Method of purification

Abstract

The invention provides a chromatographic method for separating pre-contraction state PVC Nanosyringes from post-contraction state PVC Nanosyringes, the method comprising: (a) providing a sample containing both pre- and post-contraction state PVC Nanosyringes; (b) loading the sample on a monolith anion exchange column having (preferably quaternary amine) binding moieties, under conditions (e.g. pH 7-8, temperature 19-25°C) wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities; (c) applying a gradient of ionic strength to the column that provides for differential elution of the pre- and post-contraction state PVC Nanosyringes, wherein the gradient is generated by incrementally (e.g. linearly) increasing the salt concentration of a mobile phase elution buffer that is applied to the column; and (d) eluting the pre- and post-contraction state PVC Nanosyringes into separate elution fractions under the gradient based on their differing affinities to the anion exchange column, and collecting fractions having the pre-contraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes.

Description

[0001] METHOD OF PURIFICATION

[0002] Field of the invention

[0003] The present invention relates to methods of purifying an active form of an extracellular contractile injection system, more particularly the Photorhabdus Virulence Cassette (PVC) Needle Complex (aka Nanosyringe).

[0004] Background

[0005] Bacterial secretion systems have been explored as potential delivery systems, given their natural ability to secrete (or more particularly ‘inject’) molecules into target cells. The most studied of such secretion systems is the Type III secretion system (T3SS), a “protein appendage” found in several Gram-negative bacteria. However, a significant drawback of these systems is that they remain associated with the bacterial membrane at all times, requiring use of actual bacterial cells (comprising the secretion system) as the delivery system. As such, it is difficult to fully control what molecules are transferred from the bacteria to the target cell (even when the biologic of interest is overexpressed), as these secretion systems function by providing a connection (e.g. channel) between the bacteria’s cytosol and the target cell’s cytosol, through which other components (potentially harmful to the host) may flow.

[0006] Alternative systems that have gained prominence in recent years are the extracellular contractile injection systems (eClSs), a class of bacteria-derived (secreted) protein delivery systems that present as syringe-like macromolecular complexes that bind to target cells and inject protein payloads into the cells by driving a spike through the cellular membrane.

[0007] A notable example of an eCIS is the Photorhabdus Virulence Cassettes (PVC) Needle Complex (also known as a “Nanosyringe”), a system which becomes loaded with so-called effector proteins of Photorhabdus bacteria before being secreted from the bacterial cells. The secreted Needle Complex / Nanosyringe subsequently delivers the PVC effector to a target cell where it exerts its effect(s), the PVC effectors representing a payload of such Nanosyringes.

[0008] The present inventors previously made significant progress toward adapting this natural injection system toward / for human clinical use, by developing technology allowing for packaging the Needle Complex / Nanosyringe with bespoke payloads, typically in place of the natural effector. This involved identifying that effector proteins comprise a “leader sequence” (or “leader peptide”), which functions to package (or “load”) PVC effectors into the PVC Needle Complex / Nanosyringe. The inventors found that such leader sequences could be practically utilized to direct a payload linked thereto to be packaged into a PVC Needle Complex (and related / homologous complexes). Thus, the leader sequence functions to load the PVC Nanosyringe with a payload, and further details are outlined in WO2020 / 245611A1 (incorporated herein by reference).

[0009] However, to-date there exist limited purification methodologies for preparing purified samples of such complexes, let alone that are amenable to scalable GMP manufacture, a key step in developing any protein molecule for therapeutic use.

[0010] With particular reference to the PVC Needle Complex / Nanosyringe, the ‘gold standard’ method for purifying such complexes relies on the use of ‘ultracentrifugation’ for protein extraction from cell lysate. That is, following conventional expression, host cell lysis and debris removal steps, lysate is subjected to exceptionally high centrifugation speeds (e.g. 150,000xg or more) to separate the PVC Needle Complex / Nanosyringe macromolecules, based on size difference from host cell proteins, into a pellet. A recent academic publication put forth such protocol (based on ultracentrifugation) as a recommended purification methodology for eCIS complexes - see the abstract of Wang et al (Bio Protoc. 2024 Apr 5;14(7):e4966). The paper just cited here shares authors with Jiang et al (Cell. 2019; 177: 370-383) that will be cited below.

[0011] However, the present inventors, have identified a problem associated with this prior art methodology, in that they have found that Nanosyringe complex preparations derived from such prior art methods include a mixed population of PVC Nanosyringes, containing not only the (fully assembled and functional) target “pre-contracted” state complex structure, but nonfunctional (in terms of payload delivery) “post-contracted state” complexes- see Figure 6. Such preparations are adequate for use in research and general development. However, in terms of progressing to broad clinical use and compliance with GMP manufacturing, the ability to select for the target “pre-contraction” state complex structure (e.g. in preference to the “post-contraction” state) now achieved by the present invention represents a significant contribution.

[0012] Thus, the inventors have found that their exists deficiencies in the prior art in terms of methods that would allow for purification of a preparation that is concentrated for fully assembled and functional (pre-contraction) PVC Needle Complexes, aka PVC Nanosyringes as they will be more regularly referred to in this disclosure.

[0013] The present invention solves one or more of the above-mentioned problems.

[0014] Detailed Description

[0015] The present invention is predicated on the use of a particular anion exchange chromatography (AEX) step, in conjunction with an advantageous elution strategy, in the purification of target PVC Nanosyringes that represent a correctly formed and functional complex of PVC proteins.

[0016] As outlined above, the inventors have found that preparations of recombinant PVC Nanosyringes are heterogenous, such that preparations (e.g. resulting from recombinant expression host cells) will comprise not only the active, appropriately formed “pre-contraction state” Nanosyringes, but also non-functional (in terms of the ability to deliver payload) “postcontraction” stated forms alluded to above. Having identified the problem, the inventors sought a solution.

[0017] A first attempt involved use of anion exchange chromatography as a fractionation / separations means, the most typical anion exchange chromatography approach being to load protein onto resin beads, before subsequent elution therefrom. However, previously, when passing purified Nanosyringes through columns containing beads (e.g. in one case Sepharose, Superdex, HisTrap), it was found that up to 50% of recombinant protein was lost (see Example 2) - without wishing to be bound by theory, it is thought that this is due to the Nanosyringes being physically stuck inside the pores of the resin due their large size and unique aspect ratio (large / long, rod-shaped Nanosyringe particles).

[0018] The inventors therefore attempted to resolve the issue of poor yield following anion exchange chromatography, which led to identifying an advantageous use of “monolith” columns here, finding that monolith columns having in particular quaternary amine moieties (as a protein binding moiety) could be used to good effect. Thus, a key part of the solution was to switch to “monolith” columns which utilise a “single block of homogenous stationary phase with many interconnected channels” (e.g. rather than beads or porous particles). These columns are commercially available for use in GMP manufacturing. For example, the column may be a CIM Monolith QA column (Sartorius BIA Separations). Further examples of columns include Toyopearl GigaCap Q-650M (Tosho Bioscience) and Fractogel TMAE (Merck Millipore).

[0019] Having identified an appropriate basis for conducting chromatography, the inventors went on to investigate how this could be further improved upon to yield separation of the “target” (precontraction) Nanosyringes away from the alternative forms (e.g. post-contraction).

[0020] As can be seen in the figures, by employing an elution approach based on applying a gradient of increasing ionic strength to the loaded column, resulting chromatograms (from plotting A280) show alternative peaks. This demonstrates that different forms (pre- vs post contracted) of Nanosyringe can be differentially eluted - indeed, electron microscopy analysis of fractions eluting under these alternative peaks shows differences in the Nanosyringe ‘forms’ across these peaks (see Figure 3). It was surprising that such gradient could be used to effectively separate the alternative forms, in that conditions allow for sufficiently different retention times (under an ionic strength gradient) for separation into different fractions. For example, PyMol structure analysis suggests the whole Nanosyringe to be negatively charged at neutral pH (~pH 7) and it is not known what part of the complex (sheath, base, cap, tail fibers, or combination thereof) interacts with the matrix / monolith. Thus, the inventors have discovered that active pre-contracted forms and post-contracted forms can be resolved as distinct peaks (of A280nm) and distinct fractions of elution using anion exchange chromatography. It was not previously appreciated that mixed populations would have differential affinities to anion exchange medium, let alone of sufficient difference for advantageous separation under an ionic strength gradient. Transmission electron microscopy analysis of preparations resulting from prior art methods can be seen in Figures 3 and 4C, which show the mixed population. Figure 5 provides a striking image of how ‘clean’ the fractions enriched for the ‘target’ Nanosyringe can be versus purified samples generated with prior art methods. The ability to produce a homogenous drug product (e.g. uniform in composition) is particular advantageous for patient safety and treatment efficacy.

[0021] Advantageously, collecting fractions that elute under a particular peak (under which the target pre-contracted Nanosyringes elute) allows the target Nanosyringes to be separated from the post-contraction forms. Under elution profile conditions described herein, there are typically two-to-three resolvable peaks (e.g. first, second and third peaks, numbering according to appearance with increasing ionic strength), with the ‘second’ peak typically being for pre-contracted Nanosyringes. A “second” peak derives from monitoring A280 values of eluate having Nanosyringes that elute under higher ionic strength than those that elute to provide the “first” peak. That being said, the invention embraces collection of “first peak” fractions that contain pre-contracted Nanosyringes.

[0022] Another advantage is that methods described herein can also be used to purify precontraction state Nanosyringes that incorporate modifications to individual PVC proteins. For example, as described in Kreitz et al (Nature. 2023 Apr;616(7956):357-364), the tail fibre can be modified for retargeting. The inventors have found the separation methods described herein continue to apply even to such modified Nanosyringes (Figure 4B).

[0023] This opens up the possibility for conducting purification processes that are amenable to scalable GMP manufacture, a key step in developing any protein molecule for therapeutic use.

[0024] In more detail, the inventors have demonstrated that an elution approach based on increasing ionic strength in a gradient (e.g. linear gradient) can be used to elute the different forms of the complexes into separate fractions. Thus, pre- and post-contraction state forms are surprisingly eluted into separate fractions from the anion exchange resin using a mobile phase buffer with a gradient of increasing ionic strength.

[0025] In one aspect, the invention provides a chromatographic method for separating precontraction state Photorhabdus Virulence Cassettes (PVC) Nanosyringes from postcontraction state PVC Nanosyringes, the method comprising: a. providing a sample having both pre- and post- contraction state PVC Nanosyringes; b. loading the sample on a monolith anion exchange column (e.g. CIM Monolith QA) under conditions wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities; c. applying a gradient of ionic strength to the column that provides for differential elution of the pre- and post-contraction state PVC Nanosyringes (from the monolith column), wherein the gradient is generated by incrementally (e.g. linearly) increasing the salt concentration of a mobile phase elution buffer that is applied to the column; and d. eluting the pre- and post-contraction state PVC Nanosyringes into separate elution fractions under the gradient based on their differing affinities to the anion exchange column, and collecting fractions having the pre-contraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes. The monolith anion exchange column comprises positively charged binding moieties (which may be referred to as stationary phase binding moieties) for binding the pre- and postcontraction state PVC Nanosyringes. Step b. may be worded as “loading the sample on a monolith anion exchange column having binding moieties, under conditions (e.g. pH 7-8, 19- 25°C) wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities”.

[0026] The term “binding moieties” and “anion exchange membrane” (or “anion exchange membrane”) may be used interchangeably herein.

[0027] The binding moieties may be strong anion exchangers. It is particularly preferred that the binding moieties are quaternary amine binding moieties. For example, quaternary ammonium salts having a functional group such as (CHsh N+CH2O' represent advantageous binding moieties.

[0028] The term “chromatographic method” may be used synonymously with the term “anion exchange chromatography method”.

[0029] The term “CIM” refers to Convective Interaction Media.

[0030] The PVC Nanosyringes (e.g. pre- and post-contraction state PVC Nanosyringes) are preferably recombinant PVC Nanosyringes.

[0031] The post-contraction state PVC Nanosyringes may comprise a higher net negative charge (than pre-contraction state PVC Nanosyringes) such that they have greater binding affinity for the column (e.g. binding moieties thereof) than pre-contraction state PVC Nanosyringes, when the sample is loaded under conditions wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities.

[0032] The term “under conditions wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities” preferably refers to the pH in the column during loading. The pH may be about 6.5-8.5, with pH 7-8 being preferred (e.g. about pH 7.4), such that the sample is loaded on the column under conditions wherein pre- and postcontraction state PVC Nanosyringes become bound to the column with differing affinities. For example, the column may have been equilibrated (e.g. with equilibration buffer) to such pH, and the sample at step b) may be comprised within a buffer (e.g. loading buffer) having such pH.

[0033] Additionally or alternatively (preferably additionally), the term “under conditions wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities” may preferably include the temperature in the column during loading. The temperature may be about 5-25°C, more preferably 18-25°C (with 20-23°C being particularly preferred), such that the sample is loaded on the column under conditions wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities. For example, the column may have been equilibrated (e.g. with equilibration buffer) to such temperature, and / or the sample at step b) may be comprised within a buffer (e.g. loading buffer) having such temperature.

[0034] The term “chromatographic method” is employed because the method involves chromatography, a technique for separation of mixtures. Typically, the proteins / complex of interest are present in a fluid / buffer which may be called the “mobile phase” which carries it through a structure holding another material which may be called the “stationary phase” (where stationary ligands for the protein / complex of interest are bound).

[0035] The skilled person can readily confirm that collected fractions have pre-contracted state PVC Nanosyringes separated from post-contraction state PVC Nanosyringes, for example by transmission electron microscopy.

[0036] The “fractions having the pre-contraction state PVC Nanosyringes separated from the postcontraction state Nanosyringes” may be substantially free of post-contraction state PVC Nanosyringes (e.g. as confirmable by electron microscopy such as transmission EM). The “fractions having the pre-contraction state PVC Nanosyringes separated from the postcontraction state PVC Nanosyringes” may be referred to as fractions that are enriched for pre-contraction state PVC Nanosyringes. A fraction that is “enriched for” pre-contraction state Nanosyringes may be substantially free of post-contraction state PVC Nanosyringes (e.g. as confirmable by electron microscopy such as transmission EM).

[0037] The term “substantially free of post-contraction state PVC Nanosyringes” may mean that less than 10%, 8%, 6%, 4%, 2% or 1% (preferably less than 2%) of the PVC Nanosyringes in the fraction are post-contraction state PVC Nanosyringes. Preferably, the term “substantially free of post-contraction state PVC Nanosyringes” may mean that none of the PVC Nanosyringes in the fraction are post-contraction state PVC Nanosyringes.

[0038] The term “PVC subunit proteins” refers to the proteins of PVC 1-16 that assemble to provide PVC Nanosyringes (e.g. active extracellular contractile injection system (eCIS) complexes), the Nanosyringes being typically of 100-140 nm or 100-120 nm in length, preferably 110-120 nm in length (e.g. 115-120 nm). That being said, it is possible that functional Nanosyringes of alternative sizes (e.g. about 40-60 nm in length, or up to about 500 nm in length) may become assembled (e.g. under overexpression conditions in a heterologous expression host cell) to be purified in the pre-contraction state according to methods of the invention.

[0039] The structure of PVC Nanosyringes is well described in the prior art. For example, Jiang et al (Cell. 2019; 177: 370-383) describes a cryo-EM structure and the assembly of a PVC Nanosyringe into an eCIS. In said Jiang et al, its figure one in particular shows how sixteen types of PVC proteins (PVC1-16), with varying ‘copy number’ amongst the PVC proteins, assemble into a complete PVC particle (Nanosyringe).

