Sequences and methods for the production of recombinant biomolecules in vesicles

Abstract

Methods for expressing biomolecules and components of recombinant expression systems for expressing biomolecules are described. In particular, isolated vesicle nucleating polypeptides (VNps) comprising amphipathic α-helical polypeptides derived from the amino termini of synuclein isoforms and their variants are used to promote, enhance or increase the formation of vesicles in expression systems to enable purification of biomolecules.

Description

[Technical field]

[0001] The present invention relates to a technology for enhancing the production of biomolecules in cellular expression systems. In particular, a system for exporting recombinant proteins from genetically engineered cells into derived extracellular vesicles is described. [Background technology]

[0002] E. coli is an attractive system for recombinant protein production in both academic and industrial settings. (Jan,AT(2017)Front.Microbiol.8,7692-11;Gerritzen,MJHet al(2017)Biotechnology Advances 35,565-574). Not only is E. coli cheap and easy to grow in large quantities to high densities, but a variety of strains, reagents, promoters, and tools have been developed to facilitate the production of functional proteins. The application of synthetic biology strategies is overcoming limitations commonly associated with the application of post-translational modifications and correct folding of complex proteins. (Kim,JHet al(2015)Semin.Cell Dev.Biol.40,97-104). For all expression systems, there is a constant need to improve protein yields and improve protein purification efficiency.

[0003] Gram-negative bacteria produce outer membrane vesicles (OMVs) to facilitate extracellular communication and interaction (e.g., quorum sensing). OMVs are small, spherical lipid bilayers (diameter -100 nm) that are released from the outer membrane. Detailed mass spectrometry and chromatographic analysis have revealed the composition of natural OMVs and shown them to contain DNA / RNA, proteins or other molecules from the cell wall, periplasmic or cytoplasmic regions of the cell (Jan, AT supra). Natural OMVs are enriched in proteins on their surface that enhance their ability to fuse with host cells, making them attractive vessels for drug and vaccine delivery applications (Gerritzen, MJ Het al supra). OMVs are equivalent to exosomes and extracellular vesicles in mammals and Gram-positive bacteria (Kim, J Het al supra). OMVs result from a local loss of interaction between the asymmetric lipid bilayer of the outer membrane (OM; Konovalova, A. et al (2017) Annu. Rev. Microbiol. 71, 539-556) and the peptidoglycan (PG) layer, brought about by local curvature of the outer membrane caused by turgor pressure, temperature, or local protein enrichment interactions (Jan, AT supra; Kim, J Het al supra). OMVs are naturally produced in low yields during the bacterial growth cycle, but increase slightly during stationary phase. It is possible to directly package proteins into native OMVs. However, due to the low abundance of OMVs, only relatively low protein yields have been observed so far (Alves, NJ et al (2015) ACS AppL Mater. Interfaces 7, 24963-24972). Large-scale deletion and overexpression screens have revealed that it is possible to alter the composition of the outer membrane itself, improving membrane protein yields and modulating OMV production.(McBroom, AJ et al (2006) J. Bacteriol. 88, 5385-5392; Miroux, B. & Walker, JE (1996) Journal of Molecular Biology 260, 289-298; Baker, JL et al (2014) Curr. Opin. BiotechnoL 29, 76-84). Interestingly, expression of caveolin-1 in E. coli cells led to the accumulation of small round vesicles (40-50 nm in diameter) in the cytoplasm of each cell, likely formed by inward budding of the intracellular membrane.

[0004] The ability to reprogram cells to package specific molecules into individual membrane vesicles is a major goal of synthetic biology. This controlled packaging into membrane vesicles allows biologists to create many new technologies with applications in both biotechnology and the medical industry. These include generating new metabolic factories within cells for energy production, packaging toxic proteins in a contained environment quickly before they can harm normal metabolic activity so that they can be purified for subsequent pharmaceutical use, creating protective packages that encapsulate difficult-to-isolate biomolecules so that they can be stored and purified in a stable environment, and even creating simple vehicles for delivering drugs and vaccines to patients. Summary of the Invention

[0005] It is against this background that the present invention was devised.

[0006] Thus, the present invention encompasses polypeptide sequences and methods that provide a simple mechanism for the targeted release of specific membrane-packaged biomolecules, such as proteins, into cell media that can be continuously isolated from active cultures, resulting in significantly increased yields of functional soluble molecules, such as proteins, from the cultures and facilitating efficient downstream processing for a wide range of biotechnology applications.

[0007] In particular, the present invention relates to isolated vesicle nucleating polypeptides (VNps) comprising amphipathic α-helical polypeptides derived from the amino termini of synuclein isoforms and variants thereof. Stated another way, the present invention resides in short amphipathic α-helical polypeptide variants and engineered variants thereof based on the amino termini of synuclein isoforms.

[0008] The synuclein family includes three proteins: α-synuclein, β-synuclein, and γ-synuclein. All synucleins share a highly conserved α-helical lipid-binding motif that resembles the exchangeable class A2 lipid-binding domain of apolipoproteins. Full-length endogenous Hsα-synuclein has the following sequence (GenBank sequence: AAL15443.1): [ka]

[0009] The VNp may have from 9 to 140 amino acids, for example, from 15 to 140 amino acids, preferably from 16 to 40 amino acids, more preferably about 20 amino acids or about 38 amino acids.

[0010] For example, the amino terminus of Hs α-synuclein has the sequence: [ka] The amino terminus of Hs β-synuclein has the following sequence: [ka] The amino terminus of Hs γ-synuclein has the following sequence: [ka]

[0011] Mutations in the amino-terminal sequence may involve changing one or more basic lysine residues at the membrane interface to acidic or neutral residues. Such changes stabilize the interaction with the membrane. Examples of such sequences are as follows, with differences from the wild-type protein sequence in bold and underlined: [ka]

[0012] While the mutations and changes may be designed, sequence mutations can include mutations and changes generated in random mutagenesis screens, for example, by specifically looking for mutations that increase the excretion of a particular protein or marker in the medium.

[0013] Examples of mutants identified using this method include the following sequences: [ka]

[0014] Other examples of suitable variants are those that take one of the mutated sequences and add further mutations such as the following sequences: [ka]

[0015] Examples of suitable shorter variants include the following sequences: [ka]

[0016] It is understood that the invention encompasses any number of residues within the stated ranges, for example, the sequences can specifically have 20, 21, 24, 28, 33 or 38 amino acid residues.

[0017] Alternatively or in addition, the amino-terminal sequence may contain modifications and / or mutations to (a) alter the stability of the helix termini and / or (b) prevent amino-terminal acetylation. The latter mutations may be particularly beneficial in eukaryotic (especially mammalian) expression systems, since in these cells it is often not possible to prevent amino-terminal acetylation without creating knockout cell lines. In embodiments using E. coli, if desired, amino-terminal acetylation of the target protein may be achieved using an inducible recombination system developed in the Mulvihill lab to induce this post-translational modification, which may have beneficial effects on the structure and function of many eukaryotic proteins, as described in Eastwood TA et al (2017) FEBS Letters. 591, 833-44; Johnson M. et al (2010) PLoS One. 5, e15801.

[0018] Alternatively, or in addition, the sequence may include one or more synthetic amino acids.

[0019] Optionally, the polypeptide may be acetylated at its amino terminus, a feature that promotes rapid formation and internalization of vesicular structures into the cytoplasm of the host cell, thereby sequestering toxic and / or insoluble proteins from the cytoplasm.

[0020] The VNp may further comprise a leucine zipper sequence. Preferably, the leucine zipper sequence is at the carboxyl terminus of the polypeptide. The addition of a dimerizing leucine zipper sequence, in some embodiments, facilitates the excretion and / or expression of, for example, difficult to express (e.g., toxic or insoluble) proteins. Examples of suitable leucine zipper sequences are as follows: [ka]

[0021] The present invention also resides in fusion proteins comprising the above-mentioned vesicle nucleation polypeptides fused to the amino terminus of a biomolecule, such as a recombinant protein. Although fusion proteins with vesicle nucleation polypeptides and a single biomolecule are exemplified herein, the present invention encompasses, for example, tandem fusions (such as those with a fluorescent protein along with one or more target proteins), or the ejection of separate VNp-protein complexes (as indicated by bimolecular fluorescence complementation (BiFC fluorescence) in vesicles), or the ejection of multiple proteins through vesicles by leucine zipper dimerizing proteins. For the avoidance of doubt, BiFC fluorescence is obtained by i) fusing the front half of a fluorescent protein to VNp, ii) fusing the carboxyl half of a fluorescent protein to VNp, and iii) expressing the two fusions together. When the two halves of the fluorescent protein are brought together, they fluoresce. If fluorescence is observed in the vesicle, it can be concluded that the vesicle contains both individual proteins.

[0022] In many embodiments, the vesicle nucleating polypeptide may be directly fused to a biomolecule. The VNp interacts with the host cell membrane to promote the formation of a vesicular structure that originates from the outer cell membrane and contains a biomolecule fused to the VNp sequence. In certain preferred embodiments, the VNp may be fused to a biomolecule via a protease cleavage site. Protease cleavage sites include sites for viral proteases, such as etch virus proteases, such as Tev (tobacco etch virus), viral 3C-like proteases, such as human rhinovirus HRV3C, enterokinase, factor Xa, and thrombin. In many preferred embodiments, the protease cleavage site may be intact, with all recognition sites removed. Examples of such cleavage sites include signal peptidases, such as E. coli LepB, and in particular lanthpeptidases, such as Bacillus licheniformis LicP, as disclosed in WO2015 / 175576. Also, eukaryotic signal peptidases or further processing enzymes such as Pichia Kex2 or Ste13 can be used. Membrane-bound, preferably cytosolic, protease cleavage sites can be employed. In many embodiments, the protease recognition site comprises a sequence of 5 or 6 amino acids. Such arrangement allows proteolytic cleavage and processing of the biomolecule within the vesicle.

[0023] In some embodiments, additional VNps comprising a leucine zipper sequence may be co-expressed with a fusion protein comprising a vesicle nucleation polypeptide comprising a leucine zipper sequence fused to the amino terminus of a biomolecule. The additional VNps may be expressed from the same vector as the vesicle nucleation polypeptide fused to the amino terminus of the biomolecule, or the additional VNps may be expressed from a different vector, e.g., a plasmid or episome, or may be expressed chromosomally.

[0024] In a further embodiment, a vesicle nucleation polypeptide comprising a leucine zipper sequence may be fused to the amino terminus of a protease selected to correspond to the protease cleavage site described above. The VNp may be expressed from the same vector as the vesicle nucleation polypeptide fused to the amino terminus of the biomolecule or may be expressed from a different vector, such as a plasmid or episome, or may be expressed chromosomally. It will be appreciated that the protease will be selected for its activity and ability to be expressed in the host cell of choice.

[0025] Biomolecules that may be employed in the present invention include, for example, carbohydrates such as sugars, antigens, nucleic acids, and in particular recombinant polypeptides.

[0026] Polypeptides, particularly recombinant polypeptides, include therapeutic proteins and peptides, including cytokines, growth factors, antibodies, antibody fragments, immunoglobulin-like polypeptides, enzymes, vaccines, peptide hormones, chemokines, receptors, receptor fragments, kinases, phosphatases, isomerases, hydrolases, transcription factors, and fusion polypeptides.

[0027] Antibodies include monoclonal antibodies, polyclonal antibodies, and biologically active antibody fragments, including multivalent and / or multispecific forms of any of these.

[0028] A naturally occurring antibody typically comprises four polypeptide chains, two identical heavy (H) chains and two identical light (L) chains, inter-connected by disulfide bonds. Each heavy chain contains a variable region (V H ) and a constant region (CH), which in its native form contains three domains, CH1, CH2 and CH3. Each light chain contains a variable region (V L ) and a constant region comprising one domain, CL.

[0029] V HThe V and V regions are further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions of greater conservedness called framework regions (FRs). H and each V L consists of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0030] The antibody fragments that can be expressed include a portion of an intact antibody, which portion has the desired biological activity. Antibody fragments generally contain at least one antigen-binding site. Examples of antibody fragments include: (i) V L (ii) a Fab derivative, such as a Fab' fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain, which can form a bivalent fragment by disulfide bridges between two Fab derivatives; (iii) a V H and an Fd fragment having a CH1 domain; (iv) an Fd derivative, such as an Fd derivative having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) a V L and V H (vi) Fv fragment having a V domain; L and V H (vii) single chain antibody molecules, such as single chain Fv (scFv) antibodies, in which the domains are covalently linked; (vii) separate variable domains (V H or V domain polypeptide) without a constant region domain linked to the V domain H or a V domain polypeptide (e.g., V H -V H , V H -V L , or V L -V L )(viii)V H Domain or V L Domain antibody fragments, such as fragments consisting of domains, and V domains, such as isolated CDR regions. H Domain or VL (ix) an antigen-binding fragment of any of the light chain variable domains; (ix) two antigen-binding sites in the same polypeptide chain, e.g., a light chain variable domain (V L ) linked to a heavy chain variable domain (V H ), the so-called "diabodies," which contain a pair of tandem Fd segments and, together with complementary light chain polypeptides, form a pair of antigen-binding regions, called linear antibodies.

