. In certain embodiments, the scaffold binding protein is connected, e.g., via the linker, to the C-terminus of the major vault protein.

[0029] The major vault protein of the major vault protein conjugate of the instant invention can be natural or non-natural. The major vault protein of the major vault protein conjugate of the instant invention can be a variant or wild-type. The major vault protein of the major vault protein conjugate of the instant invention can be bioengineered. The major vault protein of the major vault protein conjugate of the instant invention can be from any species. In certain embodiments, the major vault protein is from a eukaryote. In certain embodiments, the major vault protein is from Leishmania. In certain embodiments, the major vault protein is from yeast. In certain embodiments, the major vault protein is from Drosophila (e.g., Drosophila melanogaster). In certain embodiments, the major vault protein is from any mammalian species. In certain embodiments, the major vault protein is human.

[0030] Gene ID: 9961 and GenBank Accession Nos. NM_001293204.1, NP_001280133.1, NM_001293205.1, NP_001280134.1, NM_005115.5, NP_005106.2, NM_017458.3, and NP_059447.2 provides examples of the amino acid and nucleotide sequences of wild-type human major vault protein. The human major vault protein may be any isoform. In certain embodiments, the human major vault protein is isoform 1 (e.g., NM_005115.5, NP_005106.2, NM_017458.3, NP_059447.2), isoform 2 (e.g., NM_001293204.1, NP_001280133.1), or isoform 3 (e.g., NM_001293205.1,

[0031] NP 001280134.1). In certain embodiments, the human major vault protein is isoform 1. In certain embodiments, the major vault protein comprises SEQ ID NO: 1 :1 MATEEFIIRI PPYHYIHVLD QNSNVSRVEV GPKTYIRQDN ERVLFAPMRM 51 VTVPPRHYCT VANPVSRDAQ GLVLFDVTGQ VRLRHADLEI RLAQDPFPLY 101 PGEVLEKDIT PLQWLPNTA LHLKALLDFE DKDGDKWAG DEWLFEGPGT 151 YIPRKEVEW EIIQATIIRQ NQALRLRARK ECWDRDGKER VTGEEWLVTT 201 VGAYLPAVFE EVLDLVDAVI LTEKTALHLR ARRNFRDFRG VSRRTGEEWL 251 VTVQDTEAHV PDVHEEVLGV VPITTLGPHN YCVILDPVGP DGKNQLGQKR 301 WKGEKSFFL QPGEQLEQGI QDVYVLSEQQ GLLLRALQPL EEGEDEEKVS 351 HQAGDHWLIR GPLEYVPSAK VEWEERQAI PLDENEGIYV QDVKTGKVRA 401 VIGSTYMLTQ DEVLWEKELP PGVEELLNKG QDPLADRGEK DTAKSLQPLA 451 PRNKTRWSY RVPHNAAVQV YDYREKRARV VFGPELVSLG PEEQFTVLSL 501 SAGRPKRPHA RRALCLLLGP DFFTDVITIE TADHARLQLQ LAYNWHFEVN 551 DRKDPQETAK LFSVPDFVGD ACKAIASRVR GAVASVTFDD FHKNSARIIR 601 TAVFGFETSE AKGPDGMALP RPRDQAVFPQ NGLWSSVDV QSVEPVDQRT 651 RDALQRSVQL AIEITTNSQE AAAKHEAQRL EQEARGRLER QKILDQSEAE 701 KARKELLELE ALSMAVESTG TAKAEAESRA EAARIEGEGS VLQAKLKAQA 751 LAIETEAELQ RVQKVRELEL VYARAQLELE VSKAQQLAEV EVKKFKQMTE 801 AIGPSTIRDL AVAGPEMQVK LLQSLGLKST LITDGSTPIN LFNTAFGLLG 851 MGPEGQPLGR RVASGPSPGE GISPQSAQAP QAPGDNHWP VLR

[0032] In certain embodiments, the major vault protein has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with a natural or wild-type major vault protein, particularly at least 90%, 95%, 97%, 99%, or 100% identity. In certain embodiments, the major vault protein has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO: 1, particularly at least 90%, 95%, 97%, 99%, or 100% identity. In certain embodiments, the major vault protein (e.g., a major vault protein variant) comprises at least one mutation compared to the wild-type sequence. The major vault protein variant may comprise one or more substitution mutations, insertions, and / or deletions.

[0033] As stated hereinabove, the major vault protein and scaffold binding protein may be connected by a linker. As used herein, a linker is generally a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches two compounds. The linker can be linked to any synthetically feasible position of the two compounds. In certain embodiments, the linker is flexible. In certain embodiments, the linker is a peptide / polypeptide. In certain embodiments, the length of the linker is at least 1, at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least7G, at least 80, at least 90, at least 100 or more amino acids. In certain embodiments, the length of the linker is fewer than about 200, about 175, about 150, or about 125 amino acids. In certain embodiments, the length of the linker is about 1 to about 100 amino acids, about 25 to about 75 amino acids, about 40 to about 60 amino acids, or about 50 amino acids. In certain embodiments, the polypeptide linker is rich in glycine and serine amino acids to increase flexibility (e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the linker is composed of glycine and / or serine amino acids). For example, a flexible linker may comprise repeating units comprising four glycine amino acids and one serine amino acid. In certain embodiments, the majority of the amino acids of the linker are glycine. In certain embodiments, the majority of the amino acids of the linker are glycine or serine. In certain embodiments, the linker comprises SEQ ID NO: 5:

[0034] GGGGGSLVPR GSGGGGSENL YFQSGGGGSL VPRGSGGGGG GGGGGGGGGG

[0035] In certain embodiments, the linker has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO: 5, particularly at least 90%, 95%, 97%, 99%, or 100% identity.

[0036] The linker may be non-degradable and may be a chemical structure which cannot be substantially cleaved or cleaved at all under physiological environments or conditions. In certain embodiments, the linker is degradable. In certain embodiments, the linker comprises a cleavage site. In certain embodiments, the cleavage site is a protease cleavage site. The protease which cleaves the cleavage site within the linker may be present in a cell (e.g., a cell into which a vault of the instant invention is delivered). In certain embodiments, the cleavage site of the linker is not cleaved by a protease present in a cell (e.g., a cell into which a vault of the instant invention is delivered). In certain embodiments, the cleavage site of the linker is cleaved by a protease in vitro. In certain embodiments, the cleavage site of the linker is cleaved by an exogenous protease. In certain embodiments, the cleavage sequence is not present within the major vault protein. In certain embodiments, the cleavage sequence is not present within the major vault protein or the scaffold binding protein.

[0037] The cleavage site may be at any location within the linker. In certain embodiments, the cleavage site is near or at a terminus of the linker such that it is near or adjacent to a terminus of the major vault protein in the major vault protein conjugate. In certain embodiments, the cleavage site is near or at the N-terminus of the linker such that it is near or adjacent to the C-terminus of the major vault protein in the major vaultprotein conjugate. In certain embodiments, the cleavage of the cleavage site results in the release of the major vault protein. Generally, the cleavage site will be completely within the linker. However, in certain embodiments, the cleavage site may span a terminus of the major vault protein and a terminus of the linker. In certain embodiments, the cleavage site may span the C-terminus of the major vault protein and the N-terminus of the linker. For example, the C-terminal Arg of the major vault protein may be the first Arg of a furin cleavage site. Preferably, cleavage still occurs within the linker. The released major vault protein may or may not have residual amino acids at a terminus (e.g., its C-terminus) from the linker. For example, the C-terminus of the released major vault protein may have 0, 1, 2, 3, 4, 5, or more residual amino acids from the linker and / or cleavage site after cleavage. In certain embodiments, the major vault protein is released without any additional or residual amino acids at its C-terminus. While it is preferable that at least the full-length major vault protein is released after cleavage, the instant invention also encompasses the release of the major vault protein wherein amino acids are cleaved from the C-terminus (e.g., 1, 2, 3, 4, or 5 amino acids), such as by cleavage of the protease.

[0038] Proteases and their cleavage sites are very well known in the art. In certain embodiments, the protease is specific (i.e., not a nonspecific cleaving enzyme).

[0039] Examples of proteases and their cleavage site include, without limitation: human rhinovirus (HRV) 3C (e.g., PreScission) (LEVLFQ|GP; SEQ ID NO: 8), enterokinase (DDDDK|; SEQ ID NO: 9), Factor Xa (IEGR|; SEQ ID NO: 10), tobacco etch virus (TEV) protease (ENLYFQ|(G / S); SEQ ID NO: 11), thrombin (LVPR|GS; SEQ ID NO: 12), furin (RXXR| (SEQ ID NO: 13) or RX(K / R)R| (SEQ ID NO: 14)), pepsin ((F / W / Y)|), papain ((hydrophobic)RK|), tobacco vein mottling virus (TVMV) protease (GTVAFG|S; SEQ ID NO: 15), granzyme B (IEXD|X(G / A); SEQ ID NO: 16), turnip mosaic virus protease (VXHQ|; SEQ ID NO: 17), caspases (e.g., caspase-1 (YVAD|; SEQ ID NO: 18)), and West Nile virus (WNV) protease ((K / R)R|GS; SEQ ID NO: 19).

[0040] The scaffold binding protein of the instant invention is any protein capable of binding the scaffold. A scaffold binding protein may reversibly bind a scaffold. In certain embodiments, the scaffold binding protein binds a scaffold under conditions which denature the major vault protein. In certain embodiments, the scaffold is a molecule or macromolecule having a three-dimensional structure. In certain embodiments, the scaffold is a linear scaffold. A linear scaffold is a chemical structure or molecular scaffold with a generally linear architecture (as opposed to, e.g., a branchedarchitecture). As used herein, a linear architecture does not mean a straight line. Rather, a linear architecture indicates a substantially or completely unbranched structure. The scaffold (e.g., linear scaffold) may be random, semi-circular, circular, or spherical. In certain embodiments, the scaffold (e.g., linear scaffold) is sequential. In certain embodiments, the scaffold (e.g., linear scaffold) is symmetrical. Examples of scaffolds include, without limitation, nucleic acid molecules (e.g., DNA or RNA, single-stranded or double-stranded), carbohydrates, polysaccharides, polymers (e.g., homopolymers, random copolymers, blended polymers, copolymers, or a block copolymers), other proteins or polypeptides, carbon nanotubes or nanorings, viruses, nanoparticles (e.g., gold particles), and quantum dots. As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind. In certain embodiments, the scaffold is a nucleic acid. In certain embodiments, the scaffold is DNA. In certain embodiments, the scaffold is double stranded DNA.