[0040] Broadly, it can be said that:

[0041] PVC1 provides “tube” proteins

[0042] PVC2-4 provide “sheath” proteins

[0043] PVC5-7 provide “initiator tube” proteins

[0044] PVC8 provides “spike” proteins

[0045] PVC9-12 provide “tip” / “wedge” proteins

[0046] PVC13 provides “tail fiber” proteins

[0047] PVC14 provides “tape measure” proteins

[0048] PVC15 provides “ATPase” proteins

[0049] PVC16 provides “sheath / tube terminator” proteins

[0050] A PVC Nanosyringe may be referred to as an eCIS that comprises a tubular structure having a tube enclosed by a sheath (e.g. providing a structural domain), the tubular structure being formed from PVC 1-4 (e.g. formed from layers of “tube” proteins PVC1 and layers of “sheath” proteins of PVC 2-4).

[0051] The “tube” may be formed from 20-30 layers (e.g. about 24 layers) of PVC1. The term “layers” herein in the context of PVC subunit proteins may be used synonymously with the term “stacked layers”. The sheath may be formed from 1-3 layers (e.g. 1 layer) of PVC4; 16-18 layers (e.g. 17 layers) of PVC2; and 4-6 layers (e.g. 5 layers) of PVC3.

[0052] In a typical arrangement, there may be 24 layers of PVC1 forming the tube, enclosed by a sheath that has a first layer (“L1”) of PVC4; L2 of PVC2; L3 of PVC3; L4 of PVC2; L5 of PVC3; L6 of PVC2; L7 of PVC3; L8 of PVC2; L9 of PVC3; L10 of PVC2; L11 of PVC3; and L12-23 of PVC2; e.g. said layers L1-L23 being sequential in the sheath according to their numbering.

[0053] Said first layer is typically the layer closest to a “spike” domain of the PVC Nanosyringe.

[0054] More particularly, it may be said that a PVC Nanosyringe comprises the following domains: a tubular structure having a tube enclosed by a sheath (e.g. providing a structural domain), the tubular structure being formed from PVC 1-4 (e.g. formed from layers “tube” and “sheath” proteins of PVC 1-4) a baseplate at a first end of the tube enclosed by a sheath, the baseplate being formed by PVC 11 and 12, and being connected to the tube by proteins of PVC5 and 7 (or by PVC 5-7), and being connected to the sheath by an interaction of proteins of PVC 3, 4 and 9 a spike positioned on the baseplate, the spike being formed from PVC 8 and 10 proteins a targeting domain, formed from PVC13 protein(s) a terminal cap at a second end (e.g. opposite the first end) of the tube enclosed by a sheath, the terminal cap formed from PVC16 protein(s) optionally an ATPase formed from PVC15 (e.g. the ATPase being ‘optional’ because it may be absent from ‘mature’ Nanosyringes) optionally a tape measure formed from PVC14 (e.g. the tape measure being ‘optional’ because it may be absent from ‘mature’ Nanosyringes)

[0055] Thus, a pre-contraction state PVC Nanosyringe may be said to have an appropriate copy number and assembly of at least PVC proteins 1-5, 7-13 and 16. PVC6, 14 and 15 can be absent from ‘mature’ Nanosyringes, although functional eCIS forms that comprise each of PVC proteins 1-16 are embraced. Similarly, a post-contraction state PVC Nanosyringe may also be said to have an appropriate copy number and assembly of at least PVC proteins 1-5, 7-13 and 16, albeit the Nanosyringe is in the post-contraction state. As outlined in the prior art such as in Jiang et al (Cell. 2019; 177: 370-383), PVC Nanosyringes undergo a conformational change wherein the PVC proteins of the sheath become compressed, such that the PVC Nanosyringe contracts. It is believed that this contraction helps the “spike” to puncture a target cell membrane, so that payload can be delivered to the target cell cytoplasm.

[0056] Thus, a “post-contracted state” PVC Nanosyringe refers to a Nanosyringe that has (already) contracted following compression of the PVC protein subunits of the sheath (e.g. PVC proteins 2-4), wherein said PVC protein subunits (of the sheath) are more compressed relative to the corresponding PVC protein subunits in a “pre-contracted state” Nanosyringe.

[0057] The diameter of the sheath in “post-contraction state” PVC Nanosyringes may be increased relative to the diameter of the sheath in “pre-contraction state” PVC Nanosyringes (e.g. increased by 1-6 nm in diameter, such as increased by 3-5 nm in diameter); the diameter here preferably referring to a point of highest diameter.

[0058] It may be said that contraction causes the tube, formed from layers of PVC1, to protrude from the sheath.

[0059] Thus, it may be said that the “pre-contraction state” PVC Nanosyringes comprise a “tube” enclosed by the sheath; whereas the “post-contraction state” PVC Nanosyringe comprise a “tube” that protrudes from the sheath. Additionally or alternatively, it may be said that the “pre-contraction state” PVC Nanosyringes comprise a payload; whereas the “post-contraction state” PVC Nanosyringe have released their payload (e.g. they do not comprise a payload, or comprise less payload than an otherwise corresponding “pre-contraction state” PVC Nanosyringe).

[0060] In a post-contraction state PVC Nanosyringe, at least 5, at least 10, at least 15, or at least 20 layers of PVC1 (“tube” proteins) may protrude from the sheath.

[0061] It may be said that the pre-contraction state PVC Nanosyringes comprise PVC subunit proteins (e.g. at least PVC proteins 1-5, 7-13 and 16, optionally PVC proteins 1-16) assembled into active extracellular contractile injection system (eCIS) complexes of 100-140 nm or 100-120 nm in length. The term comprise “PVC subunit proteins” may be used synonymously with the term “PVC structural proteins” herein. The amino acid sequences of PVC1-16 as found in Photorhabdus asymbiotica strain ATCC43949 PVCPnf operon (corresponding to genes PAU_03353 to PAU_03338 of the sequence of GenBank accession no. FM 162591.1) can be seen in SEQ ID NOs: 1-16. That being said, there may also be related homologues of these PVC proteins (1-16) associated with other Photorhabdus species / strains, and / or other PVC operons. The homologues for each PVC protein typically have at least 85% sequence to the corresponding PVC protein of SEQ ID NOs: 1-16.

[0062] It should be noted, however, that any one of the PVC proteins may present as a variant thereof, e.g. that additionally includes an affinity tag (e.g. a FLAG tag). One reason is because, as will be discussed below, methods of the invention may comprise a clarification purification step involving affinity chromatography. For example, PVC16 (e.g. SEQ ID NO: 16) may comprise an affinity tag (e.g. a FLAG tag), for example fused to the C-terminus. Additionally or alternatively, PVC16 (e.g. SEQ ID NO: 32) may comprise an affinity tag (e.g. a FLAG tag), for example fused to the C-terminus.

[0063] Thus, PVC1-16 may comprise the amino acid sequences of SEQ ID NOs: 1-16 (respectively), or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto. For the avoidance of any doubt, the N-terminal methionine residue of any amino acid sequence described herein is optional. Thus any amino acid sequence (e.g. amino acid SEQ ID NO) herein can read as being absent the N-terminal methionine residue.

[0064] For example, PVC1 may comprise the amino acid sequence of SEQ ID NO: 1 , or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0065] PVC2 may comprise the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0066] PVC3 may comprise the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0067] PVC4 may comprise the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto. PVC5 may comprise the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0068] PVC6 may comprise the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0069] PVC7 may comprise the amino acid sequence of SEQ ID NO: 7, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0070] PVC8 may comprise the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0071] PVC9 may comprise the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0072] PVC10 may comprise the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0073] PVC11 may comprise the amino acid sequence of SEQ ID NO: 11 , or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0074] PVC12 may comprise the amino acid sequence of SEQ ID NO: 12, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0075] PVC13 may comprise the amino acid sequence of SEQ ID NO: 13, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto, e.g. with the proviso that any variant (not 100% identical to SEQ ID NO: 13 or said sequence absent the N-terminal methionine residue) comprises a targeting domain capable of binding a target cell.

[0076] PVC14 may comprise the amino acid sequence of SEQ ID NO: 14, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0077] PVC15 may comprise the amino acid sequence of SEQ ID NO: 15, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto. PVC16 may comprise the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0078] PVC16 may comprise the amino acid sequence of SEQ ID NO: 32, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity thereto.

[0079] In Photorhabdus, downstream of the genomic cluster that encodes, PVC1-16, there exists a coding region for “PVC effector” proteins that represent the payload for the Nanosyringes. In methods of the invention, it is preferred that the Nanosyringes (e.g. pre-contraction state Nanosyringes) may comprise payload. That being said, the invention also embraces purification of ‘empty’ PVC Nanosyringes that do not comprise a payload.

[0080] A pre-contraction state PVC Nanosyringe may have a length of 100-120 nm in length, preferably 110-120nm, more preferably 115-120nm (e.g. about 117 nm). Nanosyringes of such lengths are readily identifiable by methods such as election microscopy (e.g. transmission electron microscopy), as demonstrated in the examples section herein. Such lengths may apply also to the post-contraction state PVC Nanosyringe. Thus, the postcontraction state PVC may have a length of 100-120 nm in length, preferably 110-120nm, more preferably 115-120nm (e.g. about 117 nm).

[0081] The length may also be expressed in angstrom. It may be said that a pre-contraction state PVC Nanosyringe has a length of 1000-1400, 1000-1200 angstrom in length, preferably 1100-1200 angstrom, more preferably 1150-1200 angstrom (e.g. about 1170 angstrom). Similarly, a post-contraction state PVC Nanosyringe may have a length of 1000-1200 angstrom in length, preferably 1100-1200 angstrom, more preferably 1150-1200 angstrom (e.g. about 1170 angstrom).

[0082] Preferably, a pre-contraction state PVC Nanosyringe has a diameter (e.g. at its widest point) of 25-35 nm, preferably 25-33 nm, more preferably 27-29 nm. The pre-contraction state PVC Nanosyringe may have a diameter (e.g. at its widest point) of 250-350 angstrom, preferably 250-330 angstrom, more preferably 270-290 angstrom (e.g. about 280 angstrom).

[0083] The length and diameter values are consistent with those reported in Jiang et al and identified in fractions that are enriched for functional pre-contraction state Nanosyringes that the present inventors have obtained. There may also be “aberrant” Nanosyringes e.g. beyond the “post-contraction state” Nanosyringes. Optionally, methods of the invention may also comprise separating the “precontraction state” PVC Nanosyringes (e.g. of 100-120 nm in length) from “aberrant” Nanosyringes.

[0084] The ‘aberrant’ Nanosyringes may be Nanosyringes having lengths that fall outside the range of lengths for the preferred pre-contraction state PVC Nanosyringes. They may have different affinities (versus the pre-contraction state PVC Nanosyringes) for the column in methods of the invention, e.g. therefore demonstrating different retention times in the column versus the (preferred) pre-contraction state PVC Nanosyringe under the ionic strength gradient. For example, the ‘aberrant’ Nanosyringes may comprise a lower net negative charge (than precontraction state PVC Nanosyringes e.g. of 100-120nm in length) such that they have lower binding affinity for the column (e.g. binding moieties thereof) than said (target) precontraction state PVC Nanosyringes, when the sample is loaded under conditions of step b. Additionally or alternatively, there may be ‘aberrant’ Nanosyringes that comprise a higher net negative charge (than pre-contraction state PVC Nanosyringes e.g. of 100-120nm in length) such that they have higher binding affinity for the column (e.g. binding moieties thereof) than said (target) pre-contraction state PVC Nanosyringes, when the sample is loaded under conditions of step b.

[0085] For example, aberrant Nanosyringes (e.g. short Nanosyringes) may have a length of 30-50 nm, such as 35-45 nm. In Figure 3, such aberrant ‘short’ Nanosyringes can be seen as pool / fraction D4. Without wishing to be bound by theory, it is believed that they may be ‘short’ because they have fewer ‘layers’ of a tube or sheath (or otherwise) when compared with ‘full length’ PVC Nanosyringes.

[0086] Additionally or alternatively, aberrant Nanosyringes may have a length of >120nm or >140 nm, such as >120-150 nm, for example 125-140 nm. An aberrant Nanosyringes may have a length of >200nm or 145-160 nm. For example, aberrant ‘long’ syringes have been observed.

[0087] An ‘aberrant’ Nanosyringe may not necessarily be formed into a Nanosyringe structure, but rather may be an aggregate of PVC proteins. Thus, aberrant and ‘non-target’ aggregate can in some cases be synonymous terms. For example, an ‘aberrant’ Nanosyringe may refer to an aggregate of PVC2 and PVC 13 proteins. Additionally or alternatively, an ‘aberrant’ Nanosyringe may refer to an aggregate of PVC2, PVC13 and PVC16 proteins. The “fractions having the pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length) separated from the aberrant Nanosyringes” may be referred to as fractions that are enriched for pre-contraction state PVC Nanosyringes. A fraction that is “enriched for” precontraction state Nanosyringes (e.g. of 100-120nm in length) may be substantially free of aberrant Nanosyringes (e.g. as confirmable by electron microscopy such as transmission EM). The term “substantially free of aberrant Nanosyringes” may mean that less than 10%, 8%, 6%, 4%, 2% or 1% of the Nanosyringes in the fraction are aberrant Nanosyringes. Preferably, the term “substantially free of aberrant Nanosyringes” may mean that none of the Nanosyringes in the fraction are aberrant Nanosyringes.

[0088] For example, reference to a fraction that is “enriched for” pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length) may mean that at least 90% of the Nanosyringes in the fraction are pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length). For example, a fraction that is “enriched for” pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length) may mean that at least 92%, 94%, 96%, 98% or 99% of the Nanosyringes in the fraction are pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length). Preferably, reference to a fraction that is “enriched for” pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length) may mean that all of the Nanosyringes in the fraction are said pre-contraction state PVC Nanosyringes.

[0089] It has been demonstrated that the pre-contraction state PVC and the post-contraction state (and optionally also “aberrant” Nanosyringes) have differing affinities for the monolith column e.g. CIM Monolith Quaternary Amine (QA); preferably, this means that they have differing net charges. As such, they have different retention times in the monolith column (e.g. CIM Monolith QA) under the ionic salt (elution) gradient, allowing for their differential elution and thus separation from one another (into fractions).

[0090] The skilled person understands the term monolith column in the context of anion exchange chromatography (e.g. referring to a monolith column having binding moieties suitable for binding the pre- and post-contraction state PVC Nanosyringes as part of anion exchange chromatography). A preferred example is a CIM Monolith QA.

[0091] One advantage of employing a monolith column may be that sample can be under convective liquid flow, which may allow for minimisation of shear by mitigating flow vortices and turbulent eddies, which can occur in the void space in traditional packed bead (or resin) columns. A monolith column may be said to be a monolithic porous structure with interconnected channels (e.g. of a specific size to be controlled). The sample is transported through the monolith column by “convection”, e.g. resulting in fast mass transfer between the mobile phase and the stationary phase.

[0092] Examples of monolithic solid supports include molded monoliths, synthesized, for example, from a single polymer mixture composed of poly(glycidyl methacrylate -co-ethylene dimethacrylate) or from a single polymer mixture composed of polystyrene-divinylbenzene. Particular examples are CIM monolithic chromatographic columns from Sartorius BIA Separations.

[0093] In monolithic columns, interaction between ligands (e.g. QA binding moieties) and ligates (components of the sample that is of interest and shall be isolated, and that binds to the ligand) occurs mainly in the channels, on the surface of continuous material. In “conventional”, particle-based, bulk material, this interaction occurs in the pores inside the material and it is limited by diffusion. It also means that the surface inside pores of the bulk, particle-based material influences separation performance of the bulk material. On the other hand, an increase in surface area also increases the possibility for nonspecific interactions. These interactions are responsible for non-specific bindings (that can be irreversible), but also for activation and aggregation of plasma components. Additionally, the physical interaction occurs in the pores of the bulk material. Driving force here is diffusion and the physical interactions are therefore limited by the pore dimensions, surface area, and by flow rate. In monoliths, the interaction occurs in the channels of the material rather than in pores, and the driving force here is not diffusion, but rather convection (CIM - "Convective Interaction Media"). Pores may have a random orientation and comprise dead-ends, which increase the surface area without benefiting the throughput of a column. In contrast, the channels of a continuous chromatographic material (e.g. in monolith columns) continue from the beginning to the end of the column. In general, a larger diameter of the channels results in a lower surface area of the chromatographic material. This means that the surface of the continuous chromatographic material may be a couple orders of magnitude lower, and that the level and danger of non-specific interactions and activations are likewise orders of magnitude lower.