[0031] Preferably, the biological molecule or recombinant protein is not constitutively expressed to a significant detectable level in the expression system in which the fusion protein of the present invention is expressed. For example, the recombinant protein may be a membrane-bound protein, an insoluble protein, or a protein that is normally toxic to the host cell in which the fusion protein is expressed. In many embodiments, the biological molecule or recombinant protein is heterologous to the host cell.

[0032] Biomolecules need not be limited by size, but can range from less than 1 kDa to 100 kDa. Examples of recombinant proteins include Akirin repeat protein off7 (DARP), Stefin A, uricase, erythropoietin (EPO), etanercept, FGF21, human growth hormone (hGH), DNAseI, and nanobodies. The size of the biomolecule to be excreted ultimately depends on the size of the vesicles the host cells can produce. In general, vesicle sizes of 0.16-0.4 μm in diameter are considered the most likely and suitable for optimal excretion.

[0033] The present invention also resides in the nucleotide or vector (plasmid) sequence, particularly expression cassette, that codes for the above-mentioned fusion protein herein.Suitable plasmids include plasmids of the pUC19 and pBR322 family, and plasmids such as those described in WO2007 / 088371.It is understood that the above-mentioned fusion protein can be expressed from the same vector, or from different vectors, such as plasmids or episomes, or chromosomally expressed.

[0034] The nucleotide or vector sequence preferably employs a promoter, examples of which are well known in the art. The promoter may comprise a host cell polymerase promoter or a heterologous polymerase-dependent promoter, such as a phage polymerase-dependent promoter. Suitable promoters include CMV, hEF1, rhamnose, arabinose, lambda pL, T7A3, Tac and T7-based promoters. In certain embodiments, the nucleotide or vector comprises an E. coli polymerase-dependent promoter, particularly an inducible promoter, such as a promoter comprising one or more operators, such as a lac operator comprising a perfect palindromic lac operator sequence. In many preferred embodiments, the nucleotide or vector comprises a lambda pL promoter operably linked to one perfect palindromic lac operator sequence, or a T7A3 promoter operably linked to two perfect palindromic lac operator sequences, one operator sequence located upstream and one located downstream of the promoter. Most preferably, the operator overlaps the transcription start site.

[0035] Optionally, the nucleotide or plasmid sequence further comprises a leucine zipper sequence, such as that set forth in SEQ ID NO:29.

[0036] The invention also resides in host cells that express the fusion proteins or contain the nucleotide sequences or plasmids described herein. The host cells can be prokaryotic cells such as bacteria, unicellular eukaryotes such as yeast, or cultured eukaryotic cell lines. Particular examples of prokaryotic host cells include Escherichia coli strains, Bacillus strains (including subtillis and megatarium), and Schwanella oneidensis. Suitable strains of E. coli include BL21DE3, K12, and others (DH10b, JM109, etc.). Specific examples of prokaryotic cells include bacterial cells, e.g., gram-negative bacterial cells such as E. coli, Salmonella, Shewanella, Serratia, Pseudomonas aeruginosa, and gram-positive bacterial cells such as Bacillus subtilis and Bacillus megatarium. Examples of suitable cultured eukaryotic cell lines include HEK and CHO cell lines. Preferred host cells are bacteria, particularly Enterobacteriaceae, preferably E. coli, particularly its B or K12 strain.In certain embodiments, the prokaryotic cell is engineered to be deficient in at least one native protease.In many preferred embodiments, the prokaryotic host cell is an ompT- E. coli strain, particularly the W3110 E. coli strain.

[0037] Optionally, the host cell further expresses a VNp-leucine zipper peptide, or a peptide comprising a VNp polypeptide as described herein and a leucine zipper sequence, such as those described herein. It is understood that this additional peptide can comprise, include, or have a sequence as set forth in any one of SEQ ID NOs: 2-28. Alternatively, the host cell can comprise a nucleotide sequence that expresses a VNp-leucine zipper peptide as described herein, or has the sequence set forth in SEQ ID NO: 29.

[0038] In one embodiment, the host cell alternatively or additionally co-expresses one or more VNp-tagged proteases in addition to the VNp-tagged target protein (VNp-tagged fusion protein) of the present invention and described herein. In this way, the proteolytic cleavage of the target protein from the VNp is obtained in the vesicle. It is understood that the co-expressed protease is selected to be suitable for use with the specific protease cleavage site contained in the fusion protein. If the selected protease leaves part of the protease recognition site attached to the target protein, further processing of the target protein may be required.

[0039] The invention also encompasses the use of the isolated vesicle nucleation polypeptide (VNp) fusion proteins or nucleotide sequences described herein to promote, enhance or increase vesicle formation in a host cell as part of a recombinant expression system.

[0040] Expressed alternatively, the invention also resides in a method of promoting, enhancing or increasing vesicle formation in a recombinant expression system by expressing in a host cell an isolated vesicle nucleation polypeptide (VNp), fusion protein or polypeptide comprising a nucleotide sequence described herein.

[0041] The present invention also encompasses the use of an isolated vesicle nucleation polypeptide (VNp), fusion protein, nucleotide sequence or host cell described herein to produce a soluble, functional biomolecule from a cellular expression system, where the biomolecule is not constitutively expressed in the host cell of the expression system.

[0042] The invention also encompasses the use of an isolated vesicle nucleation polypeptide (VNp), fusion protein, nucleotide sequence or host cell described herein to enhance or increase the yield of a soluble, functional biomolecule from a cellular expression system, where the biomolecule is constitutively expressed in the host cell of the expression system.

[0043] "Functional" refers to a biomolecule that has been folded in such a way that the molecule is biologically active, not necessarily in the same way or to the same degree as the native molecule, so long as the molecule has essential biological activity.

[0044] Expressed in a further aspect, the present invention resides in a method for enhancing the (overall) yield of soluble and functional forms of a biomolecule from a cellular expression system, wherein the biomolecule is not constitutively expressed in a host cell of the expression system, the method comprising expressing in the host cell a VNp, fusion protein or nucleotide sequence of the invention as described herein.

[0045] In one embodiment, the polypeptide, fusion protein or nucleotide sequence facilitates, enhances or increases the release of vesicles from a host cell into the culture medium of an expression system.

[0046] Preferably, the method further comprises the step of separating the polypeptide, fusion protein or product expressed by the nucleotide sequence from the culture medium.

[0047] The host cell can be a host cell described herein.

[0048] Vesicle formation within the host cell may be further enabled, facilitated or enhanced by acetylation of the VNp at the amino terminus of the polypeptide.

[0049] Also contemplated is the use of amino-terminal acetylation of an isolated vesicle nucleation polypeptide (VNp) or fusion protein described herein to promote, enhance or increase vesicle formation in a host cell as part of a recombinant expression system, which acetylation optionally or additionally promotes release of vesicles from the host cell into the culture (growth) medium.

[0050] The methods and uses of the present invention may further comprise the additional expression of a VNp-leucine zipper polypeptide as described herein. The addition of a dimerization leucine zipper sequence between the VNp and the biomolecule, as well as any further additional expression of a VNp-leucine zipper polypeptide, enhances the expression of a biomolecule that is otherwise difficult to express (e.g., toxic or insoluble).

[0051] Preferably, the host cells are cultured at a temperature between 20° C. and 40° C., for example between 25° C. and 37° C., ideally under aerobic conditions.

[0052] Furthermore, the methods and uses described herein may further comprise a step of storing the vesicles in the host cell of the recombinant expression system or separated from the host cell. Storage at about 4°C is considered appropriate. Storage can be for at least up to 6 months.

[0053] The method and use of the present invention may further comprise a step of purifying the biomolecule. In this way, the biomolecule encapsulated in the vesicle can be released and purified for further processing or use. Methods for releasing and purifying biomolecules from membrane-bound structures such as lipid vesicles are well known and standard in the art. Methods that have been tested and confirmed to be suitable include (but are not limited to) sonication (the simplest and most attractive, since it has the least effect on the vesicle contents), treatment with detergents, or osmotic shock. Alternatively, biological purification includes cleaving the biomolecule from the VNp within the vesicle before it is released from the vesicle, and additional purification that may be required. In one embodiment, the intravesicular cleavage is performed by a suitable protease, ideally a VNp-tagged protease co-expressed with a VNp-tagged target protein.

[0054] The invention also encompasses the co-expression and excretion of biomolecules, such as carbohydrates, antigens, nucleic acids or recombinant proteins, in combination with the isolated vesicle nucleating polypeptides (VNp) described herein as cargo in membrane-bound vesicles, with the understanding that vesicle formation is facilitated or enhanced by the VNp.

[0055] The present invention also contemplates the use of recombinant expression systems, such as E. coli, for the production of one or more biomolecules that are expressed in a host cell and, optionally, released from the host cell, where the expression system comprises an isolated vesicle nucleation polypeptide, fusion protein, nucleotide sequence or host cell described herein.

[0056] In particular, in cases where intracellular expression of cargo, e.g. recombinant polypeptides expressed in prokaryotic hosts such as E. coli, are produced in an insoluble form, the present invention allows for the export of the cargo from the cell in a soluble form.

[0057] The present invention will now be described in further detail with reference to the following non-limiting examples and figures. [Brief description of the drawings]