[0041] Generally, the size and / or length of the scaffold (e.g., linear scaffold) will be such that no more than about one vault can be assembled on the scaffold (e.g., linear scaffold). As stated hereinabove, a vault generally contains 78 major vault proteins. In certain embodiments, the scaffold (e.g., linear scaffold) is long enough or big enough to allow the binding of at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% or more of the 78 major vault proteins (major vault protein conjugates). However, as explained hereinabove, mature vault proteins comprise the assembly of two half vaults into a singular mature vault. Accordingly, in certain embodiments, the scaffold (e.g., linear scaffold) is long enough or big enough to allow binding of about half (e.g., about 39) of the major vault proteins (major vault protein conjugates). The scaffold (e.g., linear scaffold) may be bigger or longer than absolutely necessary to accommodate 39 major vault proteins (major vault protein conjugates) (e.g., long enough to bind about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more (major vault proteins (major vault protein conjugates)). The extended length or size of the scaffold (e.g., linear scaffold) allows for gaps between the binding sites of the major vault protein conjugates as well as extra scaffold (e.g., extra linear scaffold at the termini) which are too small to bind a major vault protein conjugate. For example, if the linear scaffold binding domain has a footprint of 10 nucleotides, then a linear scaffold of 390 nucleotidescould theoretically bind 39 major vault protein conjugates at the same time. However, the linear scaffold may be longer than the bare minimum 390 nucleotides in this hypothetical and may be, for example, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, or more nucleotides in length.

[0042] When the scaffold (e.g., linear scaffold) is a nucleic acid molecule, the scaffold binding protein (e.g., linear scaffold binding protein) can be, for example, a DNA binding protein or an RNA binding protein. The DNA binding protein or RNA binding protein may be any synthetic or natural DNA binding protein or RNA binding protein or a DNA binding domain or RNA binding domain thereof. DNA binding proteins or RNA binding proteins may bind single-stranded and / or double-stranded targets. DNA binding proteins or RNA binding proteins may be specific (bind a specific or consensus nucleic acid sequence) or non-specific (bind randomly to a nucleic acid molecule).

[0043] In certain embodiments, the scaffold binding protein (e.g., linear scaffold binding protein) is a DNA binding protein (or DNA binding domain thereof). DNA binding proteins may bind DNA and / or RNA. In certain embodiments, the DNA binding protein is nonspecific. DNA binding proteins are well known in the art. Examples of DNA binding proteins include, without limitation: transcription factors, histone proteins, leucine zipper proteins, helix-loop-helix proteins, homeodomain proteins, zinc finger proteins, single-stranded DNA binding proteins, peptide nucleic acids, and transcription activator-like effector proteins.

[0044] In certain embodiments, the DNA binding protein is a DNA binding protein of a thermophile or a thermoacidophile. Generally, proteins from thermophiles or thermoacidophiles are very stable and DNA binding proteins from thermophiles or thermoacidophiles can retain their DNA binding ability under conditions which can denature other proteins including other DNA binding proteins. In certain embodiments, the DNA binding protein is a 7kD DNA binding domain (e.g., from a thermophile) (e.g., Kalichuk, et al. (2016) Scientific Reports 6:37274). In certain embodiments, the DNA binding protein is from the genus Sulfolobus or Saccharolobus . In certain embodiments, the DNA binding protein is from Sulfolobus acidocaldarius . In certain embodiments, the DNA binding protein is from Saccharolobus solfataricus.

[0045] In certain embodiments, the DNA binding protein is Saccharolobus solfataricus DNA-binding protein 7d (Sso7d). Gene ID: 1454006 and GenBank Accession No.WP 009990119.1 provide examples of the amino acid and nucleotide sequences of Sso7d. In certain embodiments, the Sso7d comprises SEQ ID NO: 2:

[0046] 1 MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD 51 APKELLQMLE KQKK

[0047] In certain embodiments, the initial Met or Met-Ala is omitted from the sequence when part of the major vault protein conjugate (e.g., comprises amino acids 2-64 or 3-64 of SEQ ID NO: 2). In certain embodiments, the initial Met and Ala are omitted from the sequence when part of the major vault protein conjugate. In certain embodiments, the Sso7d has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO: 2, particularly at least 90%, 95%, 97%, 99%, or 100% identity. Notably, Sso7d is nonspecific and has a footprint or binding site on DNA of about 8 nucleotides.

[0048] In certain embodiments, the DNA binding protein is Sulfolobus acidocaldarius DNA-binding protein 7d (Sac7d). Gene ID: 78440419 and GenBank Accession No. WP 011276993 provide examples of the amino acid and nucleotide sequences of Sac7d. In certain embodiments, the Sac7d comprises SEQ ID NO: 3:

[0049] 1 MVKVKFKYKG EEKEVDTSKI KKVWRVGKMV SFTYDDNGKT GRGAVSEKDA 51 PKELLDMLAR AEREKK

[0050] In certain embodiments, the initial Met or Met-Val is omitted from the sequence when part of the major vault protein conjugate (e.g., comprises amino acids 2-66 or 3-66 of SEQ ID NO: 3). In certain embodiments, the Sac7d has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO: 3, particularly at least 90%, 95%, 97%, 99%, or 100% identity.

[0051] In certain embodiments, the DNA binding protein is a protamine, derivative thereof, or DNA-binding fragment thereof. In certain embodiments, the protamine is mammalian. In certain embodiments, the protamine is human. In certain embodiments, the protamine is sperm protamine, particularly sperm protamine Pl. GenBank Accession No. NP_002752 provides an example of the amino acid sequence of human sperm protamine Pl. In certain embodiments, the protamine comprises SEQ ID NO: 28:

[0052] 1 MARYRCCRSQ SRSRYYRQRQ RSRRRRRRSC QTRRRAMRCC RPRYRPRCRR 51 H

[0053] In certain embodiments, the protamine has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO: 28, particularly at least 90%, 95%, 97%, 99%, or 100% identity.When the scaffold is a polysaccharide, the scaffold binding protein can be, for example, a carbohydrate binding protein. Examples of polysaccharides as scaffolds (e.g., linear scaffolds) include, without limitation: homopolysaccharides (or homoglycans) and heteropolysaccharides. In certain embodiments, the scaffold (e.g., linear scaffold) polysaccharide is a homopolysaccharide of mannose, galactose, maltose, fucose, glucose, hyaluronic acid, N-acetylneuraminic acid, and N-acetylglucosamine. In certain embodiments, the scaffold (e.g., linear scaffold) comprises hyaluronic acid (e.g., a low molecular weight hyaluronic acid (e.g., about 50 kDa)). The carbohydrate binding protein may be any synthetic or natural carbohydrate binding protein or a carbohydrate binding domain thereof. In certain embodiments, the carbohydrate binding protein is a lectin (e.g., Raposo, et al. (2021) Biomolecules 11(2): 188). Examples of carbohydrate binding proteins include, without limitation, mannose binding proteins (also known as mannan-binding lectin), galactose binding proteins, maltose binding proteins, fucose binding proteins, glucose binding protein, hyaluronic acid binding protein, N-acetylneuraminic acid binding proteins, and N-acetylglucosamine binding proteins. In certain embodiments, the carbohydrate binding protein is hyaluronic acid binding protein. Examples of hyaluronic acid binding proteins include, without limitation: HABP35 (LKQKIKHVVKLKVVVKLRSQLVKRKQN; SEQ ID NO: 25), HABP42 (STMMSRSHKTRSHHV; SEQ ID NO: 26), and HABP52 (GAHWQFNALTVRGGGS; SEQ ID NO: 27). In certain embodiments, the hyaluronic acid binding protein comprises HABP35. In certain embodiments, the hyaluronic acid binding protein comprises SEQ ID NO: 25:

[0054] LKQKIKHWK LKVWKLRSQ LVKRKQN

[0055] In certain embodiments, the hyaluronic acid binding protein has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO: 25, particularly at least 90%, 95%, 97%, 99%, or 100% identity.

[0056] When the scaffold (e.g., linear scaffold) is a carbon nanotube (e.g., SWCNT or MWCNT) or nanoring, the scaffold binding protein (e.g., linear scaffold binding protein) can be, for example, a carbon nanotube or nanoring binding protein. Examples of carbon nanotube or nanoring binding proteins include, without limitation, fibrinogen, gamma globulin, transferrin, albumin (e.g., serum albumin such as bovine serum albumin), lysozyme, alpha-chymotrypsin, laccase, organophosphate hydrolase, tau protein, catalase, cytochrome c, and fibronectin.When the scaffold (e.g., linear scaffold) is a protein or a polypeptide, the scaffold binding protein (e.g., linear scaffold binding protein) can be, for example, protein which binds the protein or polypeptide. Examples of scaffold proteins or polypeptides (e.g., linear scaffold proteins or polypeptides) include, without limitation: proteins or polypeptides which form polymeric chains (such as through peptide or isopeptide bond formation). In certain embodiments, the scaffold (e.g., linear scaffold) comprises a chain of ubiquitin or ubiquitin-like (UBL) proteins. In certain embodiments, the scaffold (e.g., linear scaffold) comprises a chain of SUMO (small ubiquitin-like modifier) molecules. In certain embodiments, the scaffold binding protein (e.g., linear scaffold binding protein) is a SUMO-binding protein or a SUMO-interacting protein. In certain embodiments, the scaffold binding protein (e.g., linear scaffold binding protein) is a ubiquitin-binding protein or a ubiquitin receptor. In certain embodiments, the scaffold binding protein (e.g., linear scaffold binding protein) is an antibody or antigen-binding fragment thereof which binds the protein or polypeptide of the scaffold (e.g., linear scaffold).

[0057] When the scaffold is a virus, nanoparticle (e.g., gold particle), quantum dot, or other spherical scaffold, the scaffold binding proteins may bind to the surface of the scaffold. In certain embodiments, the scaffold binding protein binds the virus (e.g., a surface protein such as a capsid protein, envelope protein, or spike protein), nanoparticle (e.g., gold particle), or quantum dot. In certain embodiments, the scaffold binding protein binds a protein fused or anchored to the surface of the virus, nanoparticle (e.g., gold particle), or quantum dot. Examples of attaching proteins to nanoparticles and quantum dotes are well known in the art (e.g., Ellis et al. (2020) Catalysts 10(1): 83 ; Talbert et al. (2013) Food Biorpod. Process., 91(4):693-699).