[0094] Examples of particularly suitable monolith columns include CIMmultus® QA 1 mL Monolithic Column (2 pm), Item No.: 311.5113-2 (Sartorius); CIMmultus® QA 4 mL Monolithic Column (2 pm), Item No.: 904.5113-2, Sartorius; and CIMmultus® QA 8 mL Monolithic Column (2 pm), Item No.: 411.5113-2, Sartorius. Thus, reference to a monolith column herein may mean a “convective interaction media’ monolith column.

[0095] The monolith columns of the invention may have a channel (or pore) size of at least about 1 pM in diameter, preferably 1-3 pM in diameter, more preferably about 2 pM in diameter. Other potential diameters include 6 pM and 1.3 pM.

[0096] The binding moieties are most preferably “quaternary amine binding moieties” which are the stationary-phase ligands within the column to which the Nanosyringes become bound (e.g. during the loading step b). The skilled person understands that the term “quaternary amine binding moieties” in the context of anion exchange chromatography, the term embracing positively-charged polyatomic ions of the structure [NR4]+, where R is an alkyl group, an aryl group or organyl group.

[0097] The monolith column used in methods of the invention has preferably been subjected to less than 40, less than 35, less than 30, less than 25, less than 20 or less than 15 purification runs. It is particularly preferred that the monolith column used in methods of the invention has been subjected to less than 30 purification runs. A “purification run” refers to an iteration of steps a-d of a chromatographic method described herein (e.g. a second run means that the column is being used to perform steps a-d for a second time, and so on), or to an iteration of an alternative chromatographic method.

[0098] As mentioned above, the sample is loaded on the column “under conditions (e.g. pH 7-8, 19- 25°C) wherein pre- and post-contractions state PVC Nanosyringes (and optionally also “aberrant” PVC Nanosyringes) become bound to the column with differing affinities”. These conditions preferably include the pH within the column. Thus, these conditions preferably refer to a pH of 7-8 (preferably pH 7.2-7.6, such as about 7.4). These conditions may also involve conducting the loading step at a temperature of 19-25°C (preferably 20-23°C), e.g. room temperature.

[0099] Description of these ‘conditions’ may also involve describing a particular loading buffer in which the PVC Nanosyringes are present during loading (e.g. step b). A preferable loading buffer (e.g. for step b) may comprise (or consist of) 9-11 mM (preferably about 10 mM) Na2HPC>4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 130-150mM (preferably about 137 mM) NaCI; 2.5-3mM (preferably about 2.7 mM) KCI, and a pH of 7-8 (preferably a pH of 7.4). Thus, a loading buffer (e.g. for step b) may comprise or consist of 10 mM Na2HPO4; 1.9 mM KH2PO4; 137 mM NaCI; 2.7 mM KCI, and a pH of 7.4; and preferably a temperature of 20- 23°C. Thus, step a) may be read as providing a sample having both pre- and postcontraction state PVC Nanosyringes within a loading buffer as described herein.

[0100] A more preferred loading buffer (e.g. for step b) may comprise (or consist of) 18-22mM Trisbuffered saline (preferably about 20 mM Tris-buffered saline), 130-150mM (preferably about 137 mM) NaCI; and 6-10 mM MgSC (preferably about 8 mM MgSC ); and a pH of 7-8 (preferably a pH of 7.4). Thus, a loading buffer (e.g. for step b) may comprise or consist of 20 mM Tris-buffered saline; 137 mM NaCI; 8 mM MgSC ; and a pH of 7.4; and preferably a temperature of 20-23°C. Thus, step a) may be read as providing a sample having both pre- and post-contraction state PVC Nanosyringes within a loading buffer as described herein.

[0101] Throughout this disclosure, any buffer described contains the recited ingredients in a suitable water medium (e.g. double distilled water).

[0102] Establishing gradient and elution

[0103] Having now been provided with the insight that appropriate conditions allow for such separation under an ionic strength gradient, the skilled person would be in a position to put such separation technique into effect. Particular examples are discussed below. Thus, we now turn to discussing the ionic strength gradient.

[0104] In anion exchange chromatography, an ionic strength gradient is established to facilitate the selective elution of anions from the stationary phase. Initially, a mobile phase with low ionic strength is introduced, allowing the target anions to interact more strongly with the positively charged (preferably quaternary amine) ligands on the stationary phase. As the separation progresses, the ionic strength of the mobile phase is gradually increased by adding a salt solution (e.g. mobile phase elution buffer) of increasing salt concentration, which competes with the anions for binding sites on the stationary phase. This competition disrupts the interactions between the anions (e.g. Nanosyringes) and the stationary phase, leading to differential elution based on the affinities of the anions for the ligand. Anions with weaker interactions are eluted first, while those with stronger affinities are retained longer, allowing for effective separation based on their charge and size. This gradient not only enhances resolution but also allows for the targeted recovery of specific anions from complex mixtures. In the examples section below, the ability to exploit such ionic strength gradient to separate the different Nanosyringe forms is exemplified with the use of NaCI. Salts are used in the buffer to influence ionic strength. Thus, ionic strength is increased under a gradient of salt concentration.

[0105] In the examples section below, one equilibration buffer (and loading and running) buffer comprises 137mM of NaCI, within a PBS background (e.g. also comprising 10 mM Na2HPC>4, 1.9 mM KH2 O4, 2.7 mM KCI). In the examples section below, an input elution buffer had 2M NaCI, but otherwise had the same content as the equilibration buffer mentioned in the preceding sentence (e.g. also comprising 10 mM Na2HPC>4, 1.9 mM KH2 O4, 2.7 mM KCI). A representative chromatogram of the separation of pre- and post-contraction state PVC Nanosyringes by anion exchange chromatography is shown in Figure 3.

[0106] Another successful equilibration buffer (and loading and running) buffer used in the examples section below comprises 20 mM Tris; 137 mM NaCI; 8 mM MgSC ; and then an elution buffer comprising 10 mM Na2HPC>4; 1.9 mM KH2PO4; 2 M NaCI; and 2.7 mM KCI.

[0107] It was found that the pre-contraction state PVC Nanosyringes elute ‘first’ under the ionic strength gradient, and post-contraction state PVC Nanosyringes begin to elute at about 0.48M NaCI. Thus, collecting fractions having the (e.g. ‘target’) pre-contraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes can be achieved by collecting fractions that elute at a concentration below 0.48M NaCI (or below an ionic strength equivalent to that of 0.48M NaCI).

[0108] It was found that fractions that elute, when the NaCI concentration during elution is between about 0.28-0.47M NaCI (or equivalent ionic strength), are enriched for pre-contraction state PVC Nanosyringes. Collecting fractions that elute between about 0.37-0.47M NaCI is preferred (e.g. fractions in such range may be enriched for 100-120nm length pre-contraction state PVC Nanosyringes). This was seen in the context of using an elution buffer (and loading / running buffer) that additionally comprised about 2.7 mM KCI, about 10 mM Na2HPC>4, and about 1.9 mM KH2PO4. Thus, the ionic strength of about of an NaCI concentration described herein (e.g. 0.29M-0.36M NaCI), about 2.7 mM KCI, about 10 mM Na2HPC>4, and about 1.9 mM KH2PO4. For example, because the NaCI was provided within the ‘background’ of phosphate buffered saline (PBS) buffer. However, these NaCI concentrations also provided for elution of the pre-contraction state PVC Nanosyringes (separated from the post-contraction state ones) in the ‘background’ of an alternative buffer (e.g. Tris buffer) that did not contain the other salts of PBS. For example, where the loading / running buffer comprised 20 mM Tris; 137 mM NaCI; and 8 mM MgSC .

[0109] For all values described herein with reference to the ionic strength gradient, the skilled person would understand that the values are intended to embrace alternative values that continue to solve the problem (i.e. of separating pre- and post-contraction state PVC Nanosyringes), for example alternative values that emanate from switching e.g. to a different column or buffer, or purification machine.

[0110] The skilled person can readily calculate the ionic strength of a buffer based on its salt content, and thus would have no issue in providing alternative buffers (e.g. having alternative salts) toward achieving an equivalent ionic strength. In other words, the ionic strength provided by the NaCI concentration that causes elution of the pre-contraction state PVC Nanosyringes in the present examples can be readily calculated, such that the skilled person can use this knowledge to employ other suitable salts to yield an equivalent ionic strength.

[0111] Advantageously, therefore, the elution buffer conditions used in the examples of this disclosure can be used as a reference elution buffer, allowing substitution of the salts used (e.g. NaCI) for any other salt buffer to achieve a similar ionic strength gradient.

[0112] Methods of the invention may comprise collecting fractions that elute when the ionic strength of the mobile phase elution buffer in the column is equivalent to that of a buffer comprising (or consisting of) about 0.28M-0.47M NaCI (preferably about 0.37M-0.47M NaCI), optionally when at a pH of 7-8 (preferably a pH of about 7.4), optionally when at a temperature of 19- 25°C (preferably 20-23°C).

[0113] Methods of the invention may comprise collecting fractions that elute when the ionic strength of the mobile phase elution buffer in the column is equivalent to that of a buffer comprising (or consisting of) about 0.28M-0.47M NaCI (preferably about 0.37M-0.47M NaCI), about 2.7 mM KCI, about 10 mM Na2HPC>4, and about 1.9 mM KH2PO4, optionally when at a pH of 7-8 (preferably a pH of about 7.4), optionally when at a temperature of 19-25°C (preferably 20- 23°C).

[0114] Throughout this disclosure, the ionic strength may be calculatable using the following formula: where q is the molar concentration of ion i (M, mol / L), Zj is the charge number of that ion i, and I is taken over all ions in the solution.

[0115] Methods of the invention may comprise collecting fractions that elute under an ionic strength equivalent to that of a buffer comprising (or consisting of) a concentration of about 0.37M- 0.47M NaCI, about 2.7 mM KCI, about 10 mM Na2HPO4, and about 1.9 mM KH2PO4, optionally when at a pH of 7-8 (preferably a pH of about 7.4), optionally when at a temperature of 19-25°C (preferably 20-23°C).

[0116] With reference to the (e.g. reference) buffer mentioned in the paragraph directly above, when the NaCI concentration is 0.37M, the ionic strength (e.g. at the start of the preferred elution fractions for pre-contraction state PVC Nanosyringes) may be calculated as 0.4036M.

[0117] For an NaCI concentration of 0.47M (but the same amount as above for KCI, Na2HPC>4, and KH2PO4), the ionic strength may be about 0.5036M.

[0118] For an NaCI concentration of 0.28M (but same amount as above for KCI, Na2HPC>4, and KH2PO4), the ionic strength may be about 0.3141 M.

[0119] As mentioned above, the inventors have also eluted the pre-contraction state PVC Nanosyringes (as per the examples) where NaCI was the only salt in the mobile phase elution buffer, again preferably at concentrations (in the gradient) between about 0.28M- 0.47M NaCI (more preferably 0.37M-0.47M NaCI).

[0120] Thus, methods of the invention may comprise collecting fractions that elute when the ionic strength of the mobile phase elution buffer in the column is about 0.31-0.5M, preferably about 0.4M-0.5M.

[0121] Taking the ionic strength range of the NaCI as the only salt, with a starting value about 0.28M (e.g. when not in the context of a PBS background), it can be seen that a wider range of ionic strength can be attributed to elution of the pre-contraction state PVC Nanosyringes. Methods of the invention may comprise collecting fractions that elute when the ionic strength of the mobile phase elution buffer in the column is about 0.28M-0.47M, preferably about 0.37-0.47M.

[0122] When using NaCI, its concentration typically starts at 137 mM (as per its concentration in PBS), increasing toward about 2M. In the examples, at close to neutral pH (e.g. pH 7-8 such as 7.4), preferred pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length) begin to elute at 0.37M of NaCI. Thus, an ionic strength equivalent to that of 0.37M of NaCI can begin to cause elution of the preferred pre-contraction state PVC Nanosyringes (e.g. of 100-120nm in length).

[0123] Reference to “incrementally increasing the salt concentration of a mobile phase elution buffer” preferably means that the gradient is generated by linearly increasing the salt concentration of a mobile phase elution buffer that is applied to the column.

[0124] It is preferred that the concentration of salt (e.g. NaCI) in the mobile phase elution buffer at the beginning of the gradient is at least two times lower than the salt concentration that causes elution of pre-contraction state PVC Nanosyringes.

[0125] The skilled person understands that gradients of ionic strength (e.g. salt concentration) in elution steps of anion exchange chromatography are typically established by mixing an increasing amount of high salt elution buffer (e.g. input elution buffer) with a running / loading buffer (of lower salt content) over time, the admixture being passed over the column as a mobile phase elution buffer.

[0126] Thus, the “mobile phase elution buffer” that is applied to the column is preferably an admixture of an input elution buffer and a running buffer (the former having a higher salt concentration / ionic strength than the latter). The running buffer can be the same as a “loading buffer” or “equilibration buffer” described herein.

[0127] Thus, the ionic strength gradient may be established by admixing an input elution buffer (having a salt concentration that is higher than that at which the pre- and post-contraction state PVC Nanosyringes elute from the column) and a running buffer (aka loading buffer) before applying the resulting admixture (e.g. as mobile phase elution buffer) to the column, wherein the amount of the input elution buffer in the admixture is incrementally (e.g. linearly) increased over 6-14 (preferably 8-12, more preferably about 10) column volumes (e.g. for a column volume of about 1-10ml, such as about 1 ml, 4ml or 8 ml). The “admixture” can be referred to as the “mobile phase elution buffer that is applied to the column”.

[0128] For example, the ionic strength gradient may be established by admixing an input buffer and a running buffer (aka loading buffer) before applying the resulting admixture (e.g. mobile phase elution buffer) to the column, wherein the amount of an input elution buffer (having a salt concentration that is higher than that at which the pre- and post-contraction PVC Nanosyringes elute from the column) is incrementally (e.g. linearly) increased to provide from 0-30% of the admixture over 6-14 (preferably 8-12, more preferably about 10) column volumes (e.g. for a column volume of 1 ml or 8 ml), and preferably collecting fractions that elute when the input buffer provides about 14-24% (preferably about 18-23%, more preferably about 18-23%) of the admixture / mobile phase elution buffer in the column (e.g. the remainder provided by running buffer).

[0129] While using the elution buffer (e.g. input elution buffer) and running / loading buffers in the Examples, the inventors achieved preferential elution of the pre-contraction state PVC Nanosyringes when the elution buffer provided between about 14%-24% (e.g. about 14-23%) of the admixture (e.g. mobile phase elution buffer) in the column, the running / loading buffer providing the remainder. The range of about 18%-24% (e.g. about 18-23%) is particularly advantageous.

[0130] Thus, methods of the invention may comprise collecting fractions that elute when the input elution buffer provides about 18-24% (preferably 18-23%) of the admixture / mobile phase elution buffer in the column (e.g. the running / loading buffer providing the remainder).

[0131] The salt concentration in the mobile phase elution buffer at the start of (or immediately prior to) the gradient may be that having an ionic strength equivalent to a buffer comprising (or consisting of) 10 mM Na2HPO4, 1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, e.g. pH 7.4). In other words, the salt concentration in the mobile phase elution buffer at the start of (or immediately prior to) the gradient may be that having an ionic strength of 171.6 mM.

[0132] The ionic strength gradient may be provided by an increasing concentration of NaCI in the mobile phase elution buffer (e.g. the admixture), preferably starting at a NaCI salt concentration of about 120-150 mM (e.g. 137mM). The ionic strength gradient may be provided by an increasing NaCI concentration in the mobile phase elution buffer (e.g. the admixture), and the method may comprise collecting fractions that elute under a NaCI concentration of about 0.28-0.47M (preferably about 0.37- 0.47M); preferably at pH conditions of pH 7-8 (more preferably about pH 7.4), preferably at a temperature of 19-25°C (more preferably 20-23°C).