[0058] [Figure 1-1] Figure 1 shows the formation of recombinant vesicles. Figure 1a shows OmpA-GFP SIM fluorescence. Figures 1b and 1c show negative stain EM serial section images of VNp-induced membrane curvature in E. coli. Figure 1d shows EM of E. coli cells cultured on grids generating vesicles. Figure 1e shows SIM fluorescence of mCherry (magenta) and -mNeongreen (green) of VNp2-mNeongreen OmpA-mCherry expressing E. coli cells. Figure 1f shows anti-mNeongreen immuno-EM image of serial section of E. coli with VNp-mNeongreen induced vesicles. Figure 1g shows EM image of isolated VNp-mNeongreen containing vesicles. Figure 1h shows fluorescent image of isolated VNp-mNeongreen containing vesicles. Figure 1i shows Coomassie stained gel of cell culture fluid and filtered medium of VNp2-mNeongreen expressing cells. Figure 1j shows a schematic of VNp-induced cargo-containing vesicles, and Figure 1k shows Coomassie stained samples of uninduced and induced cultures of VNp2-DARP, VNp2-uricase and VNp2-stefin A expressing cells, or filter-induced cultures. [Figure 1-2]Figure 1l shows the average yield per liter of culture of unlabeled mNeongreen-labeled fusions of target proteins obtained from cell extracts (open) and medium (solid). Figure 1m shows C-terminal mNeongreen-labeled fusions of target proteins obtained from cell extracts (open) and medium (solid). Error bars are s.d. from ≥3 experimental replicates. [Diagram 2] Enrichment of VNp fusions within recombinant vesicles. Figure 2A is a widefield fluorescence image of a universal (top right) containing centrifugal medium from an overnight culture of VNp-mNeongreen expressing cells (top left) concentrated through a 0.1 μm filter and associated mNeongreen-containing vesicles. Figure 2B shows a BSA quantification control and an SDS-PAGE gel of 8 μl centrifugal medium fractions from VNp fusion-inducing cultures obtained from independent biological replicates. These samples were then subjected to trypsin digestion and proteomic analysis. Figure 2C is a pie chart showing the typical composition of VNpfusion-containing vesicles as determined from proteomic analysis. [Diagram 3] Wide-field fluorescence images of VNp-induced recombinant protein packaging vesicles using different E. coli strains and promoters: (Fig. 3a) K12, (Fig. 3b) λ DE3 W3110, (Fig. 3c) JM109, and (Fig. 3d) BL21 DE3 E. coli strains expressing VNp-mNeongreen from the T7 promoter (Fig. 3a, b, c) or the rhamnose promoter (Fig. 3d). [Figure 4]VNp tags allow expression and isolation of heterodimeric soluble functional antibodies from E. coli. Figures 4A-D show VNp-fAb purification from E. coli. Figure 4A shows bimolecular fluorescently complemented VNp-fAb fusions allowing detection of fAb Hc / Lc heterodimer formation. Figure 4B shows stained SDS-PAGE. Figure 4C shows anti-His Western blots (recognizing HIS6-tagged Lc) of samples from VNp-Hc7VNp-Lc** expression, nickel affinity purification and protein G binding. Figure 4D shows VNp-Hc / Lc heterodimer-dependent BiFC fluorescence from cell lysates, nickel eluates and subsequent protein G-bound fractions. Figures 4E-F show VNp-mAb expression and purification from E. coli. FIG. 4E shows stained SDS-PAGE of samples from VNp-mAB-Hc / VNp-mAB-Lc expression, nickel affinity purification, and protein A binding, and FIG. 4F shows anti-His Western blot (recognizing HIS6-tagged He). [Diagram 5] Figure 2. VNp enables vesicle-targeted proteolysis of recombinant proteins. E. coli cells containing constructs expressing the VNp-mNeongreen-DARPinoff7 fusion alone or in combination with the VNp-maltose binding protein (MBP)-TEV protease fusion were cultured and expression was induced in overnight flask cultures. The VNp-mNeongreen-DARPinoff7 fusion contained a TEV cleavage site between the mNeongreen and DARPin sequences. Expression of VNp-mNG-DARP alone resulted in expression of the full-length protein (50.8 kDa in size) and vesicle excretion. In contrast, when the same protein was coexpressed with the TEV protease fusion, VNp-mNG-DARP was cleaved at the TEV site and the VNp-mNG (32 kDa) and DARPinoff7 (18.8 kDa) fragments were excreted in excreted vesicles in the culture medium. The predicted protein sizes are 75.4 kDa for VNp-MBP-TEV, 50.8 kDa for VNp-mNg-DARP, 32 kDa for VNp-mNG, and 18.8 kDa for DARP. [Figure 6-1]Figure 6: VNp dimer fusions generate fusions containing plasma membrane packaging.Figure 6a shows negative staining EM serial section images of VNp2-etanercept induced inward membrane curvature in E. coli. [Figure 6-2] Figure 6b shows serial anti-mNeongreen immunostained EM images of VNp2-etanercept-induced inward membrane curvature in E. coli. Figure 6c shows SIM imaging of MinD-mScarlet-labeled inner membranes of E. coli co-expressing VNp2 and the NatB amino-terminal acetylation complex, which generate cytoplasmic membrane-bound structures. Figure 6d shows negatively stained serial EM sections of E. coli co-expressing VNp2 and the NatB amino-terminal acetylation complex, which generate cytoplasmic membrane-bound structures. Figure 6e shows that amino-terminal acetylation of VNp2-mNeongreen promotes incorporation of mNeongreen into intracellular membrane-bound structures. [Figure 6-3] Figures 6f and 6g are anti-mNeongreen immuno-EM images of serial sections of E. coli expressing VNp2-LZ-mNeongreen. Figure 6h is a SIM image of CydAB-mNeongreen-labeled inner membranes of E. coli expressing VNp2-LZ. VNp2-LZ dimers are shown to be concentrated within the lumen of inner cytoplasmic membrane-bound vesicles. [Figure 7] Figure 2 shows that VNp fusions remain soluble even when the VNp tag is cleaved with the target protease. Purified VNpmNG-TEV-DARP and VNp-mNG-TEV-uricase were digested with TEV protease. The cleaved proteins were not detected in the pellet fraction (P) but remained in the supernatant (S / N) fraction after centrifugation at 13,200 RCF. [Figure 8] Figure 1: VNp fusions can be excreted from cells cultured in various media volumes. The excretion yield of VNp-mNeongreen was measured by culturing cells in various volumes in different sized flasks to control the surface area to volume ratio (x-axis). [Figure 9-1]Dimeric VNp-leucine fusions form cytoplasmic VNp-fusion-filled vesicles in E. coli. Figure 9a shows size-exclusion chromatography profiles of purified recombinant VNp-mNG and VNp-LZmNG proteins (inset), confirming that the introduction of a leucine zipper (LZ) motif into VNp-mNeongreen (mNG) fusions induces stable dimer formation. Each fusion protein and protein standards (29 kDa carbonic anhydrase - blue; 66 kDa BSA - red; 443 kDa apoferritin complex - yellow) were run under the same conditions. Figures 9b and 9c show anti-mNeongreen immuno-EM micrographs of sections of E. coli expressing VNp-LZ-mNeongreen. [Figure 9-2] Figure 9d shows a SIM image of CydAB-mNeongreen-labeled inner membranes of E. coli expressing VNp-LZ, demonstrating that VNp-LZ dimers are concentrated within the lumen of intracytoplasmic membrane-bound vesicles. [Figure 10] Expression of α-synuclein fusions in E. coli induces vesicular shedding into the culture medium. Figure 10a is a widefield image of E. coli expressing αSyn (60 amino acid sequence) fused to both mNeongreen and human growth hormone (αSyn-mNG-hGH), showing shedding of the αSyn fusion into extracellular vesicles. Figure 10b is an anti-FLAG Western blot of samples from bioreactor cultures of E. coli expressing αSyn-mNG-hGH showing expression and shedding into vesicles (highlighted with *) in four independent experiments. Figure 10c shows that SDS-PAGE analysis of filtered media from overnight IPTG-induced cultures of E. coli containing either an empty expression vector or vectors conferring ampicillin or kanamycin resistance confirmed the expression of αSyn-mNG-hGH. αSyN-hGH is shedding into the medium independent of antibiotic selection. [Figure 11a]VNp interacts with E. coli membranes in vivo and in vitro. Figure 11a shows thermal shift mNeongreen fluorescence curves for VNp2-mNeongreen alone (black), or in the presence of 100 nm vesicles composed of phosphatidic acid (red), or a mixture of total lipids (blue) or polar lipids (green). [Figure 11b] Figure 11b shows the fluorescence lifetime of the donor fluorophore in E. coli cells expressing VNp donor fluorophore fusions (VNp2-Cer3 / VNp2-mNG) together with an inner membrane protein (MinD-Citrine) or outer membrane protein (OmpA-mCherry) labeled with an acceptor fluorophore. *Nt-acetylated VNp2; ☆-Different at a confidence level of 99.99%. Data were obtained using a 60x water immersion objective. [Figure 11c] FIG. 11c is a histogram of mNeongreen fluorescence lifetime in E. coli cells expressing mNeongreen donor fluorescently labeled VNp fusion (VNp2-mNG) alone or in combination with mCherry acceptor fluorescently labeled CydAB inner membrane complex. [Figure 11d] Figure lld is an mNeongreen fluorescence lifetime image of a VNp2-mNG CydAB-mCherry expressing cell (from Figure 10c) showing a decrease in donor lifetime at the plasma membrane in the presence of CydAB. Figures llc and lld were acquired using a 60x oil immersion objective. The decrease in lifetime length is reflected by a change from blue (2.8 ns) to green (2.6 ns). [Figure 11e] Figure ​(e)shows circular dichroism indicating that the α-helical VNp structure is stabilized by the E. coli lipid membrane. CD spectra of VNp2 (black), whole E. coli lipid vesicles (grey) alone, or a mixture of VNp2 and E. coli lipid vesicles (red). The relatively broad negative CD peaks at 208 and 222 nm observed for the mixture of VNp2 and E. coli lipid membranes are consistent with a single α-helical structure, indicating that the α-helix of VNp2 is stabilized by interactions with the E. coli membrane lipid vesicles. [Figure 12a]FIG. 12a is a growth curve generated from the average of four replicate cultures of BL21(DE3) E. coli cells containing the empty pRSFDUET vector (open black circle), pRSFDUET.mNeongreen (open green circle), pRSFDUET.VNp2 (filled black circle) or pRSFDUET.VNp2.mNeongreen (filled green circle) grown in LB at 37° C. on the same 96-well plate, demonstrating that expression of VNp or VNp fusions does not affect bacterial growth. [Figure 12b] Figure 12b shows a routine test demonstrating the exclusion of viable cells from the vesicle-containing filtrate: 10 μl of whole culture (i) and 1 ml of 0.45 μm medium filtrate (ii) from an overnight culture of VNp2-mNG-expressing E. coli cells were plated out onto LB (ii) or LB supplemented with kanamycin (i) and incubated overnight at 37°C. [Figure 12c] Figure 12c shows dynamic light scattering of filter-purified VNp-mNeongreen-derived vesicles. Graph shows average data (± standard error) from 10 independent analyses. [Figure 12d] Figure 12d shows that vesicles containing VNp2-mNeongreen that were filter purified from the medium of an overnight culture of BL21 DE3 pRSFDUET1-VNp2-mNeongreen cells were stored at 4°C. The total mNeongreen fluorescence and the percentage of mNeongreen signal within 0.2 μm filter-retained vesicles did not change over time, indicating the stability of the vesicles and folded mNeongreen protein. [Figure 12e] Figure 12e shows the fluorescence (515 nm) derived concentration of complexed bimolecular fluorescent complementation fragments contained within vesicles isolated from BL21(DE3) E. coli expressing VenusN154 and VNp2-VenusC155 (white); VNp2-VenusN154 and VNp2-VenusC155 (gray); or VNp2-LZ-VenusN154_VNp2-LZ-VenusC155 (black). [Figure 12f]Figure 12f is a gel filtration curve comparing the elution profiles of VNp2-mNG (black) and VNp2-LZ-mNG (gray), demonstrating that the leucine zipper induces dimerization of the VNp fusion within the vesicles. [Figure 13] Figure 1: VNp fusions are contained within the lumen of isolated recombinant vesicles. VNp2-mNeongreen-containing vesicles filter-purified from the medium of overnight cultures of BL21 DE3 pRSFDUET1-VNp-mNeongreen cells were mounted on EM grids and subjected to anti-mNeongreen immuno-EM analysis. mNeongreen-dependent gold-dense fragments were released from the vesicles upon osmotic shock from resuspension in water. These were not observed in control samples immunoanalyzed before rupture or without the primary anti-mNeongreen antibody. [Figure 14a] Figure 14 shows that vesicle-isolated VNp fusions are functional. Figure 14a shows a uricase enzyme activity assay showing the activity of uricase isolated from cell pellets using conventional methods (red) or from VNp-induced vesicles (black). Buffer only control (grey) shows the dilution-dependent change in baseline absorbance at 293 nm. Uricase enzyme / buffer was added to the substrate after 5 min equilibration (dashed line). [Figure 14b] Figure 14b shows an anti-mNeongreen Western blot demonstrating disulfide bond-dependent oligomerization (Figure 6b) and functionality (Figure 6c) of VNp2-mNG-etanercept purified from E. coli. Figure 14b shows that disulfide bond-dependent oligomers (*) of VNp2-mNG-etanercept are disrupted by the addition of DTT reducing agent (left gel). [Figure 14c]Figure 14c shows an anti-mNeongreen Western blot demonstrating disulfide bond-dependent oligomerization (Figure 6b) and functionality (Figure 6c) of VNp2-mNG-Etanercept purified from E. coli. Figure 14c: (Right gel) Affinity-purified VNp-mNG-Etanercept-Hise expressed in E. coli was bound to Protein A-Dynabeads, then washed with binding buffer and boiled in SDS-PAGE loading buffer to release bound protein. The predicted size of VNp-mNeongreen-Etanercept is 83.9 kDa. [Figure 14d] FIG. 14d shows the growth curves of E. coli expressing DNase and VNp2-Dnase. [Figure 14e] FIG. 14e shows the expression profile (predicted sizes of Dnase and VNp-Dnase are 30.2 kDa and 34.4 kDa, respectively). [Figure 15] Figure 1 shows the excretion yield of VNp-mNeongreen-containing vesicles from E. coli cultured in different sized flasks to change the surface area:volume ratio of the culture. Blue dots are standard Erlenmeyer flasks, red dots are baffled Erlenmeyer flasks, and black dots are standard Erlenmeyer flasks sealed with parafilm to induce anaerobic conditions. Higher oxygen concentrations increased the excretion of vesicles. [Figure 16] FIG. 13 shows Coomassie stained SDS-PAGE analysis of samples from an example of E. coli VNp-DARPinOFF7 induction in a 20 L fermenter. [Figure 17]Figure 17 shows that VNp fusion protein remains active within vesicles even after storage at 4°C in sterile PBS / medium for over 6 months. Figure 17a shows the enzymatic activity of uricase isolated from cell pellets by conventional methods (red) or from VNp-uricase-derived vesicles (black). Grey = buffer only control. Uricase enzyme / buffer was added to uric acid substrate after 5 min equilibration (dashed line). Figure 17b shows the stability of uricase enzymatic activity from protein stored within VNp-uricase vesicles. Uricase activity was measured using stopped-flow of either fresh vesicle-purified VNp-uricase (black), the same vesicle-purified VNp-uricase stored in reaction buffer for 2 months at 4°C (green), or freshly purified uricase from vesicles stored for 2 months at 4°C (blue). The rate was determined from the steady state region of the averaged curves (highlighted by the box). [Figure 18] Figure 18 shows the viability of VNp in mammalian cell lines. Figure 18A shows mammalian HEK cell lines generated containing an empty vector or a plasmid containing either VNp2-mNeongreen or VNp6-mNeongreen under the control of a CMV or SV40 promoter. Figure 18B shows that HEK cells containing a plasmid with VNp6-mNeongreen under the control of the SV40 promoter induced the formation of extracellular VNp6-mNeongreen-containing vesicles (arrows). [Figure 19] Figure 1: The VNp tag promotes recombinant protein expression under anaerobic culture conditions. The gel shows the expression profile from cells containing mNeongreen and Stefin A alone or in fusion with VNp. The predicted protein sizes are mNeg, 28 kDa; VNp-mNg-, 32.5 kDa; mNg-Stefin A, 39 kDa; and VNp-Stefin A, 14.5 kDa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] array: SEQ ID NO:1 - Full length endogenous Hs α-synuclein (VNp1) SEQ ID NO:2 - the amino-terminal 38 aa of Hs α-synuclein (VNp2) SEQ ID NO:3 - The amino-terminal 38 aa of Hs β-synuclein (VNp3) SEQ ID NO:4 - The amino-terminal 38 aa of Hs γ-synuclein (VNp4) SEQ ID NO:5 - VNp2 with 3K-E mutation (VNp5) SEQ ID NO:6 - VNp4 with 2K-D mutation (VNp6) SEQ ID NO:7 - VNp4 with 6K-A mutation (VNp7) SEQ ID NO:8 - VNp4 with A29M mutation (VNp8) SEQ ID NO:9 - VNp6 with 110A mutation (VNp9) SEQ ID NO: 10 - VNp6 with the A11P.Q24P mutation (VNp10) SEQ ID NO:11 - VNp6 with E31K mutation (VNp11) SEQ ID NO:12 - VNp6 with D2G mutation (VNp12) SEQ ID NO: 13 - VNp6 with D2P mutation (VNp13) SEQ ID NO:14 - VNp6-20 amino acid truncation (VNp14) SEQ ID NO:15 - VNp6-25 amino acid truncation (VNp15) SEQ ID NO:16 - VNp6-30 amino acid truncation (VNp16) SEQ ID NO:17 - VNp6-18 amino acid truncation (VNp17) SEQ ID NO:18 - VNp6-15 amino acid truncation (VNp18) SEQ ID NO:19 - VNp6-11 amino acid truncation (VNp19) SEQ ID NO:20 - VNp6-20 amino acid truncation with F4Y mutation (VNp20) SEQ ID NO:21 - VNp6-20 amino acid truncation with F4A mutation (VNp21) SEQ ID NO:22 - VNp6-20 amino acid truncation with D2P mutation (VNp22) SEQ ID NO:23 - VNp6-20 amino acid truncation with K6D mutation (VNp23) SEQ ID NO: 24 - VNp22 with K5D and K6D mutations (VNp24) SEQ ID NO:25 - VNp6-10 amino acid truncation (VNp25) SEQ ID NO:26 - VNp6-9 amino acid truncation (VNp26) SEQ ID NO: 27 - VNp14 with S9D mutation (VNp27) SEQ ID NO: 28 - VNp2 with S9D mutation (VNp28) SEQ ID NO:29 - leucine zipper sequence