[0058] In accordance with another aspect of the instant invention, the major vault protein conjugate comprises a major vault protein and a self-assembling protein or polypeptide. In other words, the scaffold binding protein (e.g., linear scaffold binding protein) is replaced by a self-assembling protein or polypeptide. Protein self-assembly is the spontaneous organization of protein building blocks into ordered structures (e.g., filaments or tubes). The ordered structures may be formed through covalent interactions or through non-covalent interactions including hydrophobic, electrostatic, hydrogen bonding, and van der Waals interactions. Because the self-assembling protein or polypeptide forms a scaffold or a linear scaffold, the presence of a separate scaffold or linear scaffold is not needed during formation of the vault. Thus, the features and methods of the instant invention apply to the major vault protein conjugates comprising aself-assembling protein or polypeptide except that the presence of a scaffold (e.g., linear scaffold) is unnecessary. For example, an in vitro vault formation may comprise incubating major vault protein conjugates comprising a major vault protein and a selfassembling protein or polypeptide, particularly denatured major vault protein conjugates. Examples of self-assembling proteins include, without limitation: actin, amyloid (e.g., amyloid P), collagen, elastin, and protein nanotube forming proteins (e.g., PduA and RmmH).

[0059] In certain embodiments, the major vault protein conjugate further comprises at least one polypeptide or protein of interest. In certain embodiments, the protein of interest is an enzyme. In certain embodiments, the protein of interest is a fluorescent protein. In certain embodiments, the protein of interest is a localization tag. The polypeptide or protein of interest may be connected directly to the major vault protein conjugate or via a linker. In certain embodiments, the polypeptide or protein of interest is connected directly to the scaffold binding protein or via a linker. In certain embodiments, the polypeptide or protein of interest is connected directly to the C-terminus of the scaffold binding protein or via a linker. In certain embodiments, the polypeptide or protein of interest is connected directly to the N-terminus of the major vault protein or via a linker. In certain embodiments, the polypeptide or protein of interest is present in or adjacent to (e.g., N-terminal or C-terminal) the linker connecting the major vault protein and the scaffold binding protein. When connected by a linker, the linker may comprise a protease cleavage site. The cleavage site may be the same or different than the cleavage site in the linker between the major vault protein and the scaffold binding protein.

[0060] In certain embodiments, the major vault protein conjugate further comprises at least one affinity tag. Affinity tags may also be referred to as purification tags or epitope tags and can be used to effect the purification of a protein of interest. The affinity tag may be connected directly to the major vault protein conjugate or via a linker. In certain embodiments, the affinity tag is connected directly to the scaffold binding protein or via a linker. In certain embodiments, the affinity tag is connected directly to the C-terminus of the scaffold binding protein or via a linker. In certain embodiments, the affinity tag is connected directly to the N-terminus of the major vault protein or via a linker. In certain embodiments, the affinity tag is present in or adjacent to (e.g., N-terminal or C-terminal) the linker connecting the major vault protein and the scaffold binding protein. When connected by a linker, the linker may comprise a protease cleavage site. The cleavagesite may be the same or different than the cleavage site in the linker between the major vault protein and the scaffold binding protein.

[0061] Affinity tags are well known in the art. In certain embodiments, the affinity tag is a peptide or polypeptide. In certain embodiments, the affinity tag is short (e.g., less than about 25, less than about 20, less than about 15, or less than about 10 amino acids).

[0062] Examples of affinity tags include, without limitation: polyhistidine tags (e.g. hexahistidine), polyarginine tags, glutathione-S-transferase (GST), S-tag, influenza virus HA tag, VSV-Gtag, SUMO, ubiquitin, thioredoxin, staphylococcal protein A tag, FLAG™ epitope, HaloTag®, AviTag™ epitope (for subsequent biotinylation), streptavidin-binding peptide (SBP)-tag, T7 tag, V5 tag, E2 epitope, mannose binding protein, an antibody epitope (e.g., a sequence of amino acids recognized and specifically bound by an antibody), myc epitope, and heme binding peptides.

[0063] In certain embodiments, the major vault protein conjugate further comprises at least one detectable agent. Detectable agents are well known in the art. Examples of detectable agents include, without limitation: isotopes (e.g., radioisotopes), imaging agents, fluorescent agents, and / or contrast agents. In certain embodiments, the detectable agent is a fluorescent protein (e.g., green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, etc.). The detectable agent may be connected directly to the major vault protein conjugate or via a linker. In certain embodiments, the detectable agent is connected directly to the C-terminus of the major vault protein conjugate or via a linker. In certain embodiments, the detectable agent is connected directly to the N-terminus of the major vault protein conjugate or via a linker.

[0064] Compositions comprising a major vault protein conjugate are also encompassed by the instant invention. In certain embodiments, the compositions further comprise a carrier.

[0065] Nucleic acids encoding the major vault protein conjugate are also encompassed by the instant invention. The major vault protein conjugate encoding nucleic acid molecules may be prepared by any method known in the art. Major vault protein conjugate encoding nucleic acid molecules of the invention include DNA and RNA and may be single- or double-stranded. For example, nucleic acid molecules encoding the major vault protein conjugate of the invention may be prepared by using recombinant DNA technology methods.The nucleic acid molecules may be maintained in any convenient vector, particularly an expression vector. For example, plasmids comprising the major vault protein conjugate encoding nucleic acid molecule are encompassed by the instant invention. In certain embodiment, the nucleic acids may be maintained in a vector suitable for expression in cells (e.g., E. colt). The vectors may comprise the regulatory elements necessary for expression of the major vault protein conjugate encoding nucleic acid molecule in the host cell. Such regulatory elements required for expression include, but are not limited to: promoter sequences, transcription initiation sequences, and enhancer sequences.

[0066] Compositions comprising a nucleic acid molecule encoding a major vault protein conjugate are also encompassed by the instant invention. In certain embodiments, the compositions further comprise a carrier.

[0067] Methods of synthesizing a vault are also encompassed by the instant invention. The vaults synthesized by the methods of the instant invention need not be canonical vaults or identical to vaults generated naturally in vivo. Rather, vaults synthesized by the methods of the instant invention encompass vault-like nanoparticles. The vaults synthesized by the methods of the instant invention do not need to comprise all vault components (other than MVP). The vaults synthesized by the methods of the instant invention may be misshapen compared to canonical vaults. Generally, a vault-like nanoparticle comprises an assembly of MVPs (particularly about 78 MVPs, although this number can vary) which approximates the outer shell of a canonical vault and provides a hollow interior which can hold cargo. In certain embodiments, the average radii of the vault-like nanoparticles is about 20 nm to about 30 nm. In certain embodiments, the average height of the vault-like nanoparticles is about 65 nm to about 80 nm. In certain embodiments, the average diameter or width of the vault-like nanoparticles is about 40 nm to about 45 nm. In certain embodiments, the vault-like nanoparticles comprise about 50 to about 100 MVPs, about 55 to about 95 MVPs, about 60 to about 90 MVPs, about 65 to about 90 MVPs, about 70 to about 85 MVPs, about 75 to about 80 MVPs or about 78 MVPs. In certain embodiments, the vault-like nanoparticles comprise at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 78 or more MVPs. In certain embodiments, the vault-like nanoparticles comprise less than about 100, less than about 95, less than about 90, less than about 85, less than about 80 or fewer MVPs.In certain embodiments, the methods of synthesizing vaults are in vitro methods (i.e., not performed in a cell). In certain embodiments, the method comprises contacting a major vault protein conjugate with a scaffold (e.g., linear scaffold). The major vault protein conjugate and the scaffold (e.g., linear scaffold) may be incubated together for sufficient time to allow the formation of the vault (e.g., inclusive of allowing the selfassembly of half-vaults, if present). In certain embodiments, the major vault protein conjugate and the scaffold are incubated together for at least 30 seconds, at least one minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes or more. In certain embodiments, the major vault protein conjugate and the scaffold are incubated together for less than one hour, less than 55 minutes, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, or less than 30 minutes. In certain embodiments, the method comprises concentrating half-vaults to promote self-assembly.

[0068] In certain embodiments, the method further comprises synthesizing the major vault protein conjugate. In certain embodiments, the major vault protein conjugate is produced recombinantly. In certain embodiments, major vault protein conjugate is recombinantly produced in E. coli. The method may further comprise purifying or isolating the major vault protein conjugate after synthesis. In certain embodiments, the major vault protein conjugate is purified using the affinity tag, if present.

[0069] The method may further comprise contacting and / or incubating the major vault protein conjugate with a nuclease (e.g., DNase and / or RNase) prior to contacting or incubation with the scaffold (e.g., linear scaffold). In certain embodiments, the method further comprises removing the nuclease before contacting or incubation with the scaffold (e.g., linear scaffold).

[0070] The method may further comprise denaturing the major vault protein conjugate prior to and / or during contacting or incubation with the scaffold (e.g., linear scaffold). Removal of the denaturing conditions allows for vault formation. In certain embodiments, the major vault protein conjugate is contacted or incubated with a chemical denaturant. In certain embodiments, the chemical denaturant is chaotropic agent.

[0071] Examples of chaotropic agents include, without limitation: urea, guanidine hydrochloride (GuHCl), lithium perchlorate and sodium dodecyl sulfate (SDS). In certain embodiments, the major vault protein conjugate is denatured with urea. In certain embodiments, the major vault protein conjugate is contacted or incubated with a non-chemical denaturant.Examples of non-chemical denaturants include, without limitation: heat, pressure, sheer mechanical forces, sonication or ultrasonication, and pH changes.

[0072] In another embodiment of the instant invention, the methods of synthesizing vaults comprises contacting a major vault protein as described herein with a scaffold (particularly a linear scaffold, more particularly a ring or loop shaped scaffold) as described herein, wherein the scaffold has been modified to comprise interaction (INT) domains and / or anti-MVP antibodies or antigen-binding domains thereof. In certain embodiments, the INT domains and / or anti-MVP antibodies or antigen-binding domains thereof are attached to the scaffold at intervals or are evenly spaced along the scaffold. In certain embodiments, the INT domains and / or anti-MVP antibodies or antigen-binding domains thereof are attached to the scaffold directly or via a linker as described herein. The presence of the INT domains and / or anti-MVP antibodies or antigen-binding domains thereof will bind the MVP and bring them into proximity to form vaults or vaultlike nanoparticles.