[0133] An input elution buffer described herein may have an ionic strength equivalent to a buffer comprising (or consisting of) 1.5-2.5M NaCI (e.g. with a pH of 7-8, such as about 7.4). An input elution buffer described herein may have an ionic strength equivalent to a buffer comprising (or consisting of) 9-11 mM Na2HPC>4; 1.7-2.2mM KH2PO4; 1.5-2.5M NaCI; 2.5- 3mM KCI (e.g. with a pH of 7-8, such as about 7.4).

[0134] For example, an input elution buffer described herein may have an ionic strength equivalent to a buffer comprising (or consisting of) 2M NaCI (e.g. with a pH of 7-8, such as about 7.4). For example, the input elution buffer may have an ionic strength of about 2M. For example, an input elution buffer described herein may have an ionic strength equivalent to a buffer comprising (or consisting of) 10mM Na2HPC>4; 1.9 mM KH2PO4; 2M NaCI; 2.7 mM KCI (e.g. with a pH of 7-8, such as about 7.4). For example, the input elution buffer may have an ionic strength of about 2.03M (e.g. about 2034.6 mM).

[0135] An input elution buffer (e.g. at its highest salt / NaCI concentration) may comprise or consist of 9-11mM (preferably about 10 mM) Na2HPC>4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 1.5-2.5M (preferably about 2M) NaCI; 2.5-3mM (preferably about 2.7 mM) KCI, and a pH 7-8 (preferably a pH of about 7.4). For example, and input elution buffer (e.g. at its highest salt / NaCI concentration) may comprise or consist of about 10 mM Na2HPC>4; about 1.9 mM KH2PO4; about 2M NaCI; about 2.7 mM KCI, e.g. with a pH 7-8 (preferably a pH of 7.4).

[0136] A more preferred input elution buffer may comprise or consist of: 10 mM Na2HPC>4; 1.9 mM, KH2PO4; 2 M NaCI; 2.7 mM KCI, and a pH of 7.4.

[0137] The running buffer may preferably be the same as a loading buffer used when loading the sample. Similarly, the running buffer may preferably be the same buffer as an equilibration buffer used to equilibrate the column before loading. Thus, all particular descriptions of a “running buffer” in this disclosure can also be used to describe a “loading buffer” used in methods of the invention. Additionally or alternatively, all particular descriptions of a “running buffer” in this disclosure can also be used to describe an “equilibration buffer” used in methods of the invention.

[0138] Preferably, prior to loading the sample, the column has been equilibrated with a buffer (e.g. loading buffer as described herein for example with reference to the running buffer) of pH 7- 8, preferably pH 7.2-7.6, more preferably about pH 7.4.

[0139] In methods of the invention, prior to loading the sample, the column may be equilibrated with a buffer (e.g. loading buffer as described herein for example with reference to the running buffer) of pH 7-8, preferably pH 7.2-7.6, more preferably about pH 7.4.

[0140] Preferably, prior to loading the sample, the column has been equilibrated with the same buffer as a loading buffer in which the sample is present while loading the column. In methods of the invention, prior to loading the sample, the column may be equilibrated with the same buffer as a loading buffer in which the sample is present while loading the column.

[0141] Each fraction of eluate may have a volume of 0.3-0.6 ml (preferably about 0.5 ml).

[0142] An anion exchange chromatography procedure may comprise the following steps:

[0143] 1. wash the monolith column with 10 CV (column volumes) of Endotoxin free water;

[0144] 2. equilibrate the monolith column with 10 CV loading / running buffer;

[0145] 3. load sample having pre- and post-contraction state Nanosyringes comprised in loading / running buffer;

[0146] 4. wash off unbound material with 10CV loading / running buffer;

[0147] 5. wash off Endotoxin using 30 CV of wash buffer;

[0148] 6. elute Nanosyringes under a 0-30% elution buffer gradient (e.g. 0-30% of the mobile phase elution buffer being “input elution buffer”, the remainder being running buffer) over 10 CV (e.g. collecting 0.3-0.6 ml, such as 0.5 ml, fractions);

[0149] 7. optionally remove any tightly bound material using 5 CV of 100% input elution buffer;

[0150] 8. optionally analyse fractions and pool by Coomassie, Western blot and EM (electron microscopy);

[0151] 9. optionally pool peak fractions and measure A280 and A280 / 260 using a suitable spectrophotometer e.g. NanoPhotometer (Implen). A more preferred anion exchange chromatography procedure may comprise the following steps:

[0152] 1 . wash the monolith column with 10 CV (column volumes) of Endotoxin free water;

[0153] 2. equilibrate the monolith column with 10 CV loading / running buffer (e.g. TBS and 8 mM MgSC );

[0154] 3. load sample having pre- and post-contraction state Nanosyringes comprised in loading / running buffer;

[0155] 4. wash off unbound material with 10CV of a first wash buffer described herein;

[0156] 5. wash off Endotoxin using 30 CV of a second wash buffer described herein;

[0157] 6. elute Nanosyringes under a 0-30% elution buffer gradient (e.g. 0-30% of the mobile phase elution buffer being “input elution buffer”, the remainder being running buffer) over 10 CV (e.g. collecting 0.3-0.6 ml, such as 0.5 ml, fractions);

[0158] 7. optionally remove any tightly bound material using 5 CV of 100% input elution buffer;

[0159] 8. optionally analyse fractions and pool by Coomassie, Western blot and EM (electron microscopy);

[0160] 9. optionally pool peak fractions and measure A280 and A280 / 260 using a NanoPhotometer (Implen).

[0161] Said first wash buffer may comprise or consist of 9-11 mM (preferably about 10 mM) Na2HPC>4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 130-150mM (preferably about 137 mM) NaCI; and preferably also 7-9 mM (preferably about 8 mM) MgSC ; such first wash buffer may have a pH of 7-8, preferably a pH of 7.2-7.4, more preferably a pH of about 7.4.

[0162] Said second wash buffer may comprise or consist of 9-11mM (preferably about 10 mM) Na2HPC>4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 130-150mM (preferably about 137 mM) NaCI; 2.5-3mM (preferably about 2.7 mM) KCI; and 7-9 mM (preferably about 8 mM) MgSC ; and 15-25 mM (e.g. about 20 mM) CHAPS; and preferably also 18-22 mM EDTA (preferably about 20 mM EDTA); and preferably also 7-9 mM (preferably about 8 mM) MgSC ; such second wash buffer may have a pH of 7-8, preferably a pH of 7.2-7.4, more preferably a pH of about 7.4.

[0163] The wash step 4 can optionally be replaced with tangential flow filtration (TFF), preferably conducted after the anion exchange chromatography (AEX) step (e.g. as the next step post AEX), that provides for buffer exchange into said buffer otherwise used in the wash step (e.g. first wash buffer). TFF may be employed using hollow fiber filters (e.g. suitable for Nanosyringe because of reduced back pressure. An example is mPES hollow fibers: 500kDa MWCO, 0.05 um and 0.1 um (Spectrum).

[0164] The method of the invention may comprise subjecting the collected fractions (having the precontraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes) to tangential flow filtration, preferably providing for buffer exchange into said first wash buffer.

[0165] A chromatographic method (e.g. anion exchange chromatography method) described herein is preferably performed on an Akta Pure 25 (Cytiva), e.g. using its built in conductivity monitor; preferably using a CIM Monolith QA column, such as CIMmultus® QA 1 mL Monolithic Column (2 pm) [Item No.: 311.5113-2, Sartorius], CIMmultus® QA 4 mL Monolithic Column (2 pm), or CIMmultus® QA 8 mL Monolithic Column (2 pm) [Item No.: 411.5113-2, Sartorius], “Akta” can be referred to as “AKTA”.

[0166] A 280 values

[0167] Multiple peaks are sequentially resolvable under a gradient of ionic strength. Advantageously, it is possible to collect fractions that elute under the area of a peak that is provided by elution of pre-contraction state PVC Nanosyringes, which may be the base peak (e.g. of highest A280).

[0168] The term main peak may be used synonymously with the term “base peak”. This is because the base peak (or main peak) can be referred to as the most intense peak in a chromatogram e.g. the base peak being the peak with the highest relative abundance, or the tallest peak on the y-axis. The base peak (or main peak) can be set to 100% relative abundance, and all other peaks recorded relative to it.

[0169] In step (d), the method may comprise monitoring the eluate for ultraviolet absorbance at a wavelength of about 280 nm (A280) and collecting fractions that elute under a peak of A280 value that is detectable during elution of the pre-contraction state PVC Nanosyringes, the collected fractions having the pre-contraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes.

[0170] In step (d), the method may comprise monitoring the eluate for ultraviolet absorbance at a wavelength of about 280 nm (A280) and collecting fractions that elute under a second peak of A280 value, the collected fractions having the pre-contraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes.

[0171] A “second” peak derives from monitoring A280 valuates of eluate having Nanosyringes (e.g. pre-contraction state Nanosyringes e.g. of 100-120nm) that elute under higher ionic strength than those that elute to provide a “first” peak. That being said, the invention embraces collection of such “first peak” fractions that contain pre-contracted Nanosyringes (albeit preferential collection of fractions under the “second” peak is preferred).

[0172] For example, in step (d), the method may comprise monitoring the eluate for ultraviolet absorbance at a wavelength of about 280 nm (A280) and collecting fractions that elute under a base peak (e.g. of highest A280 value, the highest A280 being relative to any other A280 value detected across the eluate profile), the collected fractions having the pre-contraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes.

[0173] Conductivity

[0174] Conductivity is proportional to ionic concentration. Thus, the conductivity value of the column (e.g. in the elution buffer) at the time the pre-contraction state PVC Nanosyringes elute may be employed generically, independent of the elution ‘salt’ used.

[0175] The unit of ionic strength used herein can thus be a unit of conductivity, millisiemens. The conductivity of a buffered solution is a function of the ionic strength. The term "millisiemens ionic strength" as used herein preferably refers to the buffer solution ionic strength as measured by conductivity in a 1 cm path length cell at 20-23°C (e.g. room temperature) in units of millisiemens. It is preferred that all conductivity values referred to herein are measured at 19-23°C, preferably 20-23°C (e.g. room temperature) in a 1 cm path cell. For example, with a conductivity monitor such as that found in an Akta Pure 25 (Cytiva) purifier.

[0176] As mentioned above, it was found that the pre-contraction state PVC Nanosyringes elute ‘first’ (vs post-contraction state PVC Nanosyringes) under the ionic strength gradient, and post-contraction state PVC Nanosyringes begin to elute at about 49 mS / cm. Thus, collecting fractions having the pre-contraction state PVC Nanosyringes separated from the postcontraction state PVC Nanosyringes can be achieved by collecting fractions that elute below a conductivity (in the monolith column) of about 49 mS / cm. As demonstrated in the Examples, the pre-contraction state PVC Nanosyringes elute when the conductivity (along the ionic strength gradient) is between about 34 mS / cm-48 mS / cm, with a range of about 40 mS / cm - 48 mS / cm being particularly advantageous for specifically collecting the (e.g. 100-120nm length) pre-contraction state PVC Nanosyringes.

[0177] Throughout this disclosure, any reference to the range “about 40 mS / cm - 48 mS / cm” may more particularly be “about 40.6 mS / cm - 48 mS / cm”.

[0178] Thus, the methods of the invention (e.g. at step d) may comprise applying an increasing salt concentration gradient to the loaded (and optionally washed anion exchange column), and collecting fractions that elute when the conductivity of the elution buffer in the column is between about 34 mS / cm and 48 mS / cm, more preferably between about 40 mS / cm and 48 mS / cm (preferably when the elution is conducted under pH conditions of pH 7-8, more preferably about pH 7.4).

[0179] In other words, methods of the invention (e.g. at step d) may comprise collecting fractions that elute when the conductivity of the elution buffer in the column is between 34 mS / cm and 48 mS / cm, more preferably between 40 mS / cm and 48 mS / cm; preferably when the elution is conducted under pH conditions of pH 7-8, more preferably about pH 7.4; preferably wherein the elution is conducted at a temperature of 19-25°C (preferably 20-23°C); preferably wherein the conductivity values correspond to conductivity measured at 20-23°C in a 1 cm path cell.

[0180] Conductivity values described herein are preferably those detected when performing the chromatographic method (e.g. anion exchange chromatography method) on an Akta Pure 25 (Cytiva), e.g. using its built in conductivity monitor; preferably using a CIM Monolith QA column, such as CIMmultus® QA 1 mL Monolithic Column (2 pm) [Item No.: 311.5113-2, Sartorius], CIMmultus® QA 4 mL Monolithic Column (2 pm), or CIMmultus® QA 8 mL Monolithic Column (2 pm) [Item No.: 411.5113-2, Sartorius],

[0181] Conditions (pH and temperature)

[0182] The elution is preferably conducted at a pH of 7-8, more preferably about pH 7.2 to 7.6, with about pH 7.4 being the most preferred pH.

[0183] It is preferred that all steps of the chromatographic method are conducted under a pH of 7-8, preferably 7.2-7.6, with pH 7.4 being particularly preferred; thus, all buffers used may have said pH values. It is particularly preferred that the pH is maintained at a stable value (e.g. at the same value) throughout all steps of the chromatographic method.

[0184] Methods of the invention are preferably carried out a temperatures of 19-25°C, more preferably 20-23°C (e.g. room temperature). It is preferred that all steps of the chromatographic method are carried out a temperatures of 19-25°C, more preferably 20- 23°C (e.g. room temperature). It is particularly preferred that the temperature is maintained at a stable value (e.g. at the same value) throughout all steps of the chromatographic method.

[0185] Additional steps before and after anion exchange chromatography

[0186] The skilled person would understand that cell lysate can be expected to be subjected to initial ‘clarification’ steps (e.g. filtration and / or affinity chromatography) prior to anion exchange chromatography, such that the input sample for step (a) is a “purified sample containing both pre- and post-contracted state PVC Nanosyringes (and optionally also aberrant Nanosyringes)”. Such clarification steps are particularly advantageous in obtaining a chromatogram (in the anion exchange chromatography step) in which elution of the precontraction state PVC Nanosyringes provides a base peak (e.g. of highest A280 value, the highest A280 being relative to any other A280 value detected across the eluate profile).

[0187] It may be said that the sample (for step a)) has been subjected to a pre-anion exchange chromatography purification step e.g. to remove host cell protein. The pre-anion exchange chromatography purification step may be any suitable purification technique (e.g. other than anion exchange chromatography).

[0188] Thus, the sample (for step a)) is most preferably a sample (e.g. “pre-enriched” sample) that has been subjected to a clarifying purification step to provide a purified sample containing both pre- and post-contraction state PVC Nanosyringes (and optionally also aberrant Nanosyringes), wherein a purified sample containing both pre- and post-contraction state PVC Nanosyringes (and optionally also aberrant Nanosyringes) comprises less (e.g. at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, preferably at least 60% less) non-PVC Nanosyringe (of any state / form) polypeptide compared with the sample before it was subjected to the clarifying purification step. A sample that has been subjected to a clarifying purification step to provide a purified sample containing both pre- and post-contraction state PVC Nanosyringes may be referred to as a “pre-anion exchange chromatography purified sample”.

[0189] As will be discussed in more detail below, it is preferred that the sample in step a) is a “purified sample containing both pre- and post-contraction state PVC Nanosyringes (and optionally also aberrant Nanosyringes)” provided by subjecting a lysate comprising the Nanosyringes to one or more clarifying purification step(s) such as centrifugation, PEG (polyethylene glycol) precipitation, flocculation, affinity chromatography and / or filtration (e.g. tangential flow filtration, which may use hollow fibers) to remove (e.g. non-Nanosyringe) host cell material, thereby providing purified sample containing both pre- and post-contraction state PVC Nanosyringes (and optionally also aberrant Nanosyringes) (e.g. as the sample in step a)). Said “filtration” may be membrane filtration. Additionally or alternatively, said filtration may be diafiltration.

[0190] A flocculation step may comprise admixing lysate with polyethyleneimine (PEI), preferably PEI of about 25kDa, preferably PEI at a concentration of <0.1% (e.g. 0.010 to <0.1%); optionally followed by depth filtration.