[0060] The present invention relates to a method to program simple cells to generate membrane packages that can be loaded with a variety of molecules of interest. This method not only provides a way to manufacture membrane packages loaded with target proteins in vivo, but also reveals how inducible synthetic post-translational modifications can control whether the packages preferentially remain in the cytoplasm or are released extracellularly.

[0061] material E. coli strains used in this study: BL21DE3F - ompThsdS B (r B - , m B - )galdcm(DE3). DH10β F - mcrA Δ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15ΔlacX74 recA1 endA1 araD139Δ(ara-leu)7697galUgalKλ - rpsL(Str R )nupG W3110F - λ - IN(rrnD-rrnE)1rph-1 CLD1040F - λIN(rrnD-rrnE)1rph-1OmpT JM109 F traD36 proAB laql qZΔM15 endA1 recA1 gyrA96 thi hsdR17(r k - , m k + ) relA1 supE44 Δ(lac-proAB)

[0062] Plasmids: pRSFDuet-1_VNp-His6; pRSFDuet-1_VNp-mNeonGreen; pRSFDuet-1_VNp-mNeonGreen OmpA-mCherry;pRSFDuet-1_VNp-mNeonGreen_mScarlet-minD; pDuet-1_VNp-mCerulean3; pDuet-1_VNp-mNeonGreen; pDuet-1_VNp-mNeonGreen_VNp-mCherry; pDuet-1_VNp-mNeonGreen_VNp-mCherry; pDuet-1_VNp-mCerulean3_Citrine-minD; pACYCDuet-1_naa20_naa25(pNatB)(Johnson,M.et al(2010)PLoS ONE 5,e15801); pRSFDuet-1_VNp-LZ-mNeonGreen; pRSFDuet-1_DARPinOFF7; pRSFDuet-1_VNp-DARPinOFF7; pRSFDuet-1_uricase; pRSFDuet-1_VNp-uricase; pRSFDuet-1_Stefin A; pRSFDuet-1_VNp-Stefin A; pRSFDuet-1_FGF21; pRSFDuet-1_VNp-FGF21; pRSFDuet-1_hGH; pRSFDuet-1_VNp-hGH; pRSFDuet-1_VNp-LZ-hGH; pRSFDuet-I mNeonGreen; pRSFDuet-1_mNeonGreeN-DARPinOFF7; pRSFDuet-1_VNp-mNeonGreeN-DARPinOFF7; pRSFDuet-1_mNeonGreeN-uricase; pRSFDuet-1_VNp-mNeonGreeN-uricase; pRSFDuet-1_mNeonGreeN-stefin A; pRSFDuet-1_VNp-mNeonGreeN-stefin A; pRSFDuet-1_VNp-mNeonGreeN-etanercept; pRSFDuet-1_VNp-mNeonGreeN-etanercept; pRSFDuet-1_mNeonGreeN-erythropoietin; pRSFDuet-1_VNp-mNeonGreeN-erythropoietin; pRSFDuet-1_VNp-LZ_VNp-LZ-etanercept; pRSFDuet-1_VNp-LZ_VNp-LZ-hGH. .

[0063] Plasmid sequences and constructs have been deposited at addgene.org. Uniprot accession numbers of cargo full-length protein sequences tested in this study: - DARP - alkylin repeat protein off7 (Agrobacterium radiobacter) - B9JMD9 - DNase L- Deoxyribonuclease I (Bos taurus) - P00639 - EPO - Erythropoietin (Human (Homo sapiens)) - P01588 - Etanercept - Tumor necrosis factor receptor superfamily member 1B (Homo sapiens) - P20333 - FGF21 - Fibroblast Growth Factor 21 (Human (Homo sapiens)) - Q9NSA1 - hGH - somatotropin (Human (Homo sapiens)) - P01241 - mNeonGreen (Lancelet (Branchiostoma lanceolatum)) - A0A1 S4NYF2 - Stefin A - Cystatin-A (Human (Homo sapiens)) - P01040 - Uricase (Torula yeast (Cyberlindnera jadinii)) - P78609 method

[0064] Bacterial cell culture and protein induction: All bacterial cells were cultured at 37°C in Lysogeny broth (10 g tryptone; 10 g NaCl; 5 g yeast extract per liter) and Terrific broth (12 g tryptone; 24 g yeast extract; 4 ml 10% glycerol; 17 mM KH2PO4; 72 mM K2HPO4 per liter) media. Five ml of LB starter from freshly transformed bacteria was grown to saturation at 37°C and used to inoculate 100-500 ml volumes of TB medium. Flask cultures were grown overnight at 37°C with orbital shaking at 200 rpm. Expression of recombinant proteins from the T7 promoter was confirmed when the cultures reached an OD of 0.8-1.0. 600At the time of reaching 100 μg / ml, the VNp was induced by adding isopropylthio-p-galactoside (IPTG) to a final concentration of 20 μg / ml (except for etanercept, which was used at 10 μg / ml). To generate amino-terminally acetylated VNp, the target constructs were co-transformed with pNatB into E. coli to co-express the fission yeast amino-α-acetyltransferase complex B, Naa20 and Naa25 (Johnson, M. et al (2010) PLoS ONE 5, e15801). Amino-terminal acetylation was confirmed by electrospray mass spectrometry of purified VNp fusion proteins.

[0065] Growth curves were plotted at OD at the start of the proliferation analysis experiment. 600 The OD was prepared from a 96-well plate culture prepared from a late logarithmic phase culture diluted in fresh medium to 0.1 nm. 600 Absorbance values ​​were obtained using a Thermo Scientific Multiscan Go1510-0318C plate reader and recorded using Skanlt Software 4.0. OD 600 Values ​​were measured every 15 min for the duration of the experiment, and growth curves were constructed from the average of four individual biological replicates.

[0066] Soluble protein extracts: Cell pellets from 50 ml cultures were resuspended in 5 ml soluble extraction buffer (20 mM tris(hydroxymethyl)aminomethane (TRIS), 500 mM NaCl, pH 8.0), sonicated for a total of 2 min (6x20 sec pulses), and centrifugation at 18,000 rpm (4°C) for 30 min to remove cell debris. Target protein concentrations were determined using fluorescence of mNeonGreen fusions or gel densitometry. To determine equivalence, both methods were directly compared on the same samples.

[0067] Recombinant vesicle isolation: Vesicles were isolated directly from bacterial cell cultures by passing the culture medium through a sterile, detergent-free 0.45 μm polyethersulfone (PES) filter. Typical purity and concentration from equal culture and filter passages are shown in Figure 1. Exclusion of viable cells from vesicle containing filtrates was routinely tested by plating on LB plates without antibiotics and incubating overnight at 37 °C (examples are shown in Figure 12).

[0068] Measurement of protein concentration: Fluorescence scanning was used to determine the concentration of mNeongreen-labeled proteins in vesicle-containing medium and soluble protein extracts. Absorbance was measured at 506 nm using a Varian Cary® 50Bio UV-Vis spectrophotometer, using measurements from equivalent empty vector cultures for baseline correction, and was 116,000 nm. -1 Concentrations were determined using the extinction coefficient in cm-1. Concentrations of mNeongreen-unlabeled proteins were determined by gel densitometry analysis of triplicate samples run in parallel with bovine serum albumin (BSA) loading standards on Coomassie-stained SDS-PAGE gels. Gels were scanned and analyzed using ImageJ software. Concentrations were determined by both UV and densitometry for three independent VNp-mNeongreen samples to ensure parity between analytical techniques. Average yields in Figure 1 and Table 2 were calculated from a minimum of three independent biological replicates from cultures of BL21 DE3 E. coli grown in TB medium (mg soluble target protein / liter culture).

[0069] Protein isolation from vesicles: Purified VNp-derived vesicles were resuspended in ice-cold 1x PBS and then sonicated to disrupt the vesicle membrane and release the VNp fusion protein. To further purify the carboxyl His6-tagged recombinant VNp fusion protein (all recombinant proteins expressed in this study contain a carboxyl-terminal His6 affinity tag), the solution was mixed with a 1:5 dilution of 5x binding buffer (250 mM TRIS, 2.5 M NaCl, 5% Triton-X, 50 mM imidazole pH 7.8) and then sonicated with Ni.2+ -agarose resin gravity column. Cytoplasmic recombinant proteins were purified by eluting soluble protein extracts (with imidazole added to 20 mM) with Ni 2+ -Agarose resin gravity columns were used to purify the His-tagged proteins. In each case, the matrix-bound His-tagged proteins were washed, eluted (with imidazole), and dialyzed into the appropriate storage or assay buffer. The identity and amino-terminal acetylation of the isolated proteins were confirmed by electrospray mass spectrometry.

[0070] Measurement of cytoplasmic VNp concentration: VNp-mNeongreen expression was induced in BL21 DE3 E. coli for 4 hours at 37 °C (when extracellular vesicle production is observed), and images of ≥120 cells were acquired using widefield imaging (described below) from three independent sample preparations. The average mNeongreen intensity was determined from a 5 × 5 pixel area within each E. coli cell. A calibration curve was generated from multiple images of slide-mounted VNp-mNeongreen solutions of known concentrations, using the same imaging conditions as for cell image acquisition. This was used to calculate the average cytoplasmic mNeongreen concentration from the sum of the average intensities of all cells measured (18.76 ± 0.14 μM).

[0071] Affinity purification of His-tagged proteins: To isolate recombinant proteins from vesicles, medium from an overnight induced culture expressing a VNp-tagged protein was passed through a 0.45 μm polyethersulfone (PES) filter, and the ensuing vesicle-containing flow-through was sonicated and mixed with a 1:5 dilution of 5× binding buffer (250 mM TRIS, 2.5 M NaCl, 5% Triton™-X, 50 mM imidazole, pH 7.8) and then filtered with Ni. 2+ -agarose resin gravity column. Cytoplasmic recombinant proteins were purified by eluting soluble protein extracts (with imidazole added to 20 mM) with Ni 2+-Agarose resin gravity columns were used to purify the His-tagged proteins. In each case, the matrix-bound His-tagged proteins were washed, eluted (with imidazole), and dialyzed into the appropriate storage or assay buffer. The identity and amino-terminal acetylation of the isolated proteins were confirmed using electrospray mass spectrometry.

[0072] Circular dichroism (CD): Measurements were performed in 2 mm quartz cuvettes using a Jasco 715 spectrophotometer. VNp protein and 100 nm extrusion vesicles were diluted in CD buffer (10 mM potassium phosphate, 5 mM MgCl2, pH 7.0) to concentrations of 0.4 mg / ml and 0.2, respectively. Broad negative peaks at 208 nm and 222 nm and a positive peak below 200 nm were consistent with an α-helical structure.