[0073] The synthesis methods of the instant invention may further comprise adding other vault components to the vault. In certain embodiments, the method comprises adding one, two or all three of telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VP ARP, also known as PARP4), and small vault RNAs to the incubation step of the major vault protein conjugate with the scaffold (e.g., linear scaffold). In certain embodiments, the method comprises contacting the synthesized vault with one, two or all three of TEP1, VP ARP, and small vault RNAs. In certain embodiments, the method comprises adding TEPE In certain embodiments, the method comprises adding VP ARP. In certain embodiments, the method comprises adding small vault RNAs. In certain embodiments, the method comprises adding TEP1 and VP ARP. In certain embodiments, the method comprises adding TEP1 and small vault RNAs. In certain embodiments, the method comprises adding VP ARP and small vault RNAs. In certain embodiments, the method comprises adding TEP1, VP ARP, and small vault RNAs.

[0074] The TEP1, VP ARP, and / or small vault RNAs can be a variant or wild-type. The TEP1, VP ARP, and / or small vault RNAs can be bioengineered. The TEP1, VP ARP, and / or small vault RNAs can be from any species. In certain embodiments, the TEP1, VP ARP, and / or small vault RNAs are from the same species as the major vault protein. In certain embodiments, the TEP1, VP ARP, and / or small vault RNAs are from a species other than that of the major vault protein. In certain embodiments, the TEP1, VP ARP, and / or small vault RNAs are from a eukaryote. In certain embodiments, the TEP1,VP ARP, and / or small vault RNAs are from Leishmania. In certain embodiments, the TEP1, VP ARP, and / or small vault RNAs are from yeast. In certain embodiments, the TEP1, VP ARP, and / or small vault RNAs are from Drosophila (e.g., Drosophila melanogaster). In certain embodiments, the TEP1, VP ARP, and / or small vault RNAs are from any mammalian species. In certain embodiments, the TEP1, VP ARP, and / or small vault RNAs are human.

[0075] The methods may further comprise adding peptides, polypeptides, or proteins of interest to the vault. The peptides, polypeptides, or proteins of interest may be added during the incubation step of the major vault protein conjugate with the scaffold (e.g., linear scaffold). In certain embodiments, the method comprises contacting the synthesized vault with the peptides, polypeptides, or proteins of interest.

[0076] In certain embodiments, the method comprises adding a saber protein (e.g., from Leishmania') to the incubation step of the major vault protein conjugate with the scaffold (e.g., linear scaffold). In certain embodiments, the method comprises contacting the synthesized vault with a saber protein (e.g., from Leishmania).

[0077] Saber proteins are proteins that are generally encoded by a gene within the gene cluster containing the other proteins in vaults. The saber protein has been shown to be able to incorporate into vaults. The saber protein may stabilize the synthesized vault and can add functionality by conjugation to another protein or affinity tag or detectable tag (e.g., at the N- or C-terminus of the saber polypeptide). The saber polypeptide typically localizes to the top and bottom of the barrel-shaped vault and can provide a structural barrier aiding the sequestration of molecules within the

[0078] vault’s cavity.

[0079] The saber protein can be a variant or wild-type. The saber protein can be bioengineered. The saber protein can be from any species. In certain embodiments, the saber protein is from the same species as the major vault protein. In certain embodiments, the saber protein is from a species other than that of the major vault protein. In certain embodiments, the saber protein is from a eukaryote. In certain embodiments, the saber protein is from Leishmania. In certain embodiments, the saber protein is from yeast. In certain embodiments, the saber protein is from Drosophila (e.g., Drosophila melanogaster). In certain embodiments, the saber protein is from Trypanosomal (e.g., Trypanosomal brucei). In certain embodiments, the saber protein is from any mammalian species. In certain embodiments, the saber protein is human.The Leishmania saber polypeptide comprises three distinct domains - 1) an alphahelical domain (high flexibility and the bulk of the N-terminal region), 2) a globular domain, and 3) a shorter alpha helical domain at the C-terminus. The dynamic nature of the N-terminal helix reveals a role in facilitating and / or controlling vault opening and closing - and thus effect the loading and release of cargo in the vault. The C-terminal globular and alpha-helical regions within the dome of the vault indicates a role in structural stabilization. In certain embodiments, the Leishmania saber polypeptide comprises SEQ ID NO: 4:

[0080] 1 MQPSPRGKTT GVYGAERIPG NYVKTLTGW RGEELFCRPD LERPWRSADK 51 QGSAATIALK EKEIEELMRL CSNLRTELSN TKSDSLFLEY SLTERTKLQL 101 AEKQNEVDAL QRRVEVLEKE KKEAAAATES QIRLLRSQAE NESIGFKNEA 151 RVAIERCAEL VKTFQKQLED VKAAGVREVE FLEARFAAKV NEVTTELKSV 201 RESKKALEEH YRAVEAQSSN MVHRVEADYK ERLRSLEQRI EEQRLEYEAQ 251 LAKERKEKEA WGESKRTTE KVAVLIADKE EFLQKQKLWN SYILSHLDTF 301 YHSFIAVAPE LAVDPTGVQL QQVPELYAPR CVLEDPESKV NVERIAYRVA 351 QLKLLKALQP LGEAAARRGA KPPSAAQLDQ WESQERLWR ALRELEDQQS 401 ELEATVRNFT SRLYFFSDNL EESLRCGPRP VPPPLRDWF LCLGVPNGRV 451 LWAANTELMR TSVQLLYSTL RLKMGEYGAY ECYSDDVSML LAFADATAAC 501 RFCTESQEWL MRLPWPAALL KLPEAQEERA EGGRLVYRGL RVSMALHAGE 551 AYVEPSGIPL NGVYRNHYYG KAVSQLVHLC SLTQGGQIIV SIAAWNLCMR 601 RQHELGAWA KSLGTLPIVS FNSQSGVQEK QSIELLQILP IELEGRTFTP 651 PGKAWPPVS SLTGVKESVL AAEVAAVEAQ RERVHDALAI LQEECNSIQS 701 SMGTLVARSR AALPHFHLLP PSEMVAQLND LYSVMERVAV RAEELYTDLQ 751 DVAHAQEELG AQAQSIKDYF QQQEASAARE DDLRTKTEW RRSMNHTLQL 801 EKDRRRAETE RLQLALQDRD QLIRKLHQES QSS

[0081] In certain embodiments, the saber polypeptide has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO: 4, particularly at least 90%, 95%, 97%, 99%, or 100% identity.

[0082] The methods may further comprise isolating or purifying the synthesized vaults. The synthesized vaults may be purified using size exclusion filtration or sucrose gradient or other means. In certain embodiments, the vault is purified using the affinity tag, if present. Purified or isolated vaults may be placed in a new composition. The composition comprising the vaults may be frozen, lyophilized, and / or freeze-dried.The methods may further comprise contacting the major vault protein conjugate and / or vault with a protease which cleaves the cleavage site. In certain embodiments, the cleavage occurs during and / or after vault formation. In certain embodiments, the cleavage occurs after vault purification or isolation.

[0083] In certain embodiments, the methods of the instant invention generate heterogenous vaults. The physiological formation of vaults on a polyribosome translating a single gene inherently makes the physiological vault proteins homogenous. The method of the instant invention method allows for the production of heterogeneous vaults. In certain embodiments, the method comprises more than one (e.g., at least 2, 3, 4, 5, or more) unique major vault protein conjugate with the scaffold (e.g., linear scaffold). In certain embodiments, the method comprises incubating a first major vault protein conjugate and a second major vault protein conjugate with the scaffold (e.g., linear scaffold), wherein the first major vault protein conjugate and the second major vault protein conjugate are not the same. In certain embodiments, the first major vault protein conjugate and the second major vault protein conjugate are different isoforms. In certain embodiments, the first major vault protein conjugate and the second major vault protein conjugate are from different species. In certain embodiments, the first major vault protein conjugate and the second major vault protein conjugate comprise a different scaffold binding protein and / or a different scaffold binding protein location (e.g., C-terminus or N-terminus). In certain embodiments, the different scaffold binding proteins can bind the same scaffold. For example, the first major vault protein conjugate comprises Sso7d (e.g., SEQ ID NO: 2; e.g., at the C-terminus) and the second major vault protein conjugate comprises protamine (e.g., SEQ ID NO: 28; e.g., at the N-terminus). In certain embodiments, the first major vault protein conjugate and the second major vault protein conjugate comprise a different tag (e.g., a detectable tag). In certain embodiments, the first major vault protein conjugate and the second major vault protein conjugate comprise the same major vault protein but have a different tag (e.g., a detectable tag) or one lacks a tag. The presence of the tags can allow for cell tissue localization and / or fluorescent visualization.

[0084] As explained hereinabove, vaults may be used as delivery vehicles based on their size and hollow nature. The cargo sequestered inside the vault may be retained within the delivery vehicle and may be slowly released at the target tissue and / or cell. The methods of the instant invention may further comprise adding cargo to the vault. The cargo can be any entity or molecule including, without limitation: a protein, polypeptide, antibody,drug, small molecule, biological, pharmaceutical, nucleic acid molecule, and the like. In certain embodiments, the method comprises adding the cargo to the incubation step of the major vault protein conjugate with the scaffold (e.g., linear scaffold). In certain embodiments, the method comprises contacting or incubating the synthesized vault with the cargo. In certain embodiments, the vault can have a targeting moiety such as an antibody, nanobody, or receptor ligand which targets a cell structure such as an extracellular cell structure to achieve delivery of the cargo to desired cells and / or tissue.

[0085] Compositions comprising a vault of the instant invention are provided. In certain embodiments, the compositions further comprise a carrier such as a pharmaceutically acceptable carrier. In certain embodiments, the vault comprises or is loaded with a cargo (e.g., a protein, polypeptide, antibody, drug, small molecule, biological, pharmaceutical, nucleic acid molecule, and the like).

[0086] In certain embodiments, the invention is defined by the following numbered paragraphs:

[0087] 1. A maj or vault protein conjugate comprising a maj or vault protein and a scaffold binding protein,

[0088] wherein the scaffold binding protein is connected to the major vault protein via an amino acid linker.

[0089] 2. The major vault protein conjugate of paragraph 1, wherein the scaffold binding protein is connected to the C-terminus of the major vault protein via an amino acid linker

[0090] 3. The major vault protein conjugate of paragraph 1 or 2, wherein the major vault protein is human.

[0091] 4. The major vault protein conjugate of any one of paragraphs 1-3, wherein the amino acid linker comprises a protease cleavage site.