[0191] A PEG precipitation step may comprise (i) admixing a sample (e.g. lysate) with PEG (such as PEC 6000 and / or PEG 8000), optionally at a PEG concentration of 60-100g / L (preferably 80 g / L), optionally in the presence salt such as NaCI (e.g. 0.3-0.6M, or 0.5M salt); (ii) incubate the admixture e.g. for 4-24 hours optionally on a roller or shaker; (iii) centrifuge the incubated admixture e.g. at 7000-1 OOOxg (e.g. about 8000xg), optionally for 10-30 minutes (e.g. about 20 minutes); (iv) resuspend the pellet from (iii) to provide a resuspension, optionally wherein the pellet is resuspending in a loading buffer described herein; (v) centrifuge the resuspension, e.g. at 10000-14000xg (e.g. about 12000xg), optionally for 10-30 minutes (e.g. about 20 minutes) and collect the supernatant; (vi) filter the supernatant (e.g. with a 0.2 urn filter, such as an SFCA filter). Said supernatant may then be subjected to affinity chromatography, prior to an anion exchange step described herein. Said step (i) may include shaking the admixture e.g. at 100-300 rpm. Said step (iii) may include mechanical disruption of the pellet (e.g. with a stripette via pipetting up and down) following by shaking e.g. 100-300 rpm (such as 250 rpm) which may be conducted at room temperature (e.g. 19- 23°c) optionally for 1-3 hours (e.g. 2 hours). Where “affinity chromatography” is described, the skilled person would understand that at least one PVC protein (e.g. of the pre-contraction state PVC Nanosyringe complex) comprises an affinity tag (e.g. a FLAG tag).

[0192] For example, prior to conducting the chromatographic method described here (e.g. prior to step a), a (the) lysate comprising the Nanosyringes may be subjected to one or more clarifying purification step(s) such as centrifugation, PEG precipitation, affinity chromatography and / or filtration (e.g. tangential flow filtration) to remove (e.g. nonNanosyringe) host cell material, thereby providing a purified sample containing both pre- and post-contraction state Nanosyringes (e.g. said purified sample providing the sample in step a)). For example, affinity chromatography and / or filtration (e.g. tangential flow filtration). Said “filtration” may be membrane filtration. Additionally or alternatively, said filtration may be diafiltration.

[0193] A clarifying purification step described throughout this disclosure may comprise a combination of PEG precipitation and tangential flow filtration. A clarifying purification step described throughout this disclosure may comprise a combination of PEG precipitation and centrifugation.

[0194] The clarifying purification step described herein may comprise binding the Nanosyringes (in embodiments wherein an affinity tag such as a FLAG tag is associated with at least one of the PVC proteins) to a resin (or column) having a moiety that binds the affinity tag, washing through unbound material, and subsequently eluting the Nanosyringes thereby providing a purified sample containing both pre- and post-contraction state PVC Nanosyringes (e.g. said purified sample providing the sample in step a)); optionally wherein the affinity tag is fused to the c-terminus of PVC16.

[0195] Reference above to “washing through unbound material” (during affinity chromatography) may refer to washing the resin (or column) with a buffer (e.g. such as PBS or more preferably with a first wash buffer described herein), preferably having a pH of 7-8 (e.g. about pH 7.4); for example with 20-40 column volumes; preferably such that endotoxin in the elution (from the affinity resin / column) comprises endotoxin at < 102EU / ml.

[0196] Preferably, in step a) said sample is a sample that was previously processed to remove expression (e.g. host) cell debris following expression cell lysis. Prior to a clarifying purification step, a cell lysate is preferably incubated in the presence of a nuclease (e.g. for 30 min at 37°C).

[0197] The column may be equilibrated with buffer as described above. The skilled person would understand that wash steps can typically also be employing during anion exchange chromatography.

[0198] Prior to equilibrating the column, the column may be washed with a suitable preparation of water (e.g. endotoxin free water), preferably with 8-12 (e.g. 10) column volumes of said water.

[0199] Prior to the elution steps (e.g. prior to step c)), unbound material may be washed from the column using a running buffer (e.g. which may be the same as a loading buffer described above), preferably using 6-14, more preferably 8-12 (e.g. about 10) column volumes of running buffer. It is particularly preferred that the loading buffer for washing may have an equivalent ionic strength (e.g. salt concentration) and pH to the loading buffer used to load the sample; preferably wherein both the running and loading buffers are the same.

[0200] Concentration steps may then be conducted. Column fractions containing pre-contraction state PVC Nanosyringe may be pooled, filtered through a 0.22 pm filter, aliquoted, and stored e.g. at -70°C.

[0201] It is preferred that a wash step may be conducted prior to the elution steps (e.g prior to step c) to remove endotoxin from the column using a wash buffer comprising an endotoxin removal agent (e.g. a detergent such as CHAPS); preferably wherein the wash buffer has an equivalent ionic strength (e.g. salt concentration) and an equivalent pH to a loading buffer used to load the sample; more preferably wherein the wash buffer additionally comprises said endotoxin removal agent but is otherwise equivalent to the loading buffer e.g. thereby providing a washed monolith column. A wash step to remove endotoxin preferably comprises washing the monolith column with 20-40 (e.g. 30) column volumes of wash buffer.

[0202] Such wash step to remove endotoxin from the column is preferably performed subsequent to a wash step as described above in which the column may be washed with a suitable preparation of water (e.g. endotoxin free water). Endotoxin removal agents may be incorporated into a buffer at any appropriate stage of the separation / purification process. For example, with reference to the examples section, a wash buffer used to wash the loaded column (prior to elution) can comprise an endotoxin removal agent. Examples of suitable endotoxin removal agents include detergents, such as CHAPS. For example, CHAPs may be present in a buffer (e.g. wash buffer) at a concentration of 10-30 mM, 15-25 mM, preferably about 20mM.

[0203] A second wash buffer (e.g. to remove / elute endotoxin) may comprise or consist of 9-11mM (preferably about 10 mM) Na2HPO4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 130- 150mM (preferably about 137 mM) NaCI; 2.5-3mM (preferably about 2.7 mM) KCI; and 15-25 mM (e.g. about 20 mM) CHAPS; and preferably also 18-22 mM EDTA (preferably about 20 mM EDTA); such wash buffer may have a pH of 7-8, preferably a pH of 7.2-7.4, more preferably a pH of about 7.4. For example, a wash buffer (e.g. to remove / elute endotoxin) may comprise or consist of about 10 mM Na2HPO4; about 1.9 mM KH2PO4; about 137 mM NaCI; about 2.7 mM KCI; and about 20 mM CHAPS; and preferably also about 20 mM EDTA; such wash buffer may have a pH of 7-8, preferably a pH of 7.2-7.4, more preferably a pH of about 7.4.

[0204] The collected fractions (e.g. enriched for the pre-contractions state PVC Nanosyringes) may be subjected to a volume reduction step (e.g. concentration), preferably by using a centrifugal filter and / or tangential flow filtration. Such step is typically performed to yield a concentration of the pre-contraction state PVC Nanosyringes.

[0205] Preferably, methods of the invention may further comprise analysing the collected fractions (e.g. by transmission electron microscopy) to confirm the fractions are enriched for the precontractions state PVC Nanosyringes.

[0206] In step b) of methods of the invention, the loading flow rate may be about 1 mL / min for a 1ml column. Similarly, the loading flow rate may be about 8 mL / min for an 8ml column.

[0207] In methods of the invention, the mobile phase elution buffer flow rate may be about 1 mL / min for a 1ml column. Similarly, the mobile phase elution buffer flow rate may be about 4 ml / min for a 4ml column. Similarly, the mobile phase elution buffer flow rate may be about 8 ml / min for an 8ml column. Subsequent to step d), the eluate (e.g. collected fractions) may be passed through an endotoxin removal column. Examples of such column include CIMmultus Prima T, Sartorius (e.g. a multimodal column combining H-bond and AEX properties (metal affinity)) and EtoxiClear, Astrea (e.g. PuraBead®, synthetic ligand).

[0208] Buffers

[0209] Particularly preferred buffers that can be used in methods of the invention will now be described.

[0210] During chromatographic methods described here, particularly preferred buffers are as follows: o Loading buffer: 10 mM Na2HPO4,1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, pH 7.4; a buffer with equivalent ionic strength / pH can substitute; o Running buffer (e.g. as per loading buffer): 10 mM Na2HPO4, 1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, pH 7.4; a buffer with equivalent ionic strength / pH can substitute; o Equilibration buffer (e.g. as per loading and running buffer): 10 mM Na2HPO4, 1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, pH 7.4; a buffer with equivalent ionic strength / pH can substitute; o Wash buffer (e.g. first wash buffer) for washing off unbound material: 10 mM Na2HPO4, 1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, and preferably also 8 mM MgSO4, pH 7.4; o Wash buffer (e.g. second wash buffer) for endotoxin removal: 10 mM Na2HPO4,1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, 20 mM CHAPS, 20 mM EDTA, pH 7.4, and preferably also and 7-9 mM (preferably about 8 mM) MgSO4; a buffer with equivalent ionic strength / pH (while comprising the CHAPS and EDTA) can substitute; o Input elution buffer: 10 mM Na2HPO4,1.9 mM KH2PO4, 2 M NaCI, 2.7 mM KCI, pH 7.4; a buffer with equivalent ionic strength / pH can substitute.

[0211] A more preferred loading buffer is: 20 mM Tris-buffered saline; 137 mM NaCI; and 8 mM MgSO4. More preferred running and equilibration buffers are also 20 mM Tris; 137 mM NaCI; and 8 mM MgSO4.

[0212] Thus, in methods of the invention: the column may be (or may have been) equilibrated with an equilibration buffer as described herein prior to the loading step; the sample having the Nanosyringes may be within loading buffer as described herein during the loading step; optionally the column may be washed to remove endotoxin as described herein, prior to the elution step, using a wash buffer for endotoxin removal as described herein; and / or the elution may be conducted by incrementally (e.g. linearly) increasing the amount of input elution buffer as described herein, relative to running buffer as described, in the mobile phase elution buffer passing over the column.

[0213] In methods of the invention, each of the loading buffer, equilibration buffer and running buffer are preferably the same (e.g. in terms of content, pH and temperature).

[0214] Preferably, PVC proteins 1-16 are expressed in a host cell under the control of an inducible promoter (e.g. arabinose inducible or IPTG inducible). For example, expression preferably occurs under controlled conditions.

[0215] The following steps may be carried out prior to step a): i. culturing prokaryotic host cells (preferably E. coli) comprising a nucleotide sequence encoding the PVC structural proteins (PVC 1-16), wherein the nucleotide sequence is operably linked to an inducible promoter; ii. inducing expression of the PVC proteins (e.g. with arabinose of IPTG) by the host cells and allowing the PVC proteins to assemble into Nanosyringes in the host cells; and iii. lysing the cells (e.g. by homogenisation) thereby providing a lysate comprising said pre- and post-contraction state PVC Nanosyringes.

[0216] In methods of the invention, cell lysis may be conducted with detergent, by homogenization or by enzymatic lysis. Lysis with detergent may be particularly suitable.

[0217] Said “nucleotide sequence encoding the PVC structural proteins (PVC 1-16)” may comprise a sequence having at least 75% sequence identity (preferably at least 85% sequence identify; more preferably at least 95% sequence identity) to a sequence selected from SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19, e.g. with the proviso that the sequence encodes proteins having sufficient structural and functional similarity to PVC 1-16 proteins, and that assemble into a PVC Nanosyringe that is an active extracellular contractile injection system (eCIS) complex (e.g. of 100-120 nm in length).

[0218] For example, said “nucleotide sequence encoding the PVC structural proteins (PVC 1-16)” may comprise a sequence having at least 75% sequence identity (preferably at least 85% sequence identify; more preferably at least 95% sequence identity) to a sequence of SEQ ID NO.: 17, e.g. with the proviso that the sequence encodes proteins having sufficient structural and functional similarity to PVC 1-16 proteins, and that assemble into a PVC Nanosyringe that is an active extracellular contractile injection system (eCIS) complex (e.g. of 100-120 nm in length).

[0219] Preferably, said “nucleotide sequence encoding the PVC structural proteins (PVC 1-16)” may comprise or consist of a sequence of SEQ ID NO.: 17.

[0220] An “active extracellular contractile injection system (CIS)” refers to Nanosyringe complex capable of binding a target cell and injecting a suitable payload (e.g. effector) of the eCIS (where present) into the target cell (e.g. into the target cell’s cytosol).

[0221] Host cells can be cultured to an optical density at 600 nm (ODeoo) of 35-65; for example, host cells can be cultured to an optical density at 600 nm (ODeoo) of 40-60. In methods of the invention, it is preferred that the host cells may be cultured to an ODeoo (e.g. observed ODeoo in a shake flask) of 6-8.

[0222] A cassette (operon) encoding the PVC Needle Complex (Nanosyringe) may be operably linked to a first promoter, and a gene encoding the fusion / effector (payload) may be operably linked to a second (preferably different) promoter. Said first and / or second promoter may be an inducible promoter (e.g. an arabinose inducible promoter such a pBAD, and / or an IPTG inducible promoter). Thus, the invention embraces an expression system wherein an operon encoding the PVC is present within a first vector / plasmid (optionally operably linked to a first promoter), and the sequence encoding the effector (leader sequence fused to payload) is present within a second (preferably different) plasmid (optionally linked to a second promoter). That being said, an operon encoding the PVC Nanosyringe (e.g. PVC1-16) and a sequence encoding a payload may be present on the same vector / plasmid. The PVC Needle Complex and / or (preferably and) effector may be expressed in one or more host cell selected from a bacterial cell, a yeast cell, an insect cell and / or a mammalian cell. In a preferable embodiment, the PVC Needle Complex and effector may be expressed together in a host cell selected from a bacterial cell, a yeast cell, an insect cell and a mammalian cell (preferably a bacterial cell). Suitable mammalian cells include a HEK293 cell and / or a CHO cell.

[0223] A preferred expression host cell is E. coli.

[0224] Thus, the PVC Needle Complex and / or (preferably and) the effector (payload) may be expressed in a heterologous bacterial expression system (preferably E. coli).

[0225] The PVC Needle Complex and / or (preferably and) the PVC effector may be expressed in a Photorhabdus cell, optionally wherein the PVC operon of the Photorhabdus cell is endogenous to the cell (and optionally wherein the PVC operon is operably linked to an inducible promoter which may be incorporated into the genome to be operably linked to the PVC operon via genetic engineering). For example, an inducible promoter may be introduced into the genome of a Photorhabdus cell 5’ to a PVC (operon), preferably by recombineering as described previously.

[0226] After expression, centrifuged pellets can be lysed to release recombinant protein. In the Examples, an E. coli host is lysed by homogenisation. It is expected that a similar extraction of soluble target from an E. coli host will be obtained when the E. coli are lysed by other conventional methods such as homogenization, digestion with lysozyme, pressure cycling or detergent.

[0227] Any reference to a PVC effector herein embraces an “effector fusion” as described in WO2020 / 245611A1.

[0228] Isolated sample

[0229] The invention also provides an isolated sample (or fraction) of (e.g. recombinant) precontraction state PVC Nanosyringes obtainable by a method described herein, e.g. wherein the isolated sample (or fraction) is enriched for pre-contraction state PVC Nanosyringes. An isolated sample (or fraction) that is enriched for pre-contraction state PVC Nanosyringes may be substantially free of post-contraction state PVC Nanosyringes. The invention provides an isolated sample (or fraction) of (e.g. recombinant) pre-contraction state PVC Nanosyringes, wherein the isolated sample (or fraction) is substantially free of post-contraction state PVC Nanosyringes.

[0230] The term “substantially free of post-contraction state Nanosyringes” may mean that less than 10%, 8%, 6%, 4%, 2% or 1% (preferably less than 2%) of the Nanosyringes in the sample (or fraction) are post-contraction state Nanosyringes. Preferably, the term “substantially free of post-contraction state Nanosyringes” may mean that none of the Nanosyringes in the fraction are post-contraction state Nanosyringes.