[0073] Electrospray LC-MS of proteins: Electrospray mass spectra were recorded on a Broker micrOTOF-Q II™ mass spectrometer. Samples were desalted on-line by reversed-phase HPLC using a short water, acetonitrile, 0.05% trifluoroacetic acid gradient at a flow rate of 0.2 ml / min on a Phenomenex® Jupiter® C4 column (5 μm, 300 Å, 2.0 mm x 50 mm) run on an Agilent® 1100 HPLC system. The eluent was monitored at 214 nm and 280 nm and directed to the electrospray source operating in positive ion mode at 4.5 kV, and mass spectra were recorded from 500 to 3000 m / z. Data were analyzed with Broker's Compass Data Analysis software and deconvoluted to obtain the masses of uncharged proteins.

[0074] In-gel trypsin digestion and proteomic analysis of recombinant vesicles: Samples of purified VNp-DARP-induced vesicles (shown in Figure 7 ) were subjected to SDS-PAGE followed by Coomassie staining, and the entire sample lane was excised into small pieces, which were transferred to 1.5 ml microcentrifuge tubes and stored in distilled water at 4°C until processing.

[0075] The gel particles were washed with 150 μl of fresh 50 mM NH4HCO3:acetonitrile (1:1 ratio) for 15 min. The liquid was removed and the gel pieces were resuspended in 150 μl of acetonitrile for 15 min, after which the liquid was removed again and the gel pieces were resuspended in 100 μl of 10 mM DTT in 50 mM NH4HCO3 and incubated at 56 °C for 30 min. The gel pieces were centrifuged and excess liquid was removed, followed by incubation in 100 μl of acetonitrile for 1 min, after which the acetonitrile was removed again and the gel pieces were suspended in 100 μl of 55 mM chloroacetamide in 50 mM NH4HCO3 and incubated at room temperature in the dark for 20 min. The pellet was then centrifuged and the chloroacetamide solution was removed. The gel pieces were then washed with 150 μl of 50 mM NH4HCO3:acetonitrile (1:1) for 15 min, followed by 150 μl of 50 mM NH4HCO3 for 15 min, removing the liquid by centrifugation after each wash. The gel pieces were then washed with 200 μl of acetonitrile for 15 min, rehydrated with 50 μl of digestion buffer (12.5 mM NH4HCO3, 10% acetonitrile) containing 5 ng / μl trypsin, and left at room temperature overnight.

[0076] After digestion was complete, 15 μl of acetonitrile was added to the sample and sonicated in an ultrasonic bath for 15 min. The gel fragments were isolated by centrifugation and the supernatant was collected in a 0.5 ml microcentrifuge tube (A). The gel fragment pellet was resuspended in 30 μl of 50% acetonitrile containing 5% formic acid and sonicated in an ultrasonic bath for 15 min, the pellet was again isolated by centrifugation and the supernatant was collected in a new 0.5 ml microcentrifuge tube (B). The contents of tubes A and B were combined, dried in vacuum and resuspended in 20 μl of 5% acetonitrile, 0.1% TFA. Samples were passed through Pierce C18 Spin Tips and analyzed by nano-LCMS.

[0077] Expression of recombinant proteins in E. coli grown under anaerobic conditions: An airtight anaerobic culture vessel was filled with LB medium supplemented with antibiotics and 50 mM sodium fumarate or 100 mM sodium nitrate (fumarates and nitrates act as electron acceptors instead of oxygen). Oxygen was purged from the medium and airtight culture vessel with argon gas for 20 min. A fresh BL21 DE3 E. coli transformant containing the expression construct was used to inoculate a 5 ml overnight culture in LB + selective antibiotic. A 1:50 dilution of this starter culture was added (using a syringe and needle) to the oxygen-purged culture vessel. The anaerobic culture was incubated with shaking at 37 °C for 2 h, when oxygen-purged IPTG (final concentration 20 μg / ml) was added to induce T7 promoter-dependent protein expression. It was then grown at 37 °C for 24 h before harvesting and analysis.

[0078] Gel filtration assay: 500 μl of protein sample was loaded onto a Superdex® 200 Increase 10 / 300 GL size-exclusion column (GE Healthcare Life Sciences) equilibrated with phosphate-buffered saline (PBS) at room temperature and run at a flow rate of 0.75 ml / min. Eluted proteins were measured with a Viscotek® SEC-Mals9 and a Viscotek® RI detector VE3580 (Malvern Panalytical). Data was analyzed using OmniSEC® software.

[0079] Lipid binding assay: The affinity of VNp for E. coli membrane lipids was established using a thermal shift fluorescence binding assay adapted from Nji, E. et al (Nat. Commun. (2018) 9, 4253-12). Equivalent assay sample: 65 μl of 3 mg / ml VNp-mNeongreen, 65 μl of 1 mM 100 nm extruded vesicles consisting of the lipid mixture to be tested, 15 μl of 10% N-octyl-β-D-glucopyranoside (OGP), and 5 μl of 20 mM Tris-HCl, pH 7.0 were prepared in PCR tubes and kept at the specified temperature for 10 min in a gradient PCR device. Samples were centrifuged at 18,000xg and fluorescence of the supernatants was measured in black 96-well plates (BRAND®, Germany) using a BMG Clariostar® (BMG Labtech). Fluorescence measurements were normalized and used to generate melting curves and melting temperatures (Tm) were determined using Origin software (OriginLab). Final Tm values ​​were the mean (±sd) calculated from three independent sample repetitions.

[0080] Uricase assay: 500 μl of 200 mM uric acid in 100 mM Tris, pH 8.5 was placed in a cuvette and the OD 293 Measurements were performed for 4 or 5 min. Then, 500 μl of 4.2 mg / ml purified VNp2-uricase (dialyzed into 0.1 M Tris, pH 8.5) or dialysis buffer alone was added to the cuvette and the OD was measured for 25 min. 293 was measured. (Adapted from Huang, S.-H. & Wu, T.-K. (2004) Eur. J. Biochem. 271, 517-523).

[0081] Wide-field fluorescence microscopy: Cells were mounted on coverslips under circular LB-agarose (2%) pads less than 1 mm thick, attached to glass slides with appropriate spacers, and then observed under an inverted microscope. (Mulvihill, DP (2017) Cold Spring Harb. Protoc. 2017, 761-773). Live-cell imaging of each sample was completed within 30 minutes of mounting the cell samples on the coverslips.

[0082] Structured illumination microscopy (SIM) was performed as previously described (Periz, J. et al (2019) Nat. Commun., 1-16; Qiu, H. et al (2016) Science 352, 697-701) using a Zeiss Elyra PS.1 microscope with a 100x NA1.46 oil immersion objective (Zeiss a Plan-Apochromat). Briefly, cells were mounted on precision No. 1.5 coverslips (Zeiss, Jenna, Germany) under a thin LB-agarose pad. 488 nm and 561 nm lasers were irradiated on mNeonGreen and mCherry / mScarlet fusions, respectively. The optical filter set consisted of a laser blocking filter MBS405 / 488 / 561 as a dichroic mirror and a dual-band emission filter LBF-488 / 561. A total of three rotations of the illumination pattern were performed to obtain two-dimensional information. Super-resolution SIM image processing was performed using Zeiss Zen software. After calibration with a pre-mounted MultiSpec™ bead sample, the two color images were aligned using the same software.

[0083] Fluorescence Lifetime Imaging Microscopy (FLIM): The one-photon and two-photon systems used in this study have been described previously (Botchway, SW et al (2015) J. Microsc. 258, 68-78). Prior to data acquisition, protein expression was confirmed using a confocal microscope. Here, a Nikon Eclipse C2-Si confocal scan head attached to an inverted Nikon TE2000 or Ti-E microscope was used. mNeongreen was excited at 491 nm (emission wavelength 520 / 35 nm) and mCherry was excited at 561 nm (emission wavelength 630 / 50 nm) using an NKT supercontinuum laser. FLIM images were obtained as follows. A mode-locked titanium sapphire laser (Mira F900, Coherent Laser Ltd) generated two-photon (950 nm) wavelength light and pulsed 180 fs at 76 MHz. The laser was excited by a solid-state continuous wave 532 nm laser (Verdi 18, Coherent Lasers Ltd). Fluorescence was collected through a BG39 filter for the donor fluorophore. The acceptor was not excited.

[0084] For one-photon excitation FLIM, the system was equipped with a SuperK EXTREME NKT-SC 470-2000 nm supercontinuum laser (NKT Photonics) operating at a repetition rate of 80 MHz with a pulse width of 70 ps. The desired wavelength was selected using a SuperK SELECT 29 multi-line tunable filter (NKT photonics). Images were collected through either a 60X 1.2 NA water immersion lens (Figure 11b and Figure 14b) or a 60X 1.49 NA oil immersion lens (Figure 11c). For both one-photon and two-photon excitation, emission was collected with the same objective through filters (mentioned above) and detected with an external hybrid GaAsP (HPM-100-40, Becker & Hickl, Germany) coupled to a time-correlated single-photon counting (TCSPC) module (SPC830, Becker and Hickl, Germany). At least 1000 photon counts were used for multi-exponential analysis. Raw time-correlated single photon count decay curves at each pixel of the image (256x256 or more) were analyzed using SPCImage software v.6.9 (Becker and Hickl, GmbH). An incomplete monoexponential fit model with a laser repetition time value of 12.5 ns was used to fit the decay curves. Lifetime values ​​with χ2 between 0.8 and 1.3 were considered to be a good exponential decay fit. Lifetimes were calculated from the average of a minimum of 15 fields, with at least 5 fields taken from three separately prepared slide samples.

[0085] Transmission electron microscopy (TEM) analysis of cells and isolated vesicles: 10 μl of E. coli cells expressing VNp-mNeongreen from an overnight culture were placed on a 400 mesh gold grid coated with formvar / carbon and incubated in a humidified chamber at 37 °C to allow vesicle formation. Recombinant vesicles isolated from a culture of E. coli expressing VNp-mNeongreen were placed on a 400 mesh gold grid coated with formvar / carbon and incubated in a humidified chamber at 37 °C to allow vesicle formation. Recombinant vesicles isolated from a culture of E. coli expressing VNp-mNeongreen were placed on a 600 mesh copper grid coated with formvar / carbon and left at room temperature for 5 min to allow vesicles to settle to the surface. Both samples were then fixed with 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer pH 7.2 (CAB) for 10 min. The grids were then washed with 100 mM CAB and milliQ water, then dried and negatively stained with 2% uranyl acetate in water for 5 seconds.

[0086] TEM thin section analysis of E. coli cells: E. coli expressing VNp-mNeongreen were cultured as above and harvested by centrifugation at 3,000g for 10 min. The cell pellet (approximately 100 μI) was resuspended in 2 ml of 2.5% (w / v) glutaraldehyde in CAB and fixed for 2 h at room temperature with gentle rotation (20 rpm). Cells were pelleted by centrifugation at 6,000g for 2 min and washed twice for 10 min with 100 mM CAB. Cells were post-fixed for 2 h with 1% (w / v) osmium tetroxide in 100 mM CAB and then washed twice with ddH2O. Cells were dehydrated by incubation in an ethanol gradient of 50% EtOH for 10 min, 70% EtOH overnight, and 90% EtOH for 10 min, followed by three washes of 100% dry EtOH for 10 min. The cells were then washed twice for 15 min with propylene oxide. The cell pellet was resuspended in 1 ml of a 1:1 mixture of propylene oxide and agar LV resin and incubated for 30 min with rotation. The cell pellet was infiltrated twice (2x2h) with 100% agar LV resin. The cell pellet was resuspended in fresh resin, transferred to a 1-mL BEEM embedding capsule, centrifuged at 1100 rpm for 5 min in a swing-out rotor to concentrate the cells at the tip of the capsule, and the sample was polymerized at 60°C for 20 h.

[0087] Ultrathin sections were cut using a Leica EM UC7 ultramicrotome equipped with a diamond knife (Diatome 45°). Sections (70 nm) were collected on uncoated 400 mesh copper grids. Grids were incubated for 45 min in 4.5% (w / v) uranyl acetate in 1% (v / v) acetic acid and then washed with a stream of ddH2O. Grids were stained with Reynolds lead citrate for 7 min and then washed with ddH2O. Electron microscopy was performed using a JEOL-1230 transmission electron microscope equipped with a Gatan One View digital camera at an accelerating voltage of 80 kV.