[0092] 5. The major vault protein conjugate of paragraph 4, wherein the protease cleavage site is at the N-terminus of the amino acid linker.

[0093] 6. The major vault protein conjugate of any one of paragraphs 1-5, further comprising at least one affinity tag.7. The major vault protein conjugate of any one of paragraphs 1-6, further comprising at least one detectable agent.

[0094] 8. The major vault protein conjugate of any one of paragraphs 1-7, wherein the scaffold binding protein is a DNA binding protein.

[0095] 9. The major vault protein conjugate of paragraph 8, wherein the DNA binding protein is from a thermophile or thermoacidophile.

[0096] 10. The major vault protein conjugate of paragraph 8, wherein the DNA binding protein is Sso7d.

[0097] 11. The major vault protein conjugate of paragraph 8, wherein the DNA binding protein is protamine.

[0098] 12. The major vault protein conjugate of any one of paragraphs 1-7, wherein the scaffold binding protein is a hyaluronic acid binding protein.

[0099] 13. The major vault protein conjugate of any one of paragraphs 1-7, wherein the scaffold binding protein is a linear scaffold binding protein.

[0100] 14. A nucleic acid molecule encoding a major vault protein conjugate of any one of paragraphs 1-13.

[0101] 15. A vector comprising the nucleic acid molecule of paragraph 14.

[0102] 16. An in vitro method for synthesizing a vault-like nanoparticle, the method comprising contacting a major vault protein conjugate of any one of paragraphs 1-13 with a scaffold,

[0103] wherein the scaffold binding protein of the major vault protein conjugate binds the scaffold.

[0104] 17. The method of paragraph 16, wherein the scaffold is a linear scaffold.18. The method of paragraph 16, wherein the scaffold is selected from the group consisting of nucleic acid molecules, carbohydrates, polysaccharides, polymers, proteins or polypeptides, carbon nanotubes or nanorings, viruses, nanoparticles, and quantum dots.

[0105] 19. The method of any one of paragraphs 16-18, wherein the method further comprises producing the major vault protein conjugate prior to the contacting step.

[0106] 20. The method of paragraph 19, wherein the major vault protein conjugate is recombinantly produced in E. coli.

[0107] 21. The method of any one of paragraphs 16-20, wherein the major vault protein conjugate is denatured prior to and / or during the contacting with a scaffold.

[0108] 22. The method of any one of paragraphs 16-21, wherein the scaffold is a nucleic acid molecule.

[0109] 23. The method of any one of paragraphs 16-21, wherein the scaffold is hyaluronic acid.

[0110] 24. The method of any one of paragraphs 16-23, further comprising adding at least one of telomerase-associated protein 1 (TEP1), vault poly(ADP -ribose) polymerase (VP ARP), and small vault RNAs to the major vault protein and scaffold.

[0111] 25. The method of any one of paragraphs 16-24, further comprising adding at least one saber protein to the major vault protein and scaffold.

[0112] 26. The method of any one of paragraphs 16-25, further comprising adding at least one molecule to the major vault protein and scaffold, wherein the molecule is encompassed or encapsulated by the synthesized vault.

[0113] 27. The method of any one of paragraphs 16-26, wherein more than one unique major vault protein is contacted with the scaffold.28. The method of any one of paragraphs 16-27, further comprising purifying and / or isolating the synthesized vault.

[0114] 29. The method of any one of paragraphs 16-28, wherein the amino acid linker comprises a protease cleavage site and wherein the method further comprises cleaving the protease cleaving site.

[0115] 30. The method of paragraph 29, wherein the cleavage occurs after vault formation.

[0116] 31. A vault-like nanoparticle synthesized by the method of any one of pargraphs 16-30.

[0117] Definitions

[0118] The following definitions are provided to facilitate an understanding of the present invention.

[0119] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0120] With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. For example, the “isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote. With respect to RNA molecules of the invention, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form.

[0121] With respect to protein, the term “isolated protein” is sometimes used herein. This term may refer to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated (e.g., so as to exist in “substantially pure” form). “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence ofimpurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

[0122] The term “vector” refers to a carrier nucleic acid molecule (e.g., RNA or DNA) into which a nucleic acid sequence can be inserted for introduction into a host cell or for replication. Examples of vectors include, without limitation: plasmids, bacteriophage, viruses, cosmids, and artificial chromosomes. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions (e.g., promoter) needed for expression in a host cell.

[0123] The term “operably linked” means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.

[0124] The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.), particularly at least 75% by weight, or at least 90-99% or more by weight of the compound of interest. Purity may be measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0125] “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0126] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered.

[0127] Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0128] As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 2,000 Da or less than 1,000 Da). Typically, small molecules are organic. A macromolecule may be a substance or compound having a molecular weight greater than or equal to 50 kDa or more (e.g., more than 100 kDa, or more than 200 kDa). A macromolecule may be composed of many smaller repeating units (e.g., monomers) linked together.

[0129] An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule (e.g., antigen-binding fragment), and fusions of immunologically active portions of an immunoglobulin molecule. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (Dab; e.g., single variable light or heavy chain domain), Fab, Fab', F(ab')2, and F(v); and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv?, scFv-Fc, minibody, diabody, triabody, and tetrabody.

[0130] As used herein, the term “immunologically specific” refers to proteins / polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

[0131] The term “detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and / or measured (e.g., by quantitation).The following example is provided to illustrate various embodiments of the present invention. The example is illustrative and is not intended to limit the invention in any way.

[0132] EXAMPLE

[0133] The vault shell is composed of 78 copies of the major vault protein (MVP) and is the largest known eukaryotic protein nanocapsule, with approximately 10,000 vaults present in human cells (Taube, et al., Int. J. Mol. Sci. (2024) 25:4072; Casanas, et al., Curr. Opin. Biotechnol. (2012) 23:972-977). Despite decades of research since its initial discovery in 1986, the direct function of the vault protein remains poorly characterized, although it has been correlated with immune system activation and cancer defense (Kedersha, et al., J. Cell Biol. (1986) 103:699-709; Mossink, et al., Oncogene (2003) 22:7458-7467; Ma, et al., Biochim. Biophys. Acta Mol. Basis Dis. (2024) 1870:167441; Zhou, et al., Cancer Res. Prev. Treat. (2025) 52: 118-126). The vault is also an attractive structure for bioengineering as a drug delivery vehicle because it has a larger cargo capacity than current viral delivery vehicles, with far less danger of immune system inflammation since it is of human origin (Martin, et al., Biofabrication (2022) 14:025018; Kickhoefer, et al., Proc. Natl. Acad. Sci. (2005) 102:4348-4352; Munoz-Juan, et al., Pharmaceutics (2019) 11 : 300). In addition, it easily dissociates in a low-pH environment. The difficulty in studying and bioengineering vaults is directly linked to the difficulty of purifying vaults in sufficient quantities. Current standard vault purification protocols are long, costly, laborious, and prohibitive at scale, involving extraction from insect cells or yeast via ultracentrifugation (Demchuk, et al., Biotechnol. Adv.

[0134] (2020) 41:107547; Stephen, et al., J. Biol. Chem. (2001) 276:23217-23220; Mrazek, et al., ACS Nano (2014) 8:11552-11559; Llauro, et al., Sci. Rep. (2016) 6:34143; Wang, et al., Biotechnol. Bioeng. (2018) 115:2941-2950; Ding, et al., Structure (2018) 26:619-626; Ma, et al., Biochim. Biophys. Acta Mol. Basis Dis. (2024) 1870:167441). In addition, when assembled in vivo, the polyribosome-based assembly prohibits the bioengineering of heterogeneous vaults (Mrazek, et al., ACS Nano (2014) 8: 11552-11559). The only current successful labeling of vault nanoparticles has been through chemical modification post-purification (Benner, et al., ACS Nano (2017) 11:872-881).

[0135] The primary model for the physiological mechanism of vault formation involves the simultaneous translation of multiple MVP monomers on a polyribosome-mRNA complex (Mrazek, et al., ACS Nano (2014) 8:11552-11559). This allows vaults toassemble co-translationally while being properly aligned by the ribosomes, both translationally and rotationally, to allow for efficient vault complex assembly. This biochemical mechanism explains why attempts to refold denatured MVP monomers into self-assembled vault shells in vitro have been unsuccessful (Stephen, et al., J. Biol. Chem. (2001) 276:23217-23220.). Without the polyribosome scaffold limiting the MVP degrees of freedom, vault self-assembly is entropically less favorable. Polyribosome-based assembly in vivo also requires that any protein sequence change in an MVP monomer must be present in all 78 MVPs of a given vault. This limits bioengineering, as N- and C-terminal protein tags easily become structurally disruptive when present in such large quantities (Mrazek, et al., ACS Nano (2014) 8:11552-11559).

[0136] Herein, it was hypothesized that the polyribosome assembly mechanism could be mimicked in vitro by attaching fully translated MVP proteins to a linear scaffold using a scaffold-binding tag to form vault-like nanoparticles (VLPs). This was tested against two alternative linear scaffolds and three different scaffold-binding tags linked to MVP monomers. This approach bioengineers hollow VLPs of similar size and function to canonical vaults.

[0137] Sso7d from the thermophilic Saccharolobus solfataricus was used as a C-terminal DNA-binding tag for MVP refolding on a DNA scaffold. Sso7d binds DNA with a small footprint of 4-5 bp (Lundback, et al., J. Mol. Biol. (1998) 276:775-786), allowing for close alignment of the MVP monomers next to each other. Sso7d has also been shown to act as a refolding tag with the ability to revert protein aggregation (Guagliardi, et al., Biochem. J. (2004) 381:249-255).

[0138] Protamine was used as an N-terminal DNA-binding tag for MVP scaffold-based refolding. Protamine was chosen based on its theorized ability to unite vault halves through the simultaneous binding of two halves to the same DNA strand via the protamine. Protamine is also observed to bend DNA into a loop, thereby facilitating vault packaging (McMillan, et al., Biophys. J. (2023) 122:4288-4302). Protamine-tagged MVP is hereafter referred to as MARY-MVP. However, N-terminal tags have also been shown to disrupt vault structure and may be less ideal as a scaffold (Mrazek, et al., ACS Nano (2014) 8:11552-11559).