[0231] An isolated sample (or fraction) of recombinant pre-contraction state PVC Nanosyringes may be substantially free of endotoxin.

[0232] The term “substantially free of endotoxin” may mean that the sample (or fraction) comprises less than 40 Ell / rng, less than 30 Ell / rng, less than 20 Ell / rng or less than Ell / rng. Preferably, the term “substantially free of endotoxin” may mean that the sample (or fraction) comprises < 5 Ell / mg.

[0233] Also provided are pharmaceutical compositions comprising said isolated sample, and optionally a pharmaceutically acceptable excipient.

[0234] The term “isolated” in the context of the present invention denotes that the Nanosyringe (e.g. pre-contraction state PVC Nanosyringe) has been removed from its natural milieu and is thus substantially free of other extraneous or unwanted polypeptides. Such isolated molecules are those that are separated from their natural environment.

[0235] Such isolated sample (or fraction) may comprise 4-6% (preferably about 5%) glycerol, e.g. for freezing conditions; and optionally may additionally comprise 6-10 mM (preferably about 8 mM) MgSC ; and 200-400 mM (preferably about 350 mM) NaCI; and may be in 200-600 pl / aliquots.

[0236] Nanosyringes

[0237] Details on how PVC Nanosyringes are formed is widely published in the literature, thus the skilled person is well aware of the form that a correctly assembled Nanosyringe takes, and as such has no issue understanding what is meant by “pre-contraction state PVC Nanosyringe” as described herein, and likewise has no issue understanding what is meant by “post-contraction state PVC Nanosyringe” as described herein. One example of a paper would be Jiang et al (Cell. 2019; 177: 370-383), incorporated herein by reference. Thus, the literature describes the PVC Nanosyringe structure in detail, such that the skilled person in this field understands the meaning of this term well. For completeness, background details on the Nanosyringes are discussed below.

[0238] Photorhabdus is a bacterium of the genus Enterobacteriacae, represented by three formally recognized (to date) species - namely P. luminescens, P. asymbiotica, and P. temperata. Important strains include P. asymbiotica subsp. australis, and P. luminescens subsp laumondii. Currently available genome sequences are available on GenBank (Photorhabdus asymbiotica ATCC43949 complete genome - GenBank Accession Number: FM 162591.1; Photorhabdus laumondii subsp. laumondii strain TT01 chromosome, complete genome - GenBank Accession number: CP024901.1).

[0239] Reference to “Photorhabdus luminescens subsp. laumondii" may be used interchangeably with “Photorhabdus luminescens subsp. laumondii TT01”, “Photorhabdus laumondii subsp. laumondii strain TT01” and “P. luminescens TT01” herein.

[0240] The genome sequence for a further strain of P. asymbiotica, namely P. asymbiotica Kingscliff, is described in Wilkinson et. al. (FEMS Microbiology Letters, Volume 309, Issue 2, August 2010, Pages 136-143), incorporated herein by reference. Further genome sequences are described in Thanwisai et. al. (PLoS ONE 7(9): e43835), incorporated herein by reference.

[0241] Each of these species comprise at least one operon known as a Photorhabdus Virulence Cassette (PVC) operon, encoding a PVC Needle Complex, which may be referred to as a “Nanosyringe” herein. Given that Photorhabdus is typically found in nature as an insecticidal bacterium following regurgitation from a (symbiont) entomopathogenic Heterorhabditis sp. nematode (e.g. in order to avoid competition for food and resources from insects), it is understood that the PVC Needle Complex functions in nature to suppress insects. Indeed, it has been shown that an isolated PVC Needle Complex (holding / packaged with a natural effector toxin, such as Pnf) can be used to kill insect larvae. The Photorhabdus Virulence Cassettes represent one of at least four well-characterised toxin delivery systems of Photorhabdus. Other major classes of Photorhabdus protein insecticidal toxins include the “Toxin Complexes” (Tcs), the “binary PirAB toxins”, and the “makes caterpillars floppy” (Mcf) toxins. The term “Photorhabdus Virulence Cassette” (PVC) (used synonymously with the term “PVC operon” herein) means a discrete operon of a Photorhabdus genome comprising genes encoding for polypeptide subunits which, when expressed, assemble to provide the macromolecular PVC Needle Complex. The molecular architecture of these cassettes have been well characterized and described, for example in The Molecular Biology of Photorhabdus Bacteria (Springer International Publishing AG 2017, ISBN: 978-3-319-52714- 7, Chapter 10, pages 159-177), incorporated herein by reference. A PVC (operon) typically comprises around sixteen genes (pvc1-pvc16) encoding structural proteins which assemble to provide a “PVC Needle Complex”, which are typically followed by one or more genes at the 3’ end which encode PVC effector genes, having toxic activity (and typically being homologues of typical T3SS-like effectors). A Photorhabdus genome typically comprises a plurality of such cassettes (e.g. at least four), which are often associated with different effector payloads, or even a plurality of effector payloads.

[0242] Three classes of PVC structural operons (Classes I, II and III) have been observed in the genomes of Photorhabdus, and members of other genera. PVCs within each class are similar in terms of the number and type of genes encoding structural proteins they contain. In more detail, Class I PVCs (which may be referred to as a “prototypical PVC” herein) comprise 16 conserved genes (pvc1-16). Class II lack pvc13 host cell binding fibres and pvc3, which (without wishing to be bound by theory) the inventors believe may be a minor specialised sheath subunit that attaches pvc13 fibre proteins onto the PVC Needle Complex (Nanosyringe). As such, it is believed this class may be “non-specific”, injecting payloads into multiple (potential any) cell types. Class III is similar to Class I, but has an additional PvcO gene at the start of the operon (of unknown function) and two additional genes encoded between pvc13 and pvc14 that resemble “invasion” type protein genes. This class is typically seen in the human clinical isolate strains of Photorhabdus - the inventors have shown that optimal transcription of PVC Class III may occur when the strain (harboring the PVC operon encoding a PVC Class III operon) is grown at 37°C and exposed to human serum, suggesting this class may be a mammalian adapted version of a PVC Needle Complex.

[0243] An example cassette (PVC) is shown in Figure 1(D), which shows a map of the model “Class I” PVC operon of Photorhabdus asymbiotica ATCC43949 (obtainable from the ATCC, accession number: ATCC 43949), said operon being associated with the downstream effector gene “PAU_03332” (encoding a Pnf protein effector). This model operon is referred to as aATCC43949PVCpnf. This operon comprises sixteen structural genes (pvc1-16), and two genes (3’ end) encoding effectors (in this case the pvc17 / Rhs-like, encoding an Rhs- like effector, and pvc21, encoding a Pnf effector). Said genes pvc1-16 correspond to genes PAU_03353 to PAU_03338 of the sequence of GenBank accession no. FM 162591.1, and are represented by the sequence of SEQ ID NO.: 17.

[0244] An example PVC operon (e.g. encoding the structural genes, but not a / the PVC effector) is provided in SEQ ID NO: 17 (which is encodes the operon shown schematically in Figure1(D)), with other examples being SEQ ID NO: 18 and in SEQ ID NO: 19. These sequences begin at the ATG start codon of the first structural gene (pvc1) of the PVC cassette I operon, and end at the TAA stop codon of the final structural gene (pvc16).

[0245] A PVC Needle Complex from any one of Classes l-lll may be used for a variety of applications. However, PVC Needle Complexes of a certain class may be particularly suitable for delivery to a defined cell type. For example, a PVC Needle Complex for delivery of a payload to a mammalian cell may suitably be a member of Class III. A PVC Needle Complex for delivery of a payload to an insect cell (e.g. to an insect) may suitably be a member of Class I (such as P. asymbiotica P\ / Cpnf, encoded by SEQ ID NO.: 17, e.g. as expressed in E. coli from a cosmid clone).

[0246] Thus, as will be understood by the skilled person, the term “PVC Needle Complex” (used synonymously with the terms “PVC Needle Complex delivery system” and “Nanosyringe” herein) means a macromolecular protein complex comprising polypeptide subunits encoded by a PVC (operon) of a Photorhabdus bacterium. A PVC Needle Complex is assembled in a Nanosyringe structure, having a physical structure (superficially) similar to the antibacterial R-type pyocins (see Figure 1(c)). Functional and molecular studies have shown that a PVC Needle Complex becomes packaged (loaded) with a PVC effector protein(s) (i.e. the PVC effector proteins are packaged therein, or thereon), the packaged PVC Needle Complex is released from the bacterium, and then injects the PVC effector into a target cell such that the PVC effector protein may exert toxicity.

[0247] The term “PVC Needle Complex” preferably encompasses PVC Needle Complex-like structures / complexes, encoded by operon(s) comprising genes which are homologous to genes of a Photorhabdus PVC operon. PVC-like elements are not restricted to Photorhabdus, and a well characterized homologous operon (to a PVC operon) is present on the pADAP plasmid of the insect pathogenic bacteria Serratia entomophila. Furthermore, an analogous, and (at least partially) homologous, PVC-like ‘injectosome’ Needle Complex system is employed by the bacterium Pseudoalteromonas luteoviolacea (e.g. used to control the metamorphosis of the marine worm Hydroides elegans). Structures exist in other Enterobacteriaceae (such as Yersinia Spp.) which are encoded by operons having homology to a PVC operon, and may be used with a leader sequence described herein. Each of these (PVC-like) structures are embraced by the term “PVC Needle Complex” as used herein.

[0248] Thus, a PVC Needle Complex is a “Nanosyringe” complex, with the polypeptide encoded by the effector gene being packaged (loaded) within, or at the end (tip) of, the PVC Needle Complex, thus representing a “payload” or “warhead” of the PVC Needle Complex. The present inventors have demonstrated that the PVC Needle Complex itself (with the payload still loaded) is freely released (e.g. secreted) from Photorhabdus cells, before interacting with the membrane of a target cell and injecting the payload into the cell’s cytosol. Indeed, the inventors have successfully expressed and loaded PVC Needle Complexes in heterologous expression systems, before isolating / purifying the PVC Needle Complexes and using them to suppress (e.g. kill) insect larvae (see Example 2 of WO2020 / 245611A1). Thus, the PVC Needle Complexes act as long-range protein delivery systems.

[0249] The terms “PVC Needle Complex” and “Nanosyringe” may be used synonymously herein.

[0250] The PVC Nanosyringe (e.g. Needle Complex) may be encoded by a sequence having at least 75% sequence identity (preferably at least 85% sequence identify; more preferably at least 95% sequence identity) to a sequence selected from SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19 (for example, SEQ ID NO.: 17).

[0251] The PVC Nanosyringe (e.g. Needle Complex) may be encoded by a sequence selected from SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19 (for example, SEQ ID NO.: 17).

[0252] The eCIS may be a modified version. For example, any one of the PVC1-16 subunits (such as PVC13) may be modified.

[0253] The pre-contraction state PVC Nanosyringe may be preferably a ‘loaded’ (or ‘packaged’) Nanosyringe. That being said, the invention is not limited to purifying loaded Nanosyringes.

[0254] A Nanosyringe that is loaded contains a payload (preferably a polypeptide or peptide payload). Examples of how to provide a loaded (or packaged) Nanosyringe are described in WQ2020 / 245611A1 , incorporated herein by reference. For example, a Nanosyringe described herein (e.g. pre-contraction state PVC Nanosyringe) may be loaded / packaged with a PVC effector.

[0255] The term “packaged” (used synonymously with the terms “trans-packaged” and “loaded”) means that a payload was directed by a leader sequence (to which the payload is linked / fused) into the interior, or end (tip), of an assembled PVC Nanosyringe, such that the PVC Nanosyringe is subsequently configured for delivering (e.g. injecting) the payload into a target cell. Thus, the payload may be packaged within a PVC Nanosyringe, or may be packaged at the end (or tip) of the PVC Nanosyringe (e.g. at least a portion of the payload may be external to the PVC Nanosyringe).

[0256] The term “payload” (used synonymously with the term “warhead” herein) means a molecule which is packaged into the interior, or end (tip), of an assembled PVC Nanosyringe, and subsequently delivered (e.g. injected) into a (target) cell. In wild-type Photorhabdus, the payload is a PVC effector (more particularly, the effector portion of said PVC effector), encoded (as described above) by a gene that is downstream to (3’ to) the structural genes of a PVC operon. For example, see model PVC operon of Figure 1(D), having effector genes PAU_03337 (listed as PVCpnf 17), encoding an adenylate cyclase effector; and PAU_03332 (listed as PVCpnf 21), encoding a Pnf effector.

[0257] The term “PVC effector” (used synonymously with the term “PVC operon-encoded effector”, and “PVC effector protein”) means an effector polypeptide encoded by a Photorhabdus PVC operon, more particularly (and typically) found shortly downstream (3’) of the structural genes of said operon (preferably shortly or immediately downstream of pvc16, and typically within 5kb). The term “PVC effector” preferably embraces homologues thereof. Thus, the leader sequence may also be from a polypeptide encoded by a gene which is a homologue of gene encoding a PVC effector - see Table 1 of WO2020 / 245611A1 for examples of such homologues. Indeed, identification of PVC effectors is aided by detecting homology of a gene downstream of pvc16 with a known toxin polypeptide (e.g. a gene which encodes said toxin polypeptide). As will be understood by the skilled person, the term “homologue” preferably means a gene that descended from the same ancestral gene, and shares similar function - such gene (or polypeptide encoded thereby) is homologous to a gene encoding the PVC effector. A homologue may be from the genome of a Photorhabdus species or from a species other than a Photorhabdus species. Examples of suitable homologues are outlined in Table 1 of WO2020 / 245611A1 (incorporated herein by reference). The term “leader sequence” (synonymous with “PVC effector leader sequence”) means the leader region (polypeptide region) from a PVC effector polypeptide which is capable of packaging a payload (e.g. effector) into a PVC Needle Complex, and is preferably amino acids 1-50 of a PVC effector, or amino acids 2-50 when omitting the initial methionine. The inventors demonstrated in WO2020 / 245611A1 (incorporated herein by reference) that the leader sequence is encompassed within (or may consist essentially of) amino acids 1-50 of a multitude of identified PVC effector polypeptide sequences. However, leader sequences having alternative lengths and positioning within a PVC effector are intended to be encompassed (e.g. with the proviso that said leader sequence is capable of packaging a payload into a PVC Needle Complex).

[0258] Thus, a Nanosyringe (e.g. pre-contraction state PVC Nanosyringe) may be loaded / packaged with a PVC effector.

[0259] Additionally or alternatively, a Nanosyringe (e.g. pre-contraction state PVC Nanosyringe) may be loaded / packaged with an “effector fusion” (or simply “fusion”) that is “distinct from a (e.g. wild-type) PVC effector”. For example, the effector fusion may be a chimaera, formed of a leader sequence from a first PVC effector fused to (an / the effector portion of) a second (different) PVC effector (preferably amino acids 51 to the C-terminal amino acid of said second PVC effector), wherein said first PVC effector and said second PVC effector are different. The effector fusion may be a chimaera, comprising (or consisting essentially of) a leader sequence described herein fused to a non-PVC effector polypeptide. The effector fusion may be a chimaera, comprising (or consisting essentially of) a leader sequence described herein fused to a non-Photorhabdus polypeptide. The effector fusion may be a leader sequence-nucleic acid fusion (preferably conjugate), comprising a leader sequence described herein fused to a nucleic acid.

[0260] An effector fusion is not limited to a fusion complex comprising a leader sequence fused to a toxic payload (e.g. the leader could be fused to a therapeutic payload). Thus, the term “effector” as used in the context of “effector fusion” means the payload which is packaged into the PVC Nanosyringe (which could provide a variety of effects, including toxigenic and / or therapeutic effects). Thus, the term “effector fusion” may be used interchangeably with the term “fusion” herein.