[0088] Immuno-EM of isolated vesicles: 2 μl of filtered medium containing recombinant vesicles from a culture of E. coli expressing VNp-mNeongreen was placed on a formvar / carbon-coated 600 mesh copper grid and left at room temperature for 5 min to allow vesicles to settle. The grid was transferred to two 20 μl drops of milliQ water and osmotically shocked at room temperature for 10 min to rupture the vesicles. The sample was then fixed with 2% formaldehyde and 0.5% glutaraldehyde in CAB for 15 min at room temperature. The grid was then washed with six drops of 20 μl CAB and six drops of 20 μl TBST (20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween 20, 0.1% BSA pH 7.4). The sample was blocked with a 20 μl drop of 2% BSA in TBST for 30 min at room temperature. The grid was then transferred directly to a 20 μl drop of anti-mNeongreen rabbit polyclonal primary antibody (Cell Signalling Technology) diluted 1:100 in TBST and incubated for 1 h. The grid was washed with six 20 μl drops of TBST. The grid was then transferred to a drop of goat anti-rabbit IgG 5 nm gold (British Biocell International) diluted 1:50 and then transferred to a new drop of the same antibody for 30 min. Excess antibody was washed with six 20 μl drops of TBST solution and six 20 μl drops of milliQ water and allowed to dry.

[0089] Grids were negatively stained with 2% aqueous uranyl acetate for 5 seconds. Electron microscopy was performed on a JEOL-1230 transmission electron microscope equipped with a Gatan One View digital camera at an accelerating voltage of 80 kV.

[0090] Immunoelectron microscopy (EM) of E. coli cells: E. coli expressing VNp-mNeongreen were cultured as above and harvested by centrifugation at 3,000 g for 10 min. Cell pellets (approximately 100 μl) were resuspended in 2 ml of 2% (w / v) formaldehyde and 0.5% glutaraldehyde in CAB and fixed for 2 h at room temperature. Samples were washed 2x10 min in CAB. After incubation in an ethanol gradient of 50% EtOH for 10 min, 70% EtOH overnight, and 90% EtOH for 10 min, cells were dehydrated by three 10-min washes in 100% dry EtOH. Cells were then suspended in LR White resin medium grade (London Resin Company) for 4 h and fresh LRWhite resin overnight. After two 4-h exchanges with fresh LR White resin, samples were placed in sealed gelatin capsules and spun at 1100 rpm in a swing-out rotor to concentrate the cells. Gelatin capsules containing cell pellets were polymerized upright at 60°C for 20 h. Ultrathin sections were cut using a Leica EM UC7 ultramicrotome equipped with a diamond knife (DiATOME 45°). Sections (80 nm) were collected on uncoated 400 mesh gold grids.

[0091] Samples were blocked in a 20μl drop of 2% BSA in TBST for 30 min at room temperature. The grids were then transferred directly to a 20μl drop of anti-mNeongreen rabbit polyclonal primary antibody (Cell Signalling Technology) diluted 1:10 in TBST and incubated for 1 h. The grids were washed 6x with TBST. The grids were then transferred to a drop of goat anti-rabbit IgG 5nm gold (British Biocell International) diluted 1:50 and then transferred to a new drop of the same antibody for 30 min. Excess antibody was removed by washing with 6 drops of 20μl TBST and 6 drops of 20μl milliQ water and allowed to dry.

[0092] Grids were stained with 4.5% uranyl acetate in 1% acetic acid solution for 15 min and then washed with 6x20 μl drops of milliQ water. Grids were then stained with Reynolds lead citrate for 3 min and washed with 6x20 μl drops of milliQ water. Electron microscopy was performed using a JEQL-1230 transmission electron microscope equipped with a Gatan One View digital camera and operating at an accelerating voltage of 80 kV.

[0093] Dynamic Light Scattering Testing: All tests were performed using an Anton Paar Litesizer™ 500 and processed with Kalliope™ Professional. All vials / cuvettes / microcentrifuge tubes used for sample preparation were clean and dry. All solvent systems used were filtered to remove any particulates that may interfere with the results obtained.

[0094] VNp fusion excretion by fermentation: 160 ml of a 450 ml overnight preculture of E. coli harboring pAVE011-VNpDARPinOFF7 in LB (supplemented with 1% w / v glucose and 15 mg / L tetracycline) grown overnight in a 2 L baffled flask was used to inoculate a 16 L fermenter culture of Terrific Broth basal medium (no glucose or glycerol, supplemented with tetracycline) grown at 37°C in a shaking incubator at 200 rpm (2.5 cm throw).

[0095] Culture conditions in the fermenter: pH set to 7.0 (controlled with 25% aqueous ammonia or 8.5% w / v aqueous orthophosphoric acid); pO2 set point 30% (cascaded to the agitator and supplemented with oxygen to the inlet gas); aeration rate: 1.0 v / v / m; agitation speed: 200-630 rpm; expression feed rate of glucose / yeast extract: capped at 6 g / L / h. A feed medium consisting of 400 g / L glucose and 200 g / L yeast extract was fed to the batch culture at 96 g / h. The predicted size of VNp2-DARP is 22.5 kDa.

[0096] Expression of VNp-DARP was induced by addition of IPTG (final concentration 20 μg / mL) during the batch phase, as indicated by the first spike in dissolved oxygen tension at 5.3 h (fermentation elapsed time). After 6.6 h of depletion, linear feeding was initiated.

[0097] Fermentation duration: 18.95 h from IPTG induction (17.65 h from startof linear feed rate).

[0098] Cell pellets were collected by centrifugation at 3,000 RCF for 20 min at 10° C. An aliquot of the SN collection was passed through a 0.45 μm PES filter for analysis.

[0099] result The present invention resides in the discovery that recombinant expression of part or all of the amino terminus of human synuclein results in the continuous formation and release of extracellular vesicles from E. coli cells into the medium (Figure 1a). Specifically, for α-synuclein, the first 60 residues of the sequence represent the entire α-helical membrane interaction domain, and the first 38 residues represent the first half of this domain, truncated at a predicted loop region. Both of these sequences promoted the release of mNeongreen vesicles to the same extent.

[0100] The first 38 residues of the sequence (VNp2) interacted with the cell membrane (Fig. 10 and Fig. 11), and VNp expression after 4 h (the cytoplasmic VNp2 concentration reached 18.8 ± 0.14 μM) induced localized curvature of the bacterial membrane, which extended outward until separate vesicles were formed and released into the growth medium upon membrane cleavage (Fig. 1b-d). This process occurred repeatedly without affecting cell viability, and vesicles could be mass-produced from the same cell (Fig. 1d and Fig. 12).

[0101] Fusing the sequence encoding VNp with the sequence encoding the monomeric fluorescent protein mNeongreen (Shaner, NC et al (2013) Nat Meth, 407-409) produced fluorescent vesicles containing large VNp-mNeongreen that were excreted into the culture medium (Figure 1e, 1f). Immuno-EM analysis confirmed that the mNeongreen cargo was present only in the lumen of the vesicles (Figure 1e-1f). Immuno-electron microscopy also confirmed that the mNeongreen cargo was localized only in the lumen of the vesicles (Figure 1f, Figure 13), supporting the interpretation that VNp interacts with lipids in the inner membrane and accumulates to a critical concentration that induces localized outward curvature of the membrane and subsequent vesicle formation. VNp is then released from the inwardly curved membrane into the lumen of the formed vesicle, and is released into the medium in turn as the cell membrane sever and recloses (Figure 1j).

[0102] While centrifugation separated vesicles from the majority of bacterial cells, rapid one-step filtration using sterile 0.45 μm polyethersulfone (PES) filters efficiently and effectively separated vesicles from bacteria (Figure 1g-h, Figure 11). Remarkably, these isolated vesicles were uniform in size, resulting in a stable environment for effective long-term protein storage of functionally folded recombinant proteins (Figure 11). Proteomic and densitometric analysis of the contents of the recombinant vesicles demonstrated the relative purity of the recombinant proteins, which ranged from -40 to 60% of the total from proteomic and gel analysis (see Figure 2). The purity of the fusion proteins obtained by one-step filtration is likely sufficient for a very wide range of applications (Figure 1i) and supports further purification after sonication of the vesicles if necessary.

[0103] This system provides a simple and attractive mechanism for releasing membrane-packaged recombinant proteins into the medium to facilitate recombinant protein production and subsequent processing. mNeongreen provides rapid quantification of soluble target proteins exported into the medium with different physical properties and expression challenges (e.g., membrane-bound, disulfide bond-containing, or insoluble or toxic proteins), and we tested the applicability of this technology for the expression of a variety of molecules demanded by the life science community. Expression of each protein was tested as VNp, or as a VNp-mNeongreen amino-terminal fusion, and compared with expression of the equivalent non-VNp fusion protein (Figure 1k-m, Table 2).

[0104] Experiments have also shown that this technology can be applied to a wide range of E. coli strains (e.g., BL21, JM109, K12) using expression from a variety of inducible and constitutive promoters. The VNp system functions in W3110 cells, allowing for the production of recombinant protein-loaded vesicles with reduced immunogenic responses. VNp fusions can be expressed from a variety of plasmids (including pUC19 and pBR322-based derivatives), and expression of VNp fusions can be regulated from a variety of promoters (e.g., T7, rhamnose) and induction levels (see Figure 3 and Table 1). Table 1: W3110λDE3 E. coli cell excreted protein yield: [Table 1]

[0105] We also demonstrated that once the protein of interest was released from the vesicles, it remained soluble even when the VNp tag was removed using a protease digest (i.e., VNp was not required to maintain protein solubility). As shown in Figure 7, purified VNpmNG-TEV-DARP and VNp-mNG-TEV-Uricase were digested with TEV protease. As a result, the cleaved protein was not detected in the pellet fraction (P) but remained in the supernatant (S / N) fraction after centrifugation at 13,200 RCF.

[0106] Although fluorescent proteins allow rapid quantification of soluble target proteins excreted into the medium, a broader range of target proteins with different physical properties and expression challenges were selected to validate the technology. Tested proteins include designed ankyrin repeat protein off7 (DARP), stefin A, uricase (Figure 1k), erythropoietin (EPO), etanercept, fibroblast growth factor 21 (FGF21) and human growth hormone (hGH). As such, these proteins represent different physical properties and expression challenges (e.g. membrane-bound, dimeric, disulfide bond-containing or insoluble proteins) that are not typically expressed to detectable levels in E. coli, thus demonstrating the broad applicability of this technology. Expression of each protein was tested as a VNp, or VNpmNeongreen amino-terminal fusion protein and compared to the expression of the equivalent non-VNp fusion protein (Table 2).

[0107] Table 2: Summary of soluble protein yields. [Table 2-1] [Table 2-2]

[0108] Mean yields ± sd were calculated from >3 independent biological replicates. Yields were measured as mg soluble target protein / L. Unless otherwise stated, the T7 promoter was induced with 20 μg / ml IPTG and bacterial shake flask cultures were grown at 37°C. nd: undetectable; LZ = leucine zipper sequence; mNG = mNeonGreen

[0109] The relative excretion yields of mNeongreen fusions with a range of vesicle nucleation peptide sequences were measured. As can be seen in Table 3, the excretion yield decreased with decreasing VNp length. Although some excretion was observed with a peptide length of 9 amino acids, this shorter length significantly reduced vesicle formation activity. Table 3: [Table 3-1] [Table 3-2]

[0110] Fusion with VNp very effectively enhanced protein expression and secretion of each target protein. Thus, VNp protein expression not only supports the expression of individual proteins ranging in size from less than 1 kDa (VNp-His6) to 85 kDa (VNp-mNeongreen-etanercept), but also the expression of protein complexes, as indicated by fluorescence from pairs of bimolecular fluorescently complemented VNp fusions in excreted vesicles (Figure 11) (Kodama, Y. & Hu, C.-D. (2010) BioTechniques 49, 793-805). Importantly, VNp fusions improved the overall yield of soluble forms of each target protein examined, with a yield of nearly 1 g of soluble protein per liter of shake flask culture in the case of DARP (Table 2). The versatility of this system was demonstrated by the production of a membrane-bound protein (FGF21) and VNp-uricase with full enzymatic activity. Furthermore, VNp-etanercept exhibits appropriate ligand-binding properties (Figure 13), and mNeongreen fluorescence demonstrates how functional protein folding associated with VNp-induced vesicle packaging is maintained within isolated vesicles.

[0111] VNp fusions allow the production of soluble folded proteins (e.g., DNase, etanercept, EPO, hGH) that are otherwise insoluble or reduce bacterial cell viability. In the case of the disulfide bond-containing proteins etanercept and hGH (Goffe, B. & Gather, JC (2003) J Am Acad Dermatol 49, S105-S111; Ultsch, MH & Somers, WJ (1994) Mol. Biol. 236, 286-299), the majority of the soluble recombinant protein remained intracellular. EM data show that VNp-mNG-etanercept affects the remodeling of the VNp inner membrane to induce VNp fusions containing internalized cytoplasmic membrane structures (Figure 6a-b). Similarly, amino-terminal acetylation of VNp by recombinant NatB complex (Eastwood, TA et al (2017) FEBS Letters 106, 8157-9) led to the formation of membrane internal structures in E. coli (Figure 6c-d) and increased the proportion of mNeongreen in the cells (Figure 6e, Table 2), similar to VNp-etanercept.

[0112] The VNp2 tag also allows the expression and isolation of heterodimeric soluble functional antibodies from E. coli. Fab (fragment antigen binding; folded hen egg lysozyme) and mAB (monoclonal antibody; Muc1) antibodies consisting of heavy and light chain heterodimers (linked by disulfide bonds) were found to be soluble and capable of binding to protein G / A, demonstrating correct structure, complex formation, and functionality (see Figure 4).