[0139] To test for potential DNA-specific artifacts, an alternate scaffold-tag pair was tested: the hyaluronic acid (HA) scaffold and MVP with the HA-binding tag HABP35 (Zaleski, et al., Antimicrob. Agents Chemother. (2006) 50:3856-3860; Yang, et al., J. Biol. Chem. (1994) 13:286-296). HA is a linear carbohydrate polymer, and HABP35 is asmall 27-29-residue peptide derived from the RHAMM HA-binding receptor. Analogous to the Sso7d, this is proposed to provide scaffold binding with a small footprint.

[0140] Two MVP tags, SUMO and GFP, with solubility-enhancing and / or entropic bristle capability (Michiels, et al., Cell Rep. (2020) 30:2834-2845; Grana-Montes, et al., Biomacromolecules (2014) 15: 1194-1203), were used to investigate if these could facilitate VLP formation in the absence of any scaffold binding. An untagged MVP was also used as a negative control.

[0141] Formation of heterogeneous VLPs was tested using FRET spectroscopy on a mixture of MARY -MVP, MVP-YFP, and MVP-CFP.

[0142] Refolding vault MVPs purified from E. coli to assemble VLPs in vitro is the first method for bioengineering of heterogeneous vault-like nanoparticles at a much larger scale than current protocols based on purification from insect cells.

[0143] Materials and Methods

[0144] Transmission Electron Microscopy

[0145] TEM was used to visualize the refolded vault protein nanocapsules. The refolded samples were fixed with 2% formaldehyde in refolding buffer at room temperature.

[0146] Glow-discharged carbon-coated 400 mesh copper grids (CF400CU, Electron Microscopy Sciences, Hatfield, PA) were floated onto 10 pL of suspension for 5 minutes. The excess solution was blotted from the grid with filter paper. The sample was washed by touching the grid to drops of ultrapure water (3x) and floated onto a drop of 1% aqueous uranyl acetate for 30 seconds, blotted dry, and examined with a FEI Tecnai™ G2 Spirit Twin TEM (FEI Corp., Hillsboro, OR), and digital images were acquired with a Gatan UltraScan® 2k x 2k camera and Digital Micrograph software (Gatan Inc., Pleasanton, CA). Some samples were visualized with a ThermoFisher G2 Talos L120C TEM (Thermo Fisher Corp.,Wilham, MA) operated at 120 kV, and digital images were acquired with a Ceta CMOS 4K x 4K camera and Velox software ThermoFisher G2 Talos L120C TEM (Thermo Fisher Corp., Wilham, MA).

[0147] Transformation and Growth ofE. coli

[0148] Genes for variants of tagged MVP variant plasmids were made by GenScript. All were cloned into Kanr Pet28. These plasmids were transformed into competent BL21-DE3 E. coli cells according to the New England Biolabs protocol and grown on LB agar plates in a 37°C incubator for ~18 hours. Colonies were then introduced into seed culturesolutions of 50 mL of LB Broth and 50 pL of 50 mg / mL kanamycin (1:1000 dilution of Kanamycin). The cultures were placed in a shaking incubator for approximately 16 hours at 37°C. The seed cultures were then poured into larger cultures of 1 L LB broth and 1 mL of 50 mg / mL kanamycin and allowed to grow at 37°C in a shaking incubator. The growth of E. coli was periodically checked using a PerkinElmer UV / VIS until the optical density (OD) readings at 600 nm were within 0.5-0.8. Then, 1 mL of 0.8M IPTG was added to each 1 L culture to stop bacterial growth and to induce production of the desired protein. The cell cultures were placed overnight in a shaking incubator at 25 °C and harvested by centrifugation at 5000 rpm for 10 minutes. The supernatant was discarded, and the pellet was harvested and stored at -80°C until purification.

[0149] Protein Purification

[0150] Recombinant human MVP proteins and INT-CFP fusion were purified under denaturing conditions using the Ni-NTA Purification System protocol from Thermo Fisher Scientific. The frozen cell pellet collected from centrifugation was thawed either on ice or in the fridge and then solubilized at 37°C, pH 7.8, in Guanidinium Lysis Buffer to produce a final 40 mL of lysate. The lysate was then sonicated using a Branson Sonifier Cell Disruptor 200 at a high-intensity setting with 50% on / off bursts for 12 minutes.

[0151] Prior to the purification process, the columns were charged by running 15 mL of DI water, 10 mL of 0.05M EDTA, 10 mL of DI water, 10 mL of 0.05M cobalt, another 10 mL of DI water, and finally 10 mL of pH 7.8 8M urea binding buffer through the column resin. The batch purification method was used to bind the protein to the column beads as follows: the cobalt beads in the column were resuspended in the binding buffer within the column. The beads and residual binding buffer from the column were then allocated into two 15 mL FALCON tubes along with the prepared lysate before being centrifuged at -1000 rpm for 30 seconds. The supernatant was aspirated before resuspending the beads in additional binding buffer. The beads were gently centrifuged under the same conditions, and the supernatant was aspirated. The beads were then resuspended in residual binding buffer and added back to the column for the continuation of the Ni-NTA Purification protocol.

[0152] Stocks A and B (sodium phosphate monobasic and dibasic, respectively) were prepared without 5M NaCl. A separate 5M NaCl solution was prepared, and the appropriate amount was added directly to the buffers needed for denatured purification.Outside this exception, buffers were prepared according to the protocol instructions. A 5.3 pH wash buffer was produced or used during the purification process. A total of ~5 mL of pH 7.8 binding buffer was run through the column and collected in a waste beaker. Wash buffer (6 mL, pH 6.0) was added to the column and collected in 1 mL fractions in microfuge tubes. Finally, 8 mL of elution buffer (pH 4.0) was added to the column and collected in 1 mL fractions in microfuge tubes. Following collection of the eighth fraction, an additional elution buffer was added to the column to prevent the beads from drying. The column and collected fractions were stored at 4°C. For all MVP variants, concentration was determined using densitometry through the software ImageJ 1.54d by comparing a scale of varying BSA concentrations against MVP bands. Sso7d-MVP was determined to contain 1.2 mg / mL copurified DNA by Quanti-IT™ Picogreen assay from Invitrogen.

[0153] Sso7d tag sequence (with linker and 6x His tag) (SEQ ID NO: 20):

[0154] TVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVS EKDAPKELLQMLEKQKKGGGGGGSLVPRGSGGGGGHHHHHH

[0155] Linker sequence (SEQ ID NO: 5):

[0156] GGGGGSLVPRGSGGGGSENLYFQSGGGGSLVPRGSGGGGGGGGG GGGGGG

[0157] Linker, Sso7d, and 6xHis tag sequence (SEQ ID NO: 6):

[0158] GGGGGSLVPRGSGGGGSENLYFQSGGGGSLVPRGSGGGGGGGGG GGGGGGTVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGK TGRGAVSEKDAPKELLQMLEKQKKGGGGGGSLVPRGSGGGGGHH HHHH HABP35 tag sequence (with linker and 6x His tag) (SEQ ID NO: 21):

[0159] LKQKIKHVVKLKVVVKLRSQLVKRKQNGGGGGGSLVPRGSGGGG GHHHHHH MARY MVP tag and linker sequence (SEQ ID NO: 22):

[0160] MARYRCCRSQSRSRYYRQRQRSRRRRRRSCQTRRRAMRCCRPRY RPRCRRHGGGGGG

[0161] Superfolder Cyan fluorescent protein tag sequence (with linker and 6x His tag) (SEQ ID NO: 23):

[0162] GGGGSGGGGSGGGGSMSKGEELFTGVVPILVELDGDVNGHKFSV RGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRY PDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANF KIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKD PNEKRDHMVLLEFVTAAGITHGMDELYKGGGGGGHHHHHH

[0163] Superfolder Yellow fluorescent tag protein sequence (with linker and 6x His tag) (SEQ IDNO: 24):

[0164] GGGGGGGGGGGGGGGMSKGEELFTGVVPILVELDGDVNGHKFSV RGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYP DHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANF KIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSVLSKD PNEKRDHMVLLEFVTAAGITHGMDELYKGGGGGGHHHHHH

[0165] Refolding and Dynamic Light Scattering Experiments

[0166] Experimentation of the refolding of the vault protein using a linear scaffold was performed by mixing varying amounts of refolding buffer (100 mM sodium phosphate, pH 7.0, containing 100 mM NaCl and 1.5 mM MgCh), DNA or HA at varying concentrations, and purified denatured MVP in the cuvette of the Wyatt dynamic light scattering (DLS) nanostar instrument. The sample was then monitored in the DLS. Changes in the species within the solution over time were also monitored using the DYNAMICS software.

[0167] The mixing of the refolding process was as follows: 320 pL of refolding buffer was added to the DLS cuvette, followed by 1.1-16 pL of tagged MVP. Sso7d-MVP or MARY-MVP for DNA scaffold and HABP35-MVP for HA scaffold were added to the cuvette at 1.8-8 nM and mixed by pipetting. The final volume added to the cuvette was 40 pL.

[0168] All DLS assays were conducted at 25°C, with a 5 second acquisition time and averages of 25-50 acquisitions. The model for radius calculation was MIE spheres, with %Number readout. The correlation cutoff was 1.5 to 2zl05ps, low radius cutoff was 3.0 nm, and high radius cutoff was 10,000 nm.

[0169] Initially, to demonstrate the synthesis of vault, Sso7d-MVP was incubated with different amounts of DNA. The DNA used was a 400 bp DNA fragment. The DNA fragment came from the gene encoding the MV3 protein of the matryoshka virus (amino acid sequence of: MSGLVPRGSHMASMTGGQQMGRGSMKGNRKRLGTFHLKGG TTSAALPTETKDLCRRQITLSCPLKFRLSVFEVKKEIDNLKLEESLHLDRRRRRNRKDFFRRPRDWPDLRYRRINRKEIKALGFLSRLKLEEFSFREYKLVRYIYLKVLPR VDLVLYRYVHQLVTFSVLQLLETYLSNRELCSRLN (SEQ ID NO: 7)). Notably, Sso7d binds DNA non-specifically. Accordingly, the exact sequence of the DNA was not expected to significantly influence the ability of Sso7d to bind. The 400 bp DNA fragment was chosen as a scaffold because Sso7d has a binding footprint of -8 nucleotide base pairs. As such, a single DNA of 400 basepairs would allow enough Sso7d-MVP to bind to form only one half vault. Two half vaults, when properly formed, can then selfassemble into a mature vault.

[0170] Nuclease Controls: Eliminating Potential Contaminating DNA

[0171] Sso7d (8M urea) was incubated with or without universal nuclease (UN+ and UN-, respectively) stock (250 U / mL nuclease) at a 3:1 buffer volume ratio for 10 minutes to remove any copurified DNA / RNA polymers. UN+ samples were incubated with nuclease buffer containing 50% glycerol.