[0261] The term “effector fusion” may be used synonymously with the term “leader sequencepayload fusion”, and / or “leader sequence-payload complex”. Alternatively or additionally, the payload may be distinct from a PVC effector protein (e.g. distinct from amino acids 51 to the C-terminal amino acid of a PVC effector). For example, the payload may be a polypeptide or nucleic acid that is not found in a wild-type Photorhabdus bacterium.

[0262] The payload may be one or more selected from a polypeptide (e.g. a polypeptide payload), a nucleic acid (e.g. a nucleic acid payload), or a combination thereof. In a preferable embodiment, the payload is a polypeptide.

[0263] Examples of polypeptide payloads include an antibody (e.g. an anti-MDM antibody), a nanobody, a peptide vaccine (e.g. a tyrosinase-related protein 2 (TRP2) peptide vaccine), a nuclear factor-KB inhibitor, a T3SS payload (e.g. a T3SS payload which inhibits the NF-kB and / or MAPK pathways), an anti-apoptotic peptide (e.g. BH4), nicotinamide adenine dinucleotide quinone internal oxidoreductase (Ndi1), a PHOX complex subunit, a myotubularin, a nucleic acid (preferably DNA)-modifying enzyme, or a combination thereof. Examples of suitable nucleic acid-modifying enzymes include a recombinase (e.g. Cre recombinase), a transposase, a Cas enzyme (e.g. Cas9), and / or a Mad7 (preferably Mad7, more preferably Cre recombinase). The payload may be, for example, tBid and / or BaxBH3 peptide (aa59-73).

[0264] Any polypeptide having enzymatic activity may be a payload.

[0265] A nucleic acid payload may be conjugated / crosslinked to a leader sequence of the invention. For example, copper-free click chemistry (e.g. strain-promoted alkyne azide cycloaddition (SPAAC)) may be used to crosslink a nucleic acid to a leader sequence. Examples of nucleic acid payloads include a primer, an mRNA, a nucleic acid analogue, an aptamer, a small interfering RNA (siRNA), a microRNA therapeutic inhibitor (antimiR), a microRNA therapeutic mimic (promiR), a long non-coding RNA modulator, a single guide RNA (sgRNA), or a combination thereof.

[0266] The leader sequence may be fused directly or indirectly (e.g. by means of a spacer) to the payload. The leader sequence may be fused covalently or non-covalently to the payload. In a preferable embodiment, the leader sequence is covalently fused to the payload. For example, the fusion / effector fusion may be a (recombinant) fusion protein comprising (or consisting essentially of) a PVC effector leader sequence fused to a (polypeptide) payload. BRIEF DESCRIPTION OF THE DRAWINGS

[0267] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0268] Figure 1 shows (A) a schematic representation of one PVC operon layout (gene clusters present in varying regions of the originating genome) encoding a PVC Needle Complex / Nanosyringe. (B) A schematic representation of Class I, II and III PVC operon layouts. Homologous subunit types amongst the classes are shown as having similar shading (in grey scale). (C) An illustration of an assembled PVC Needle Complex (pre-contraction). The numbering shown is used to correlate a gene cluster in (A) with the position of the encoded proteins in the structure in (C) (e.g. the cap ‘16’ cluster in A is shown as ‘16’ in the left-most cap region of (B)). (D) A map of the model Class I PaATCC43949PVCpnf operon (e.g. encoded by SEQ ID NO.: 17), showing two effector genes in the payload region (Rhs-like adenylate cyclase, and PAU_03332).

[0269] Figure 2 shows an overview of a cloning procedure for preparation of PVC Needle Complexexpressing plasmids, based on overlapping PCR. PCR fragments (having overlapping regions) are provided from template gDNA of P. asymbioticaMCC^^ (available from the ATCC under accession no. ATCC 43949) with relevant primers targeting the PVC operon.

[0270] Figure 3. This demonstrates the ability to employ AEX purification (using a monolith column as described herein) to select for a fraction that is concentrated for the ‘target’ precontraction state PVC Nanosyringe complex of interest e.g. fully assembled and functional complex (aka intact, full-length, loaded).

[0271] Figure 4. A - This is a chromatogram where the method involves pre-clarification with affinity chromatography prior to AEX. Again, this demonstrates the ability to employ AEX purification (using a monolith column as described herein) to select for a fraction that is concentrated for the ‘target’ pre-contraction state PVC Nanosyringe complex of interest e.g. fully assembled and functional complex (aka intact, full-length, loaded). B - This is further to A, and shows an overlay of elution profiles when using either ‘loaded’ (smaller base peak in this overlay) and empty (no payload) Nanosyringes, additionally the former has a modified version of a PVC13 protein; C - EM analysis of the various fractions. The final EM image is from a fraction having post-contraction state PVC Nanosyringes, pointed out by white arrows.

[0272] Figure 5 provides a strong illustration (A v B) of the progress made with this invention. The EM image of (A) is indicative of a PVC Needle Complexes / Nanosyringes derived from a protocol using ‘ultracentrifugation’, demonstrating the presence of a mixed population of complex macromolecules (including post-contraction state complexes). Thus, (A) shows a transmission EM image of a sample prepared by methods involving ultracentrifugation (e.g. prior art methods), and (B) shows a transmission EM image of a sample purified to enrich for target pre-contraction state PVC Nanosyringes.

[0273] Figure 6 (A) shows a workflow for using a combination of PEG precipitation and TFF before AEX (e.g. as clarifying purification steps). (B) shows a gel to demonstrate removal of host cell proteins by PEG precipitation (e.g. pre-AEX). (C) shows a gel to demonstrate removal of host cell proteins by combination of PEG and tangential flow filtration (e.g. pre-AEX).

[0274] EXAMPLES

[0275] Materials and Methods

[0276] Cloning

[0277] Construction of an inducible over-expression strains for P. luminescens TT01 PVCunit4 (chassis encoded by genes plu1667 - plu1652)

[0278] Photorhabdus strains overexpressing a PVC Needle Complex were prepared using chromosomal recombineering to place a PVC (operon) of choice (operon encoding PVCunit4 Needle Complex was used here, as an example) under the control of an arabinose inducible transcription promoter. The recombineered strains are then genetically transformed with effector expression plasmids (e.g. based on the arabinose inducible expression vector pBAD30) to facilitate PVC Needle Complex over-expression, PVC effector expression, PVC effector trans-packaging, and secretion of the whole complex simply through the addition of the arabinose sugar.

[0279] The inventors have successfully excised (cloned) the reguired expression genes from the host bacterium, Photorhabdus (e.g. which are comprised within SEQ ID NO: 17, SEQ ID NO.: 18 and / or SEQ ID NO:.19), and have devised a reliable, scalable expression system in laboratory E. coli as explained above. It has been demonstrated that trans-expression on separate plasmids enables incorporation of payloads (e.g. Pnf) into the Nanosyringes, creating a multi-plasmid (modular) platform.

[0280] Plasmids encoding PVC Needle Complexes were prepared using standard molecular technigues known in the art. Briefly, genomic DNA from P. asymbioticaMCC^^ (obtainable from the ATCC under accession no. ATCC 43949) was used in PCR (with appropriate primers) to amplify multiple (e.g. four) overlapping regions of the PVC operon. Overlap / extension PCR was employed to prepare a whole operon, and fused (again using overlapping PCR) into an appropriate expression vector as detailed in Figure 1 (using the primers of SEQ ID NO: 24 - SEQ ID NO: 29). Briefly: four overlapping PVC fragments (generated with primers of SEQ ID NO: 24 (F1) and SEQ ID NO: 28 (R1); SEQ ID NO: 25 (F2) and SEQ ID NO: 29 (R2); SEQ ID NO: 26 (F3) and SEQ ID NO: 30 (R3); and SEQ ID NO: 27 (F4) and SEQ ID NO: 31 (R4), respectively) were made covering the PVC operon (e.g. of SEQ ID NO: 17). The target cloning vector was cut at the required insertion site. These 5 DNA fragments were then assembled by overlapping PCR (using primers of SEQ ID NO: 24 and of SEQ ID NO: 31), and the resulting fragment was ligated into the cloning vector. Products were transformed into laboratory E. coli and recovered with vector marker selection (e.g. due to ampicillin resistance).

[0281] The operons are typically operably linked to an inducible promoter (e.g. arabinose inducible, and / or IPTG inducible) as is known in the art. This is generally achieved by cloning into pBAD family plasmids (inducible via arabinose) (Invitrogen, catalog number: V43001) and pVTRa (inducible via IPTG) (Biomedal, S.L.) vectors (although any combination of compatible expression vector systems should suffice).

[0282] A PVC Needle Complex can be expressed independently of the payload (toxin), and vice versa. Separate expression vectors (e.g. having differing inducible promoters) may harbour the PVC Needle Complex and the payload, respectively.

[0283] Expression (e.g. laboratory scale expression of PVC Needle Complexes in E. coli

[0284] A typical process to express a PVC Needle Complex in a 1 L culture of an E. coli expression strain (transformed with an appropriate expression vector / cosmid) is as follows:

[0285] 1- An overnight culture of the bacteria (transformed with PVC Needle Complex expression vector) is prepared by picking a colony from a plate and inoculating 100 mL of LB media. The culture is grown at 37°C with shaking. a. Typically, the media may be routinely supplemented with 0.2% d-Glucose to aid repression of the genetic constructs for optimal cell health. b. The media is also supplemented with the relevant antibiotics for maintenance of the expression (PVC Needle Complex) vector. If a payload vector is also being used, the relevant antibiotic for that vector is also supplied.

[0286] 2- The next day, a 1L flask is inoculated via dilution in a 1 :100 ratio from the overnight culture. The media for the 1L flask is identical to the overnight media but typically does not contain glucose.

[0287] 3- Cultures are grown to approximately mid-to-late exponential (an QD600nm of ~0.8, more suitably about 1) at which point the plasmids are induced. a. For the PVC Needle Complex (Nanosyringe) plasmid, typically 0.2% arabinose is added to induce expression. For the payload plasmid (plasmid encoding for the payload, such as Pnf), IPTG concentrations may typically be optimised on a perprotein basis, and a typical starting figure of 0.1 mM is preferable.

[0288] 4- The cultures are returned to the incubator post-induction and cultured at 25°C until the following day.

[0289] 5- Cultures are harvested by centrifugation in appropriate centrifuges / bottles / rotors at 5000xg for 30 mins.

[0290] 6- Cell pellets are then lysed to release PVC Needle Complexes (Nanosyringes), where the purification steps outlined below can then proceed.

[0291] Transmission electron microscopy

[0292] For transmission electron microscopy (TEM) pioloform-covered 300-mesh copper grids that were coated with a fine layer of carbon were used as substrates for the protein fractions. A preferred aqueous negative stain is 2% uracyl acetate. The coated grids were exposed to UV light for 1 min immediately prior to use to ensure adequate wetting of the substrate. A 5 pl drop was applied to the TEM grid, and the protein was allowed to settle for 1 min. Liquid was absorbed with filter paper from the edge of the grid and replaced immediately with 10 pl of filtered negative stain. The drop was partially removed with filter paper, and the grids were allowed to air dry thoroughly before they were viewed with a JEOL 1200EX transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV.

[0293] Coomassie and western blotting

[0294] 1. Add 2x LDS sample buffer containing 50 mM DTT to all samples.

[0295] *Resuspend the pellet in 10 ml / g PBS or water before mixing with sample buffer (e.g. if the pellet weighed 80 mg, use 800 pl of water).

[0296] 2. Loading scheme

[0297] Marker 5 pl

[0298] Total protein (optional) 5 pl

[0299] Pellet (optional) 5 pl

[0300] Supernatant 5 pl

[0301] FT 5 pl

[0302] Wash (optional) 10 pl

[0303] E1 10 pl

[0304] E2 10 pl E3 10 pl

[0305] Example 1 - Purification of PVC Needle Complexes

[0306] An exemplary purification protocol based on anion exchange chromatography (AEX) using a monolith column will now be outlined. In addition to describing the AEX step, this protocol describes expression cell lysis, capture (e.g. pre-purification) and wash steps. The protocol is conducted over four days, simply to accommodate relevant incubation periods. The steps conducted in days 1-4 (following expression / pellet provision) will be outlined in turn.

[0307] Day 1

[0308] 1. If needed, defrost cell pellet.

[0309] 2. Resuspend the cell pellet in 5 ml / g PBS or TBS, 1 tablet / 50 ml complete EDTA-free protease inhibitor, small spatula tip of lyophilized DNase I (for up to 300 ml, product ID: DN25, Sigma). A more particular amount of DNase I is 50 pg / ml. Lysozyme can also be included at 200 pg / ml .

[0310] 3. Lyse cells using GEA Lab Homogenizer Panda 2000, 3x -900 bar. Keep -100 ul sample for QC (total protein). Alternatively can use detergent buffer (B-PER (ThermoFisher), protease inhibitor, DNase I, lysozyme), mostly for BSL-2 material. Two other successfully used lysis conditions include: (i) use of lysis buffer 50 mM Tris, 150 mM NaCI, pH 8, 0.1 mg / mL Lysozyme, a suitable DNase / endonuclease (e.g. 50 U / mL DENARASE), and lyse 3x 600 bar, GEA Lab Homogenizer, and incubate at room temperature for 30 min; and (ii) use of lysis buffer 50 mM Tris, 150 ml NaCI, pH 8.0, 0.1 mg / ml lysozyme, 50 U / mL Denarase, 0.1% EcoSurf-6, and incubate at room temperature for 30 min.

[0311] 4. Incubate lysate at 37C, 30 min, gentle shaking.

[0312] 5. Spin lysate at 20 000 g, 15 min. a. Beckman JXN26 high speed centrifuge with rotors JLA16.250 or JA25.5 or b. Eppendorf benchtop centrifuge with rotor 5804 / 581 OR

[0313] 6. Transfer the supernatant and spin again for another 15 min.

[0314] 7. Optional: Transfer a small amount of the original pellet into a pre-weighed Eppendorf tube. Note down the weight of the pellet for QC (pellet).

[0315] 8. Filter sterilise the supernatant with a 0.45 pm SFCA filter and a 0.22 pm SFCA filter (Use Fisherbrand / Nalgene filter units). Keep -100 ul of the filtrate for QC (supernatant).

[0316] When working with toxin loaded complexes, typical to alter simply for H&S practice: 1. If needed, defrost cell pellet.

[0317] 2. Resuspend the cell pellet in resuspension buffer (use Nalgene bottle and magnetic stirrer) o Resuspension buffer: o 5 ml / g B-PER (phosphate, Fisher Scientific), o 1 tablet / 50 ml complete EDTA-free protease inhibitor, o two small spatula tips of lyophilized DNase I (for up to 150 ml, product ID: DN25, Sigma), and two small spatula tips of lysozyme; or more precisely 50 pg / ml DNAse I and 200 pg / ml lysozyme.

[0318] 3. Incubate at 37C, 30 min, -100 rpm. Keep -100 ul sample for QC (total protein).

[0319] 4. Spin down at 20,000 xg, 10 min (Eppendorf benchtop centrifuge with rotor 5804 / 581 OR)

[0320] 5. Filter sterilise the supernatant with a 0.22 pm SFCA filter. Keep -100 ul of the filtrate for QC (supernatant).

[0321] Capture: toward the end of day 1, a clarification step in the form of anti-Flag resin preparation and binding procedure began (the C-terminus of the cap protein Pvc16 is fused to a Flag tag ensuring the purification of fully loaded and capped PVC molecules). To begin: batch incubate lysate with equilibrated anti-Flag resin (-0.5 ml / g cell pellet), overnight at 4°C rolling. In more detail:

[0322] 1. Use anti-Flag resin according to manufacturer’s instructions (GenScript, Anti- DYKDDDDK G1 Affinity Resin, #L00432). Use -14 ml bed volume (= 28 ml 50% slurry) per 30 g cell pellet.

[0323] ■ Rinse the gravity column once with water followed by buffer.

[0324] ■ Thoroughly resuspend the resin by gentle inversion and immediately load appropriate volume of the slurry into the column. Wide bore pipette tips are recommended for easy resin slurry transfer.