[0113] Introducing a leucine zipper sequence between VNp and cargo created stable α-helical VNp dimers (Figure 12) and induced the formation of mNeongreen containing vesicular structures associated with the inner membrane in the bacterial cytoplasm (Figure 6f-h), consistent with previous dimeric VNp fusions. Notably, the addition of a leucine zipper sequence to the carboxyl terminus of VNp led to the formation of VNp dimers and predominantly generated cytoplasmic vesicles. As seen in Figure 9, the elution profile of VNp-mNG (black) was consistent with a monomeric protein, whereas VNp-LZ-mNG (grey) eluted from the column in earlier fractions and existed predominantly as a dimer. Thus, the present invention provides an attractive and simple system for expressing toxic proteins and / or proteins containing disulfide bonds from E. coli (Figure 6i).

[0114] VNp was also generated to allow intravesicular targeted proteolytic cleavage of recombinant proteins. VNp-mNeongreen-DARPin off7 E. coli cells containing constructs expressing the fusion alone or in combination with the VNp-maltose binding protein (MBP)-TEV protease fusion were cultured and expression was induced in overnight flask cultures. off7 The fusion contained a TEV cleavage site between the mNeongreen and DARPin sequences. As can be seen in Figure 5, expression of VNp-mNG-DARP alone resulted in the expression and vesicle excretion of the full-length protein (50.8 kDa size). On the other hand, when the same protein was coexpressed with a TEV protease fusion, VNp-mNG-DARP was cleaved at the TEV site, resulting in the expression of VNp-mNG (32 kDa) and DARPin. off7 (18.8 kDa) fragment was excreted into the culture medium. Thus, the present invention provides an attractive method to generate recombinant proteins containing internal membrane-bound structures for the expression and compartmentalization of proteins that contain disulfide bonds, are insoluble (e.g., etanercept, hGH and antibody conjugates), or are toxic (e.g., DNase). However, when VNp-LZ dimer cargo fusions were co-expressed with VNp-LZ peptides (to increase the production of extracellular vesicles), proteins bound to the interior of the compartment were redirected to the excretion route as soluble cargo-packaged vesicles separated from the medium (Table 2), facilitating certain downstream processes of these proteins. Thus, the VNp system not only supports the immediate isolation of fusion proteins from the medium, but also provides an alternative internal expression system that may be advantageous in certain fields, such as the generation of enzyme cascades for complex synthesis and other aspects of synthetic biology.

[0115] We next investigated whether simple modifications to the VNp protein to modulate lipid interactions could improve the yield of excreted protein. We tested a series of VNp mutants and found that altering the charge and side chain length of targeted residues along the helical surface not only promoted vesicle excretion over a wide range of incubation temperatures (e.g., VNp6), but also reduced the size of VNp down to a length of 9 residues (e.g., VNp26) (Table 2).

[0116] Consistent with VNps influencing membrane dynamics, optimal vesicle formation was observed for VNp2 when cells were cultured at elevated temperatures (see Tables 2 and 4). However, mutants of VNp6 induced vesicle formation with high efficiency over a wide range of culture temperatures (25°C to 37°C). Table 4: Yields of VNp-mNeonGreen fusions at 25°C, 30°C and 37°C. [Table 4]

[0117] The effect of oxygen concentration was also examined. Cells were cultured in the same volume of medium with increasing flask size (hence increasing surface area), ± Parafilm (± anaerobic), ± baffles (increasing agitation of the culture). As shown in Figure 16 and presented in Table 5, increasing the surface area:volume ratio was found to directly affect the shedding of vesicle-packaged proteins. The addition of Parafilm® (i.e. anaerobic conditions) had a dramatic negative effect on vesicle formation, and agitation had a small but significant negative effect.

[0118] Table 5: Extracellular vesicle formation requires cell culture oxygen supply. [Table 5-1] [Table 5-2]

[0119] The inventors have also established that the VNp technology described herein can be applied to 1 L shake flask culture volumes when cultured in 5 L flasks to maintain surface area:volume ratio (see FIG. 8 and Table 6).

[0120] Table 6: Yield of excreted VNp-mNeongreen in various culture volumes and flask sizes. [Table 6]

[0121] It is understood that to identify optimal conditions for vesicle formation, expression levels will need to be adjusted depending on the cargo expressed. Consistent with VNp affecting membrane dynamics, optimal vesicle generation was observed when cells were cultured at elevated temperatures where membranes are more dynamic. However, expression can be supported from a variety of promoters (e.g., T7, rhamnose, arabinose, Tac) in a variety of plasmids, including pUC19 or pBR322-based derivatives, so specific expression levels can be easily tested in a variety of expression systems.

[0122] Figure 15 shows that the VNp2-DARP construct successfully excreted protein in a 20 L fermentor / bioreactor, with protein found in both the cell pellet and the supernatant, with the highest concentration of protein in the supernatant.

[0123] Experiments confirmed that the recombinant vesicles and the fusion protein therein are stable when stored at 4 °C (see Figure 17). Dynamic light scattering (DLS) confirmed the presence of two species with different intensity distribution maxima in the solution. The first one showed a hydrodynamic diameter of 166 nm, was attributed to isolated vesicles and correlates with the species present in the EM images. The second one showed a hydrodynamic diameter of ~10 nm, was attributed to vesicle fragments. The average polydispersity index of each sample was greater than or equal to 1, indicating vesicles with a broad size distribution in the medium. The average hydrodynamic diameter of the vesicles did not change (hydrodynamic diameter = 169 nm) after storage at 4 °C for 6 months in sterile phosphate-buffered saline (PBS). Furthermore, there was no significant difference in the zeta potential (calculated from the peak maximum) of day-old vesicles (-10.5 mV) compared to 4-month-old vesicles (-11.1 mV), and there was no observable significant loss of VNp-mNeongreen contained in the vesicles over the 3 months. Thus, isolated vesicles provide a stable environment for effective long-term protein storage of soluble recombinant proteins. Not only did VNp-uricase isolated from vesicles exhibit enzymatic activity to the same extent as uricase purified from cell pellets, but this activity was maintained to a greater extent for VNp-uricase stored for 2 months at 4°C in isolated vesicles compared to purified protein stored at 4°C in buffer for the same period (Figure 17), highlighting the stable environment in which vesicles provide their protein cargo.

[0124] VNp sequences can be used to induce the formation of extracellular VNp fusion-containing vesicles from mammalian cell expression systems in culture (see Figure 18). HEK293T cells were transiently transformed (Lipofectamine2000-mediated transfection) with VNp2-mNeongreen and VNp6 expression constructs under the control of either the CMV or SV40 promoter. Expression and relative induction levels were confirmed by Western blot (Figure 18A). HEK cells transfected with VNp-mNeongreen were grown on coverslips in DMEM medium containing 10% fetal bovine serum. Cells were imaged with a wide-field microscope to visualize mNeongreen expression. Arrows in Figure 18 highlight recombinant vesicles containing VNp-mNeongreen. Similar results were observed in CHO-S cells when expressing VNp6-mNeongreen under the control of the SV40 promoter.

[0125] We also demonstrated that the VNp tag technology dramatically enhanced recombinant protein expression in E. coli cultured under anaerobic conditions. off7 Conditions were identified that promote cytoplasmic compartmentalization of VNp fusions between mNeongreen and StefinA. Figure 19 shows gels of expression profiles from cells containing expression constructs of mNeongreen and StefinA alone or fused to VNp, demonstrating that in both cases the addition of VNp promotes expression of these recombinant proteins in cells cultured under anaerobic growth conditions. Consideration

[0126] As demonstrated, the simple peptide fusions described herein increase the yield of a wide range of recombinant proteins from E. coli and simplify downstream processing. Importantly, the ease of isolating insoluble or toxic proteins in milligram or gram quantities makes this method an attractive starting point for expressing recombinant proteins of interest. A highly attractive aspect of this production system is the maintenance of protein stability and enzymatic activity when the vesicles are maintained at 4 °C. As a result, this versatile system is suitable for a wide range of downstream processes and applications, including high-throughput expression screening, protein storage, recombinant bioreactor creation, environmental dispersion of biomolecules, microvesicle therapeutics, nanobody production alongside vaccines, and viral therapeutic delivery.

[0127] In the first study, the effect of amino-terminal acetylation of α-synuclein (αSyn), an essential protein in human neurons, was observed in Escherichia coli (Maltsev, ASet al (2012) Biochemistry 51, 5004-5013). As a result, it was discovered that differences in oligomerization state could be tracked within bacterial cells (Eastwood, TA et al (2017) FEBS Letters 106, 8157-9), in addition to leading to the formation of a large number of extracellular vesicles formed from the E. coli membrane (unpublished). Fluorescence imaging revealed that when membrane-bound αSyn reached a critical level, it induced outward curvature of the bacterial membrane and led to the formation of extracellular vesicles (considerably larger than natural outer membrane vesicles) containing αSyn-fluorescent protein fusions.

[0128] VNp-induced vesicle formation requires a critical concentration of VNp fusion protein that promotes vesicle formation. The vesicle nucleation peptide interacts with the cell membrane, and from 4 h onwards, expression of VNp (cytoplasmic VNp concentration was typically 18.8 ± 0.14 μM) induced a localized curvature of the bacterial membrane, which spread outward until distinct vesicles were formed and released into the growth medium upon membrane cleavage. When expressed, proteins of interest may be toxic to cells, interact with the cell membrane, or not fold correctly even at moderate cytoplasmic concentrations. Therefore, a dimerizing leucine zipper (LZ) peptide was introduced between VNp and the protein of interest sequence. The results showed that expression of this construct alone or together with additional VNp-LZ peptides promoted the packaging and export of proteins that are normally difficult to express (e.g., etanercept and hGH).

[0129] Furthermore, in vivo amino-terminal acetylation of VNp in bacterial cells (see Eastwood, TA et al (2017) FEBS Letters 106, 8157-9; Johnson, M. et al (2010) PLoS ONE 5, e15801) promoted the formation of internalized VNp-fusion vesicle structures in the bacterial cytoplasm and improved expression of the protein of interest.

[0130] From these initial observations, we established that expression of a fusion of αSyn and human growth hormone (hGH) in E. coli cells results in the release of vesicles containing αSyn-hGH into the culture medium in both shake flasks and commercial bioreactor cell culture systems, demonstrating that vesicles containing the recombinant protein of interest can be released into the growth medium, facilitating downstream purification and processing of the protein of interest.

[0131] An investigation was carried out to ascertain whether a specific region of αSyn, rather than the entire 15 kDa (140 amino acids) αSyn protein, leads to vesicle formation. The results showed that a short amphipathic α-helical polypeptide, 38 amino acids from the amino terminus of αSyn, was sufficient to interact with the cell membrane and promote the formation of vesicle structures. It was then found that this 38 amino acid vesicle nucleation polypeptide (VNp) interacts with the inner side of the E. coli cell membrane and affects the shape of the membrane. Data from spectroscopy, EM, DLS, mass spectrometry, and live-cell imaging confirmed that expression of a VNp-fluorescent protein (FP) fusion induced the excretion of vesicles containing VNp-FP into the medium. These phenomena were not observed when FP lacking the VNp amino-terminal fusion was expressed alone.

[0132] We subsequently found that equivalent polypeptides from other synuclein proteins could be used to generate comparable levels of fluorescent VNp-FP-containing vesicles. In an ongoing systematic in vitro molecular evolution approach (using the first 38 amino acids of γ-synuclein as a starting point), a number of modified VNp sequences were identified that significantly promoted (and decreased) the export of vesicle-packaged VNp fusions from E. coli cells compared to the native peptide sequence. Table 7 summarizes the effect of the mutants and each sequence on the export of mNeongreen (mNG). Table 7: [Table 7-1] [Table 7-2]

[0133] A shorter version of VNp6 works particularly well, with 95% of the recombinant protein being exported into the medium. The α-helical structure of VNp is reinforced by interactions with specific lipids, allowing VNp to interact with the bacterial inner membrane and enable expression and export of the protein of interest from the host cell into the medium.

[0134] In addition to optimizing the VNp sequence itself, various E. coli strains and culture conditions were tested and compared to identify the best conditions for production of membrane-packaged target protein. Standard E. coli culture media such as Luria Broth (LB) and especially rich media such as Terrific Broth (TB) all seemed to work well, with approximately four times more VNp fusion protein released when cultured in TB compared to LB. Not only was there more total recombinant protein produced in the medium, but a higher percentage of it was released into the medium in TB. In contrast, when cells were cultured in minimal / complex media, VNp-induced vesicle packaging was obtained, but at significantly lower levels. Some specific media components, such as citric acid, have been found to affect, reduce or inhibit release, while others, such as phosphate ion concentration, have no effect on vesicle release. In some embodiments, one or more, and preferably all, of the following have been found to be advantageous: Final K2SO4 concentration less than 4 g / l; Final MgSO4 concentration less than 10 mM; Final CaCl2 concentration less than 0.03 g / l; · NaCl concentration up to 5 g / l; a glycerol concentration of less than 0.04% (v / v); and

[0135] Final sorbitol concentration of max 1mM (preferably max 0.1mM).