[0172] Heterogeneous Vault Formation

[0173] MARY-MVP (1.8 nm) was refolded with MVP-YFP (2.4 nm) or a combination of both MVP-YFP and MVP-CFP (1.2 nm each). In addition, MVP-YFP and MVP-CFP (1.2 nm each) were refolded in the absence of MARY-MVP. These three solutions were refolded in 20 mM phosphate, pH 6.6, 50 mM NaCl, 1.5 mM Mg Ch. Solutions were allowed to refold at room temperature for 20-30 minutes prior to concentration 57-150 fold through ultrafiltration (MWCO 100,000 kDa). FRET data were collected on a Varian Cary Eclipse fluorometer with an excitation wavelength of 433 nm and 20 nm excitation and emission slits.

[0174] INT-CFP Cargo Loading

[0175] INT-CFP binding to VLPs was examined with an ultrafiltration fluorescence binding assay (Kickhoefer, et al., Proc. Natl. Acad. Sci. (2005) 102:4348-4352). MARY-MVP was refolded in pH 6.5 phosphate buffer, 50 mM NaCl, 1.5 mM MgCh. 8.28 nM INT-CFP was held fixed, simultaneously refolded in the same solution as [MARY-MVP], and was increased from 0 to 7.3 nm. After refolding for 20 minutes, refolding solution was centrifugally concentrated (MWCO 100 kDa; INT-CFP) from 15 mL to -150 pL. Concentrated refolding reactions were monitored for CFP fluorescence by excitation at 433 nm, and emission peak was monitored at 480 nm.Results

[0176] Purification of MVP and Tagged MVP Variants

[0177] Human MVP and all tagged variants were grown in BL21-DE3 E. coli and lysed with 6M Gdn-HCl. MVP and variants were purified using single-step Co-NTA resin under denaturing conditions with 8 M urea (Figure 1). The final concentrations were determined by SDS-PAGE densitometry with ImageJ 1 ,54d using a BSA ladder. SDS-PAGE was also used to assess purity.

[0178] Two DNA scaffold-binding tags were tested for the MVP: Sso7d on the C-terminus (MVP-Sso7d) and protamine on the N-terminus (MARY-MVP). In addition, an MVP was generated with a C-terminal HA-binding tag, HABP35 (MVP-HABP35) (Zaleski, et al., Antimicrob. Agents Chemother. (2006) 50:3856-3860; Yang, et al., J. Biol. Chem. (1994) 13:286-296). All MVP variants were cloned into the pet28 plasmid (Kanr). Tag sequences were added to the MVP C- or N-terminus with a linker consisting of a long poly-G sequence with a TEV protease cut site. All MVPs, including untagged, were also cloned with a 6-His affinity tag.

[0179] Dynamic Light Scattering Analysis

[0180] Time-resolved DLS was performed to observe the dynamic assembly process of VLPs. DLS measurements were performed in a 100 mM sodium phosphate buffer (pH 6.5-7.0) containing 50-100 mM NaCl, 1.5 mM MgCh, and 27-400 mM urea. MVP was maintained at 1.8-8.0 nm. Initial refolding was consistently performed at 22°C, followed by 4°C for 1-2 days if applicable. Refolding of Sso7d-tagged MVP in the presence of copurified DNA (1.2 mg / mL) resulted in the assembly of a complex with a 26.8 nm hydrodynamic radius within 20 minutes (Figures 2A, 3A, 3B, and 3F). This radius is consistent with previously reported DLS assays on intact vaults purified from eukaryotic cells (20-27 nm) (Benner, et al., ACS Nano (2017) 11:872-881; Tomaino, et al., Int. J. Mol. Sci. (2024) 25:6629). To confirm the necessity of copurified DNA, Sso7d-MVP treated with a universal nuclease enzyme (UN+) to degrade any copurified DNA / RNA prior to refolding showed no significant nanocapsule formation (Figure 3B).

[0181] Refolding of MARY-MVP with copurified DNA over two days resulted in the formation of particles similar to those observed with the Sso7d tag (hydrodynamicradius = 26.4 nm) (Figures 2B, 3C, 3D). Assembly of this VLP was observed to be pH-dependent, remaining monomeric above pH 6.8 and forming large aggregates below pH 6.2 (Figure 3D).

[0182] A 50 kDa HA scaffold at 1 nm was used with 8 nm MVP-HABP35 under the buffer conditions described above for Sso7d-MVP. Refolding into vault-sized nanocapsules (hydrodynamic radius = 30.75 nm) was observed only in the presence of exogenously added HA (Figures 2C, 3E, and 3F). In contrast to the other tagged variants, for the HABP35-MVP, nanocapsule formation was immediate, with no significant change over time observed through DLS (Figure 3E).

[0183] Wild-type MVP with no scaffold-binding tag showed rapid assembly of large bodies with a radius of -60 nm (Figures 3 A and 3F). The SUMO-MVP variant showed no DLS signal above the background, and GFP-MVP showed no significant nanoparticle formation (Figure 3 A). DLS with only aggregates was recorded as zero. All linker regions between MVP and the C-terminal tags were identical, except for the GFP tag, which used a truncated linker.

[0184] DLS was also performed on in vitro experiments involving the incubation of Sso7d-MVP with various concentrations of DNA. Specifically, 0.5 pl, 2 pl, or 3 pl of a stock solution of DNA (6.5 ng / pl) was used resulting in a final volume of 40 pl and DNA concentrations of 0.08125, 0.325, and 0.4875 ng / pl, respectively. Measurements were performed in 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 1.5 mM MgCh. The hydrodynamic radius of -20-30 nm is consistent with the formation of vaults (Fig. 3G). Indeed, the hydrodynamic radius of intact assembled vaults purified from vault-expressing cells typically ranges from 20 to 27 nm (Benner, et al. (2016) ACS Nano 10(I):872-81 ; Tomaino, et al. (2024) Int. J. Mol. Sci., 25(12):6629).

[0185] DLS controls with the scaffold alone in refolding buffer were also run. To substitute for copurified DNA of unknown length, controls were run at 30 ng / pL, using either salmon genomic DNA or a pESC-leu2d purified plasmid of 11,042 bp. HA control was run using 1 nm 50 kD MW HA, as in refolding experiments. All controls using only scaffold showed no DLS peaks other than high-molecular- weight species of radii 254.5 nm (salmon DNA), 223.5 nm (plasmid DNA), and 255 nm (HA).

[0186] To exclude the possibility that the added universal nuclease itself interfered with vault formation, the universal nuclease was added to the Sso7d-MVP (8 M urea) refolding reaction without preincubation in order to minimize the likelihood of universal nuclease activity. Refolding reactions were also performed with Sso7d-MVP without universalnuclease or with Sso7d-MVP and an inactivated nuclease (DNase II). Figure 3H shows the formation of vaults within 30 minutes in all three samples with similar kinetics.

[0187] Results shown in Figure 2 are typical for all VLP samples, where only one major peak is observed with a poly dispersity of -25-30%. Due to the hollow interior cavity of the vault, reporting species percentages based on “percent mass” is unreliable, and “percent number” is used for all samples. The MIE spheres algorithm is also used in all samples.

[0188] Transmission Electron Microscopy Imaging

[0189] TEM images of refolded Sso7d-tagged, MARY-MVP -tagged, and untagged MVP were collected to determine the shape of VLPs observed in DLS (Figure 4). Samples were refolded as described above and concentrated. Sso7d-tagged MVP was fixed with 2% formaldehyde prior to concentration. A total of 10 pL of suspension was added to each grid for 5 minutes, followed by washing with ultrapure water. Samples were then stained with 1% uranyl acetate for 30 seconds before examination with a FEI Tecnai™ G2 Spirit Twin TEM.

[0190] A distribution of different sizes was observed for MVP-Sso7d. For MVP-Sso7d, the most common species observed was a circular nanocapsule, consistent with a top-down view of a “half vault” (Figure 4A) (Mrazek, et al., ACS Nano (2014) 8:11552-11559; Tomaino, et al., Int. J. Mol. Sci. (2024) 25:6629; Tanaka, et al., Proc. Jpn. Acad. Ser. B (2012) 88:416-433; Kong, et al, RNA (2000) 6:890-900). Image analysis shows that 23% of these VLPs were -27 nm in diameter, 38% were -42 nm, and 38% were -57 nm, consistent with DLS observations (Figure 4A), with some full vaults being observed across multiple samples (top, left, n = 1). The nanocapsule diameters of (45 ± 13 nm) are consistent in size and shape with intact half-vaults previously purified from insect cells (41 nm) (Mrazek, et al., ACS Nano (2014) 8:11552-11559).

[0191] MARY-MVP was prepared similarly to MVP-Sso7d but in the absence of formaldehyde. The image of MARY-MVP refolded VLPs (Figure 4B) also shows a mixture of sizes consistent with full vaults (-40 nm by -75 nm) and half vaults (-40 nm diameter). However, there were a larger percentage of full VLPs (n = 4) vs. half (n = 8) compared to the experiment with MVP-Sso7d. The MARY-MVP VLPs appeared to be deformed relative to Sso7d, consistent with the understanding that the N-terminally tagged vault has decreased structural stability due to an increased tendency to unroll into sheets (Mrazek, et al., ACS Nano (2014) 8:11552-11559).Control wild-type untagged MVP yielded large amorphous particle-like structures consistent with MVP renaturation without a scaffold, leading to damage and / or disordered aggregation (Figure 4C) (Mrazek, et al., ACS Nano (2014) 8: 11552-11559). No separate VLP structures could be distinguished from the TEM.

[0192] Heterogeneous Vault Formation and Cargo Loading

[0193] FRET qualitative analysis was used to test the ability to form heterogeneous VLPs using the scaffold-based refolding protocol. MVP variants were produced with either yellow fluorescence protein (YFP) or cyan fluorescence protein (CFP) tags on their C-termini, MVP -YFP and MVP-CFP, respectively. Given the vault diameter of ~10 nm at the tip of the cap and total vault height of 75 nm, a CFP-YFP FRET signal should only be observable within vault halves and not between halves (Figure 5A).

[0194] MVP-CFP, MVP-YFP, and INT-CFP were purified under denaturing conditions, as described above, and the following three differing refolding solutions were prepared. Control solution MY : MARY-MVP (1.8 nM) was refolded with MVP-YFP (2.4 nm). Solution MCY: MARY-MVP (1.8 nm) was refolded with a combination of both MVP-YFP and MVPCFP (1.2 nm each; Figure 5 A. Control solution CY: MVP-YFP and MVP-CFP (1.2 nm each) were refolded in the absence of MARY-MVP. These three solutions were refolded in 20 mM phosphate, pH 6.6, 50 mM NaCl, and 1.5 mM Mg Ch. The solutions were allowed to refold at room temperature for 20-30 min prior to concentration 57-150 fold through ultrafiltration (MWCO 100,000 kDa) and emission spectrum analysis. Upon excitation at the CFP excitation wavelength (433 nm), YFP fluorescence at 530 nm was detected only in solution MCY (Figure 5B), indicating a FRET-based signal from a heterogeneous complex. The spectrum of the buffer alone was subtracted from all signals. Consistent with expectations, no fluorescence was detected in either control solution (MY (no CFP) or CY (no scaffold-binding MVP)).

[0195] DLS was used to analyze the heterogeneous refolding solutions. Following concentration, solutions MCY, MY, and CY showed hydrodynamic radii of 18 nm, 68 nm, and 10.3 nm, respectively (Figure 5C). The 18 nm radius from solution MCY is more consistent with a half-vault than with a full vault. Half-vaults may be favored here due to the use of only 40% MARY-MVP necessary for optimal FRET signal-noise ratio. The 68 nm radius from solution MY may indicate a tendency of YFP to form oligomers at the locally high concentrations caused by the scaffolding. The 10.3 nm radius observedfrom solution CY, combined with the lack of any FRET signal, is consistent with MVP monomers that are unable to form nanoparticles.

[0196] The vault-sized particles, observed with DLS and TEM, along with MVP-YFP FRET with MVP-CFP, could be explained by a misfolded MVP aggregate. An INT-CFP fusion protein was therefore generated to test the cargo loading capability of VLP. If the VLPs are MVP aggregate, burying of the INT binding domain, or aggregation-caused quenching of the CFP, would result in a significant drop in fluorescence signal of INT-CFP bound to the VLP. The INT (interaction) domain (e.g., C-terminus of VP ARP protein (e.g., aa 1563-1724)) is a well-established fusion tag to load cargo proteins to the interior hollow cavity of the vault complex (Munoz -Juan, et al., Pharmaceutics (2019) 11 :300). The standard INT-binding site faces the vault interior, spans three domains of the MVP (Yu, et al., Sci. Rep. (2017) 7:14816), and is ~30 nm from the vault cap.

[0197] INT-CFP binding to refolded VLPs was examined with an ultrafiltration fluorescence binding assay (Kickhoefer, et al., Proc. Natl. Acad. Sci. (2005) 102:

[0198] 4348-4352). Refolding was conducted as above for MARY-MVP in pH 6.5 phosphate buffer, 50 mM NaCl, 1.5 mM MgCh. INT-CFP was simultaneously refolded with MARY-MVP and was held fixed at 8.28 nm as [MARYMVP] was increased from 0 to 7.3 nM. After refolding for 20 minutes, refolding solutions were centrifugally concentrated (MWCO 100 kDa; INT-CFP MW 45.2 kDa) from 15 mL to -150 pL. Concentrated refolding reactions were monitored for CFP fluorescence by excitation at 433 nm, and emission peak was monitored at 480 nm (Figure 5E). DLS radii were also collected as above for each MARY-MVP concentration (Figure 5D).

[0199] As shown in Figure 5E, a linear increase in CFP fluorescence is observed as the [MARY-MVP] is increased. At -5 nm [MARY-MVP], DLS observes a sharp transition from monomeric MARY-MVP to VLP formation (Figure 5D). This inflection point in radius is not associated with a concordant deviation in CFP emission intensity, but instead, CFP fluorescence continues to increase linearly as a function of [MARY-MVP], This result is consistent with studies loading GFP into the hollow vault interior, where fluorescence is preserved upon loading (Kickhoefer, et al., Proc. Natl. Acad. Sci. (2005) 102:4348-4352).

[0200] Alphafold 3 simulations of fifteen Sso7d-MVP monomers support the DNA scaffold means for vault assembly. Due to computational limitations, only the pore region and a portion of the long C-terminal alpha-helical capping domain were included. DNA corresponding to the MVP mRNA 3’ terminal sequence (483 nucleotides) was usedin the simulation. Figures 6A and 6B show the Alphafold 3 simulations the absence and presence of DNA, respectively. Figure 6C shows the Alphafold 3 simulation of in vitro folding of untagged MVP monomers. The cylindrical conformation was found to be sensitive to the DNA and highlights the Sso7d tag-DNA interaction to change the quaternary structure. Notably, the MVP DNA was predicted to form a loop consistent in diameter with the vault complex pore.

[0201] The proposed scaffold-based refolding mechanism is consistent with the results. MVP-Sso7d, MARY-MVP, and MVP-HABP35 with scaffold-binding tags all showed DLS signal consistent with formation of vault-sized nanoparticles. The MVP-Sso7d VLP formation was eliminated with the addition of nuclease, consistent with reliance on a DNA scaffold. HABP35-MVP VLPs also formed only in the presence of the HA scaffold. Neither SUMO-MVP nor GFP-MVP formed nanoparticles under the same conditions because of their inability to bind the DNA scaffold. Using TEM with MVP-Sso7d, a significant number of half-vault-like particles were observed, with some observed full vaults. MARY-MVP, meanwhile, showed a significant number of VLPs consistent in size with both half and full-vaults. However, these were somewhat misshapen compared to MVP-Sso7d structures. A mixture of MVP-Sso7d and MARY-MVP may provide both well-ordered VLPs from the Sso7d tag while allowing MARY-MVP to link them together to allow a higher percentage of full vaults.

[0202] Formation of VLPs using heterogeneously tagged MVPs was tested using FRET. A FRET signal was observed between the MVP -tagged donor-acceptor pair in the presence of scaffold-binding MARY-MVP. This is consistent with proper alignment of the C-termini in the heterogeneous VLPs. CFP fluorescence intensity of INT-CFP fusion protein showed no significant deviation in intensity upon binding free MVP vs. binding MVP as part of a 25 nm radius VLP. This is consistent with proper refolding of the three-domain FNT binding site. This is also consistent with no loss of CFP signal due to aggregation-caused quenching or burying of the INT binding site as the VLP forms. The data are consistent with intact INT-binding sites on the VLPs with a hollow cavity (Kickhoefer, et al., Proc. Natl. Acad. Sci. (2005) 102:4348-4352; Yu, et al., Sci. Rep. (2017) 7:14816).

[0203] This is the first instance of VLP formation in vitro using refolded MVP monomers and the first use of a linear scaffold for the guided refolding of any multimeric complex.The in vitro assembly eliminates the need for the high-cost, low-yield production from eukaryotes, and the single-step affinity purification from E. coli allows for low-cost, large-scale purification. This protocol may be expanded to bioengineering applications such as heterogeneous VLPs with cell-localization and / or therapeutic tags for drug delivery.

[0204] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS1. A maj or vault protein conjugate comprising a maj or vault protein and a scaffold binding protein,wherein said scaffold binding protein is connected to the major vault protein via an amino acid linker.

2. The major vault protein conjugate of claim 1, wherein said scaffold binding protein is connected to the C-terminus of the major vault protein via an amino acid linker3. The major vault protein conjugate of claim 1, wherein said major vault protein is human.

4. The major vault protein conjugate of claim 1, wherein said amino acid linker comprises a protease cleavage site.

5. The major vault protein conjugate of claim 4, wherein said protease cleavage site is at the N-terminus of the amino acid linker.

6. The major vault protein conjugate of claim 1, further comprising at least one affinity tag.

7. The major vault protein conjugate of claim 1, further comprising at least one detectable agent.

8. The major vault protein conjugate of claim 1, wherein said scaffold binding protein is a DNA binding protein.

9. The major vault protein conjugate of claim 8, wherein said DNA binding protein is from a thermophile or thermoacidophile.

10. The major vault protein conjugate of claim 8, wherein said DNA binding protein is Sso7d.

11. The major vault protein conjugate of claim 8, wherein said DNA binding protein is protamine.

12. The major vault protein conjugate of claim 1, wherein said scaffold binding protein is a hyaluronic acid binding protein.

13. The major vault protein conjugate of claim 1, wherein said scaffold binding protein is a linear scaffold binding protein.

14. A nucleic acid molecule encoding a major vault protein conjugate of any one of claims 1-13.

15. A vector comprising the nucleic acid molecule of claim 14.

16. An in vitro method for synthesizing a vault-like nanoparticle, said method comprising contacting a major vault protein conjugate of any one of claims 1-13 with a scaffold,wherein the scaffold binding protein of the major vault protein conjugate binds said scaffold.

17. The method of claim 16, wherein said scaffold is a linear scaffold.

18. The method of claim 16, wherein said scaffold is selected from the group consisting of nucleic acid molecules, carbohydrates, polysaccharides, polymers, proteins or polypeptides, carbon nanotubes or nanorings, viruses, nanoparticles, and quantum dots.

19. The method of claim 16, wherein said method further comprises producing the major vault protein conjugate prior to said contacting step.

20. The method of claim 19, wherein said major vault protein conjugate is recombinantly produced in E. coli.

21. The method of claim 16, wherein said major vault protein conjugate is denatured prior to and / or during said contacting with a scaffold.

22. The method of claim 16, wherein said scaffold is a nucleic acid molecule.

23. The method of claim 16, wherein said scaffold is hyaluronic acid.

24. The method of claim 16, further comprising adding at least one of telomerase-associated protein 1 (TEP1), vault poly(ADP -ribose) polymerase (VP ARP), and small vault RNAs to the major vault protein and scaffold.

25. The method of claim 16, further comprising adding at least one saber protein to the major vault protein and scaffold.

26. The method of claim 16, further comprising adding at least one molecule to the major vault protein and scaffold, wherein the molecule is encompassed or encapsulated by the synthesized vault.

27. The method of claim 16, wherein more than one unique major vault protein is contacted with the scaffold.

28. The method of claim 16, further comprising purifying and / or isolating the synthesized vault.

29. The method of claim 16, wherein said amino acid linker comprises a protease cleavage site and wherein the method further comprises cleaving the protease cleaving site.

30. The method of claim 29, wherein said cleavage occurs after vault formation.

31. A vault-like nanoparticle synthesized by the method of claim 16.

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