[0136] The cell culture temperature may also affect the efficiency of extrusion depending on the particular VNp variant. For example, SEQ ID NO:2 is temperature dependent and is better released at 37° C., whereas in some embodiments, for VNp6 and VNp15 variants, culturing cells at temperatures ranging from 25° C. to 37° C. improves the extrusion of vesicle-packaged proteins.

[0137] We also found that increasing oxygen concentrations to maximize respiration / metabolism had a significant positive effect on the release of vesicle-packaged proteins into the medium.

[0138] This experiment demonstrated that the expression level of the VNp fusion protein is important to ensure optimal vesicle release. The core VNp constructs were examined and compared for cell viability, target protein expression and shedding at various induction levels. The best yields were obtained by inducing expression at a level that only detectably affected cell growth, balancing vesicle production against the overall stress on the bacterial cell membrane by VNp and viability. Not only did the total yield of VNp fusions decrease significantly with higher induction rates, but so did the percentage of target protein produced that was shedding into the medium. For VNp SEQ ID NO:2 fusions, optimal induction for most proteins was identified at 20 μg / ml IPTG. However, optimal yields for some toxic target proteins were achieved at 10-15 μg / ml IPTG (e.g., etanercept, hGH).

[0139] Expression of VNp fusions was controlled by modulating the promoter and / or inducer (e.g., T7 promoter expression was controlled using 10–100 μg / ml IPTG; rhamnose promoter expression was controlled by adding 0.001%–0.2% (final W / V) rhamnose to the medium). Naturally, too high an expression level would result in excessive membrane disruption (as judged by growth curves, microscopic identification of dead cells, and / or the appearance of additional proteins released into the medium from lysed dead cells observed by SDS-PAGE analysis) leading to transient high levels of vesicle production, but the effect this has on the cell membrane would cause cells to burst and the decline phase of the culture to begin. In contrast, too low an expression level of VNp fusions (i.e., ≤2 μg / ml IPTG) would result in VNp not being able to sufficiently interact with the cell membrane and vesicles not being formed. As a starting point for new VNp fusions, we used expression levels approximately 5–10-fold lower than the maximum expression level allowed by IPTG induction of the T7 promoter.

[0140] Various strategies and reagents for targeting proteins to vesicles were evaluated, including a comparison of methods for delivering target proteins to VNp-derived vesicles, including direct fusion with VNp (as described for VNp-FP fusions above), co-expression of VNp with a cytoplasmic recombinant protein of interest, co-expression of VNp with a membrane-targeted recombinant protein, and even an optogenetic approach (inducing interaction of VNp with the target protein in response to specific wavelengths of light): - Fusions of target proteins with bacterial membrane proteins (OmpA, CydAB, MinD) have been made, but this method is not as efficient as direct fusion, membrane proteins are large, and overexpression of membrane protein-target protein fusions can destroy cell viability. Overexpression (i.e., cytoplasmic localization) of the target protein increased its uptake into vesicles, but this was not nearly as efficient as direct fusion, and VNp did not promote proliferation with target proteins that were difficult to express or toxic. - Optogenetics worked more efficiently than either of the above, but the optogenetics tags were large and the relative yield of the target protein was greatly reduced.

[0141] None of the above was as efficient as direct fusion of VNp with a protein of interest, which not only targeted the protein to vesicles but also improved folding and protein yield, allowing the expression of toxic proteins as they were cleared from the bacterial cytoplasm.

[0142] Introducing a proteolytic cleavage (TEV) site between the VNp and the cargo molecule allowed for the purification of untagged, untagged proteins of interest from the vesicles. Experiments showed that etanercept remained soluble upon removal of the VNp tag.

[0143] Using short VNp sequences and under optimized conditions, not only was a significant proportion of the fluorescent protein cargo excreted into the medium, but the overall yield of the target protein in culture (i.e., both in the cells and released into the medium) was also significantly improved compared to the equivalent protein lacking the amino-terminal VNp fusion (i.e., only in the cells) (Table 2). All VNp sequences tested promoted excretion, with SEQ ID NO:10 representing a greater proportion of the protein excreted.

[0144] Under optimal growth conditions (TB medium, 37 °C, good oxygen supply, 10–20 (usually 20) μg / ml IPTG (depending on the cargo)), cargo-containing vesicles were released from bacterial cells within 10 min of the onset of their formation. Thus, it is possible to release large amounts of cargo-containing vesicles in a relatively short time from a continuous culture. Purification strategies by centrifugation require harvesting a significant number of cells as well as the medium from the culture, which is time-consuming and labor-intensive. A simple cell filtration protocol has been developed, which allows the separation of cells from the vesicle-containing medium by removing a portion of the cell culture (and replacing it with fresh medium) and passing it through a 0.45 μm filter (see Methods above). From all experiments tested, no colony-forming units were detected in 1 ml of vesicle-containing medium filtrate. Thus, this method provides a simple, efficient, automatable, high-throughput purification method.

[0145] VNp-derived vesicles and packaged proteins isolated in culture medium in this manner were stable for several months at 4°C. Not only was VNp-uricase isolated from vesicles as enzymatically active as uricase purified from cell pellets, but this activity was maintained to a greater extent by VNp-uricase in isolated vesicles stored at 4°C for 2 months compared to purified protein stored under the same conditions for the same period of time (see Figure 12d). This result again indicates that vesicles provide a stable environment for their protein cargo.

[0146] The VNp-dependent targeted excretion technique of the present invention was tested with a variety of proteins of bioindustry relevance, with different biophysical properties and functions. These proteins range in size from less than 10 amino acids to almost 100 kDa. ) VNp was directly fused to the protein of interest, and cells were cultured under the conditions described above. For each protein examined, VNp fusion not only resulted in a significant proportion of the cargo being excreted into the culture medium, but also significantly improved the overall yield of the soluble form of each target protein (Table 2). The functionality of several proteins was tested (e.g., fluorescence for FP, enzymatic activity for uricase, and protein A binding ability for etanercept), and measurable activity was expected for each.

[0147] The VNp technology was confirmed to function with different types of promoters (e.g., CMV, rhamnose, arabinose, Tac, and T7 promoters of different strengths) and backbone plasmids (e.g., pcDNA3.1, pUC19, and pBR322-based), and was also validated using various E. coli strains (e.g., BL21DE3, W3110+-DE3, K12, DH10b, and JM109).

[0148] Finally, the scale-up of the vesicle excretion system was also successfully demonstrated, suggesting that this technology is suitable for industrial-scale protein production.

[0149] In conclusion, the present invention provides a technology to increase the yield and simplify downstream processing of a wide range of recombinant proteins from E. coli using simple peptide fusions. It allows for continuous protein purification during active cultivation and also allows for simplified protein isolation and downstream processing (i.e., without the need to disrupt cells) of a wide range of recombinant proteins from E. coli. As a result, this system provides a simple and attractive mechanism for releasing membrane-packaged recombinant proteins into the medium to enhance recombinant protein production and subsequent processing.

[0150] Importantly, the method is an attractive starting point for expressing any recombinant protein of interest, as insoluble or toxic proteins can be easily isolated in milligram or gram quantities. A highly attractive feature of this production system is the retention of protein stability and enzymatic activity when the vesicles are maintained at 4°C. The technology is also suitable for a wide range of downstream processes and applications, including high-throughput expression screening, protein storage, recombinant bioreactor creation, environmental dispersion of biomolecules, microvesicle therapeutics, nanobody production alongside vaccines, and viral therapy delivery.

Claims

1. An isolated vesicle nucleating polypeptide (VNp) comprising an amphipathic alpha-helical polypeptide derived from the amino terminus of synuclein isoforms and variants thereof.

2. 2. The isolated vesicle nucleating polypeptide of claim 1 having between 9 and 140 amino acids, preferably between 16 and 40 amino acids, more preferably about 20 amino acids.

3. 3. The isolated vesicle nucleation polypeptide according to claim 1 or 2, wherein said sequence is any one of SEQ ID NOs: 2 to 28.

4. 4. The isolated vesicle nucleating polypeptide of any one of claims 1 to 3 which is acetylated at the amino terminus.

5. 5. The isolated vesicle nucleating polypeptide of any one of claims 1 to 4, further comprising a leucine zipper sequence.

6. 6. The isolated vesicle nucleating polypeptide of claim 5, wherein the leucine zipper sequence has the sequence of SEQ ID NO:

29.

7. 7. The isolated vesicle nucleation polypeptide of claim 5 or 6, wherein the leucine zipper sequence is at the carboxyl terminus of the isolated vesicle nucleation polypeptide.

8. 8. The isolated vesicle nucleating polypeptide of any one of claims 1 to 7, further comprising a protease cleavage site.

9. A fusion protein comprising a vesicle nucleating polypeptide according to any one of claims 1 to 8 fused to the amino terminus of a biomolecule.

10. The fusion protein of claim 9, wherein the vesicle nucleation polypeptide is fused to the amino terminus of the biomolecule via a protease cleavage site.

11. The fusion protein of claim 10, wherein the protease cleavage site is for a viral protease or a viral 3C-like protease.

12. A fusion protein according to claim 10 or claim 11, wherein the protease cleavage site is intact.

13. The fusion protein according to any one of claims 10 to 12, wherein the protease cleavage sites are specific for one or more membrane-bound or cytoplasmic proteases.

14. The fusion protein according to any one of claims 9 to 13, wherein the biological molecule is not constitutively expressed at a detectable level in an expression system that expresses the fusion protein.

15. The fusion protein of claim 14, wherein the biological molecule is a membrane-bound protein, an insoluble protein, or a protein that is normally toxic to a host cell expressing the fusion protein.

16. The fusion protein according to any one of claims 9 to 15, wherein the size of the biomolecule is within the range of less than 1 kDa to 100 kDa.

17. A nucleotide sequence or vector expressing a fusion protein according to any one of claims 9 to 16.

18. A host cell expressing a fusion protein according to any one of claims 9 to 16 or comprising a nucleotide sequence or a vector according to claim 17.

19. 19. The host cell of claim 18 which is a prokaryotic cell, a unicellular eukaryote or a cultured eukaryotic cell line.

20. The host cell of claim 19, which is Escherichia coli.

21. The host cell of any one of claims 18 to 20, further expressing a VNp-leucine zipper peptide.

22. 22. The host cell of any one of claims 15 to 21, further expressing one or more VNp tagged proteases.

23. 18. A method for promoting, enhancing or increasing vesicle formation in a recombinant expression system, comprising expressing in a host cell a polypeptide comprising an isolated vesicle nucleation polypeptide (VNp) according to any one of claims 1 to 8, a fusion protein according to any one of claims 9 to 16, or a nucleotide sequence or vector according to claim 17.

24. 20. A method for producing at least one soluble and / or functional form of a biomolecule from a cellular expression system, wherein said biomolecule is not constitutively expressed in a host cell of said cellular expression system, comprising expressing in the host cell a vesicle nucleation polypeptide (VNp), fusion protein, or nucleotide sequence or vector according to any one of claims 1 to 17.

25. The method according to claim 23 or 24, wherein the host cell is the host cell according to any one of claims 18 to 22.

26. 26. The method of any one of claims 23 to 25, wherein said vesicle nucleation polypeptide, said fusion protein, or said nucleotide sequence or said vector promotes, enhances or increases the release of vesicles from said host cell into the medium of said cell expression system.

27. The method of any one of claims 23 to 26, comprising culturing the host cells at between 20°C and 40°C.

28. The method of any one of claims 23 to 27, comprising culturing the host cell under aerobic or anaerobic conditions.

29. 29. The method of any one of claims 23 to 28, further comprising storing the vesicles in a host cell of a recombinant expression system.

30. 29. The method of any one of claims 23 to 28, further comprising isolating or purifying the vesicles from the cell culture prior to storage.

31. 31. The method of claim 30, wherein the vesicles are stored at about 4°C.

32. The method of any one of claims 29 to 31, wherein the vesicles are stored for up to at least 4 months.

33. The method of any one of claims 23 to 32, further comprising releasing the fusion protein and, optionally, further purifying the fusion protein.

34. 33. The method of any one of claims 23 to 32, further comprising releasing the biomolecule from the vesicle, and optionally further purifying the biomolecule.

35. A method for co-expressing and exporting a biomolecule as cargo in membrane-bound vesicles in combination with an isolated vesicle nucleating polypeptide (VNp) according to any one of claims 1 to 8.

36. 36. The method of claim 35, wherein the biological molecule is a recombinant protein.

37. 23. A method for producing one or more biomolecules which are expressed in a host cell as part of a recombinant expression system, and optionally released from the host cell, said recombinant expression system comprising an isolated vesicle nucleation polypeptide, fusion protein, nucleotide sequence or host cell according to any one of claims 1 to 22.

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