[0325] ■ Equilibrate the resin by washing with 3x 3 bed volumes of buffer. Allow the buffer to drain from the column by gravity; do not let the resin run dry.

[0326] 2. Transfer the anti-Flag resin to clarified supernatant (rinse the column 3x with some buffer and transfer to the supernatant, to include ALL resin).

[0327] 3. Incubate overnight, 4°C, gentle shaking or rolling.

[0328] Additionally or alternatively to use of affinity chromatography with anti-FLAG resin, successful capture / clarification has also been achieved with PEG precipitation using the following protocol: 1. After lysis and clarification add PEG6000 (or as an alternative PEG8000) at a concentration of 80g / L and 0.5 M NaCI (final concentration).

[0329] 2. Place on a shaker until the PEG is completely dissolved (vigorous shaking, up to 300 rpm).

[0330] 3. Incubate overnight at 4°C on a roller or shaker.

[0331] 4. Centrifuge samples at 8,000 xg for 20 minutes.

[0332] 5. Fully resuspend pellet in TBS + 8mM MgSO4. Use a pipette to break up the pellet into small pieces by pipetting up and down followed by 250 rpm at room temperature for 2 hrs to resuspend the pellet fully.

[0333] 6. Centrifuge at 12,000 xg for 20 minutes.

[0334] 7. Filter the supernatant with a 0.2 urn SFCA filter and where also using affinity chromatography, continue with binding to equilibrated anti-Flag resin.

[0335] Additionally or alternatively to use of affinity chromatography with anti-FLAG resin, examples of columns which may be used for capture / clarification may include CIMmultus OH, Sartorius; Sartobind Phenyl, Sartorius; CIMmultus SO3, Sartorius; CaptoCore 400 or 700, Cytiva; and ViralPolish, BioToolomics.

[0336] Day 2 - anti-flag continued

[0337] 1. Collect flow-through (FT) using a gravity column. Make sure to collect ALL resin by washing the vessel 3x with buffer. Keep -100 ul for QC (FT).

[0338] 2. Wash resin withminimum 30 bed volumes of buffer to remove any non-specific binding and LPS. Allow the column to drain completely and proceed to elution procedure. Optional: Keep -100 ul of very last millilitre for QC (wash).

[0339] 3. Close bottom of gravity column. Add 2 bed volumes of 500 ug / ml Flag peptide in buffer.

[0340] 4. Close the top of the gravity column (use additional parafilm).

[0341] 5. Incubate 1 hr, RT, rolling.

[0342] 6. Collect elution fraction (E1). Keep -200 ul sample for QC.

[0343] 7. Wash 2x with 2 bed volumes buffer and collect (E2 and E3). Keep -200 ul of each sample for QC.

[0344] 8. Measure A280 and A280 / 260 for each elution fraction using a NanoPhotometer (Implen).

[0345] 9. Pool E1-E3, add 8 mM MgSO4 and filter sterilise (0.2 urn SFCA). Measure A280 and A280 / 260 using a NanoPhotometer. Note: If elution pool is kept for over 1 week, please add 1 tablet / 50 ml complete EDTA-free protease inhibitor (Roche). Pool should be stable for at least 4-6 weeks.

[0346] Quality control: A280, A260 / 280, Coomassie, WB

[0347] As an alternative to affinity chromatography, PEG or a combination of PEG and TFF were used to remove host cell protein - see Figure 6.

[0348] Day 3 - Anion exchange chromatography

[0349] Run on Akta Pure 25 (Cytiva), which has a built in conductivity monitor.

[0350] • Column: o CIMmultus® QA 1 mL Monolithic Column (2 pm), Item No.: 311.5113-2, Sartorius o CIMmultus® QA 4 mL Monolithic Column (2 pm), Item No.: 904.5113-2, Sartorius, or o CIMmultus® QA 8 mL Monolithic Column (2 pm), Item No.: 411.5113-2, Sartorius

[0351] • Buffer (buffers are Endotoxin free): o Endotoxin free water o Running buffer: 11.9 mM phosphate (10 mM Na2HPO4,1.9 mM KH2PO4), 137 mM NaCI, 2.7 mM KCI, pH 7.4 (also used as loading buffer and equilibration buffer, can also be referred to as PBS) o Wash buffer: 11.9 mM phosphate, 137 mM NaCI, 2.7 mM KCI, 20 mM CHAPS, 20 mM EDTA, pH 7.4 o Elution buffer (e.g. input elution buffer): 11.9 mM phosphate (10 mM Na2HPO4,1.9 mM KH2PO4), 2 M NaCI, 2.7 mM KCI, pH 7.4 o Note: this procedure has also been successfully performed by replacing the running buffer (and loading and equilibration buffers) mentioned above with the following buffer: 20 mM Tris; 137 mM NaCI; 8 mM MgSO4

[0352] • Anion-exchange procedure

[0353] 1. Wash column with 10 CV (column volumes) of Endotoxin free water.

[0354] 2. Equilibrate column with 10 CV running buffer.

[0355] 3. Load sample.

[0356] 4. Wash off unbound material with 10CV running buffer.

[0357] 5. Remove Endotoxin using 30 CV of wash buffer.

[0358] 6. Elute Nanosyringes in 0-30% elution buffer (e.g. input elution buffer) gradient over 10 CV. Collect 0.5 ml fractions.

[0359] 7. Remove any tightly bound material using 5 CV of 100% elution buffer.

[0360] 8. Analyse fractions and pool by Coomassie, Western blot and EM (electron microscopy)

[0361] 9. Pool peak fractions and measure A280 and A280 / 260 using a NanoPhotometer (Implen).

[0362] Note: this procedure has also been successfully performed by performing step 4 using a first wash buffer described herein and performing step 5 with a second wash buffer described here.

[0363] For Figure 3, o D4 (first peak): short Nanosyringes (e.g. 30-50 nm) o D11 (second peak) and E4 (third peak): full-length Nanosyringes (pre-contraction) o E11 (fourth peak): contracted Nanosyringes

[0364] For Figure 4, fractions indicated above and Al EX pool were analysed by EM, in short: o FT (flow through of load): no Nanosyringes, mostly aggregate o E1 (first peak): short Nanosyringes (e.g. 30-50 nm) o E2 (fraction before AIEX pool):mix of short and full-length Nanosyringes o AIEX pool: full-length Nanosyringes (pre-contraction) o E3-E4: mostly full-length Nanosyringes o E5: contracted Nanosyringes o E6 (far end of elution): contracted (inactive) Nanosyringes.

[0365] The NaCI concentration / conductivity at the beginning of E1 pool: 14.4% (equates to 0.28M NaCI), 34 mS / cm.

[0366] The NaCI concentration / conductivity at the beginning of elution AIEX pool: 18.5% (equates to 0.37M NaCI), 40.6 mS / cm.

[0367] The NaCI concentration / conductivity at the peak of AIEX pool: 20% (equates to 0.4M NaCI), 43 mS / cm.

[0368] The NaCI concentration / conductivity at the end of AIEX pool: just below 24% (equates to 0.47M), 48 mS / cm.

[0369] The NaCI concentration / conductivity at start of E5: 24% (equates to 0.48M), 49 mS / cm.

[0370] Quality control: A280, A260 / 280, Coomassie, WB, Endotoxin quantification.

[0371] Day 4

[0372] Concentrate: Concentrate to 2 mg / ml or more suitably 0.5 mg / ml using Amicon (100K MWCO), filter sterilize (0.2 pm SFCA). Tangential flow filtration has also been used successfully as an alternative to Amicon filters.

[0373] Quality control: A280, A260 / 280, Coomassie, WB, Endotoxin quantification (<5 Ell / rnl and lower is possible), EM

[0374] Storage:

[0375] Store at 4°C (native Nanosyringes) or

[0376] - Add freezing buffer (PBS, 200-350 mM NaCI, 8 mM MgSO4, 50% glycerol (v / v)) if needed (targeted Nanosyringes) to a final concentration of 5% glycerol, freeze in liquid nitrogen and store at -80°C

[0377] Thus, store Nanosyringes at 0.4 mg / ml or more suitably 0.5 mg / ml in phosphate buffer containing 8 mM MgSO4, 350 mM NaCI, 200-600 pl / aliquots. After addition of the additive (see table, final concentrations are given) aliquots are flash frozen in liquid nitrogen and stored at -80°C. If needed samples are defrosted at room temperature (19-25°C)

[0378] The table below shows that such freezing storage conditions performed the best:

[0379] Example 2 (reference example) - using resin instead of monolith columns

[0380] This provides further evidence for the advantage of employing a monolith column (as above) in anion exchange chromatography of Nanosyringes. When passing purified Nanosyringes through columns containing beads (Sepharose, Superdex, HisTrap) we lose up to 50% protein, which we believe is due to the Nanosyringes being stuck inside the pores.

[0381] 1. 10 mg of purified Nanosyringe were loaded on a 1 ml HisTrap column to investigate protein loss using bead based matrix. That being said, Nanosyringes were not His- tagged. Expected volume of flow-through starting is 1 ml.

[0382] 2. Flow-through was collected and concentration measured.

[0383] 3. Following samples were imaged by EM to verify presence of Nanosyringes: load, flow-through, elution

[0384] • Observations: o Nanosyringes appeared late in flowthrough. Expected: 1 ml, observed ~5 ml, probably retention time was prolonged due to Nanosyringe size. o Nanosyringe yield in flow-through was 3.3 mg. Protein loss was 66%. o No Nanosyringes were found in the elution pool, but protein is coming off the column during the 1M NaOH wash during column cleaning.

[0385] SEQUENCE HOMOLOGY

[0386] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993); Align- M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0387] Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1 , and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).

[0388] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides I amino acids divided by the total number of nucleotides I amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

[0389] ALIGNMENT SCORES FOR DETERMINING SEQUENCE IDENTITY

[0390] A 4

[0391] R -1 5

[0392] N -2 0 6

[0393] D -2 -2 1 6

[0394] C 0 -3 -3 -3 9

[0395] Q -1 1 0 0 -3 5

[0396] E -1 0 0 2 -4 2 5

[0397] G 0 -2 0 -1 -3 -2 -2 6

[0398] H -2 0 1 -1 -3 0 0 -2 8

[0399] I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

[0400] L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4

[0401] K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5

[0402] M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5

[0403] F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6

[0404] P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

[0405] T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5

[0406] W-3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11

[0407] Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

[0408] V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

[0409] The percent identity is then calculated as:

[0410] Total number of identical matches x 100

[0411] [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

[0412] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an aminoterminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0413] CONSERVATIVE AMINO ACID SUBSTITUTIONS

[0414] Basic: arginine lysine histidine

[0415] Acidic: glutamic acid aspartic acid

[0416] Polar: glutamine asparagine

[0417] Hydrophobic: leucine isoleucine valine

[0418] Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine

[0419] In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.

[0420] Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991 ; Ellman et al., Methods Enzymol. 202:301, 1991 ; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271 :19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0421] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.

[0422] Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.

[0423] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0424] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0425] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

[0426] Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and / or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0427] Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.

[0428] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0429] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a payload” includes a plurality of such payloads and reference to “the payload” includes reference to one or more payloads and equivalents thereof known to those skilled in the art, and so forth.

[0430] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0431] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

[0432] SEQUENCES

[0433] The sequences of SEQ ID NO: 1-16 correspond to the sequences of PVC 1-16 in Photorhabdus asymbiotica strain ATCC43949 PVCPnf operon (e.g. are encoded by nucleic acid sequences corresponding to genes PAU_03353 to PAU_03338 of the sequence of GenBank accession no. FM162591.1).

[0434] The initial (N-terminal) methionine is optional, thus all polypeptide sequences described herein may be read in the absence of the initial (N-terminal) methionine.

[0435] SEQ ID NO: 1 (PVC1Z PVC-Pnf1) MSTSTSQIAVEYPIPVYRFIVSVGDEKIPFNSVSGLDISYDTIEYRDGVGNWFKMPGQSQSTN

[0436] ITLRKGVFPGKTELFDWINSIQLNQVEKKDITISLTNDAGTELLMTWNVSNAFPTSLTSPSFDA

[0437] TSNDIAVQEITLMADRVIMQAV

[0438] SEQ ID NO: 2 (PVC2Z PVC-Pnf2)

[0439] MTTVTSYPGVYIEELNSLALSVSNSATAVPVFAVDEQNQYISEDNAIRINSWMDYLNLIGNFN NEDKLDVSVRAYFANGGGYCYLVKTTSLEKIIPTLDDVTLLVAAGEDIKTTVDVLCQPGKGLF AVFDGPETELTI NGAEEAKQAYTATPFAAVYYPWLKADWAN I DI PPSAVM AGVYASVDLSRG VWKAPANVALKGGLEPKFLVTDELQGEYNTGRAINMIRNFSNTGTTVWGARTLEDKDNWR YVPVRRLFNSVERDIKRAMSFAMFEPNNQPTWERVRAAISNYLYSLWQQGGLAGSKEEDA

[0440] YFVQIGKGITMTQEQIDAGQMIVKVGLAAVRPAEFIILQFTQDVEQR

[0441] SEQ ID NO: 3 (PVC3 / PVC-Pnf3)

[0442] MSAILKAPGVYIEEDASLALSVSNSATAVPVFIGKFTPTWDSIQVCTRISNWLEFTSSFSLAP TVEIWQSNTESESESETYHYIETINLSPAVEALRLYFQNGGGACYIYPLNDAEDELVLAAIPE VIEQKGDITLLVCPELDLDYKTKIYGAVSSLLNDNKVGYFLIADSNDGESVSGVWNSAKAAAY YPQLETNLKFSTLPGDKDIRISGYQDDDETHKPKNLDELRTINEALAQDIDARLLEEKQRAVII PPSAAIAGIYCQTDNRRGVWKAPANVALTGIGSLLDKVDDERQGEMNDKGINVIRSFTDRGF

[0443] MVWGARTCVDAANISWRYIPVRRLFNSVERDIRQALRAVLFETNSQPTWVRAKAAVDQYLY TLWQKNALMGARPEEAYFVQIGQDITMSEADIKQGKMIMTVGLAAVRPAEFIILQFTQDVVQ

[0444] SEQ ID NO: 4 (PVC4 / PVC-Pnf4)

[0445] MLTVPTLTILEEVMMMERLQPGVTLTESIITMGQQEIPSAVPVFIGYTVRYPEQSEASVRIDSL AEYTSLFGDDHVMMFAVRHYFDNGGQQAFVLPLKDNMPSVEMTTAEAENLIAALRSATVSE AIGGHSQITLILVPDMARLNDSDIDDSSTQVSLWSQGWEALLQLSQVRPNLFVLLDAPDNVE QAQKCMTTLSSDYRQWGAAYWPRLETTYQKEISGKDNESQGIFQGTVLSPTAAVAAVIQRT DNDAGVWKAPANIALSQVIRPVKSYLQGSVLFNSSGTSLNVIRSFPGKGIRVWGCRTLENTD

[0446] NTQWRYLQTRRLVSYVTAHLTQLARMYVFEPNNELTWMKLKGQSYNWLRQLWLQGGLYG SQEDEAFNILLGVNETMTEDDVRAGKMIMKVELAVLFPAEFIEISLVFNTQTEALS

[0447] SEQ ID NO: 5 (PVC5 / PVC-Pnf5)

[0448] MNDYYTPVVSHRFMASFIFNRIPDPLDIRFQRISGLSRELQVTQYSEGGENARNNYLAEKIQ HGTLTLERGVMTVSPLTWM FDRVLSGEKI AYADWVM LLN ENSLPLSSWTLSNALPVRWQT SDFDANSNAILVNTLELRYQDMRWLGVKI SEQ ID NO: 6 (PVC6Z PVC-Pnf6)

[0449] MTVEIRELLIQAKVVPSTRPTESERQNHSLIQESLDEATWVETIKREVLAALRDEEGWRP

[0450] SEQ ID NO: 7 (PVC7 / PVC-Pnf7)

[0451] MSLI ERGLAKLTI NAYKDREGKI RAGTLQAMYN PDSLQLDYQTDYQQSQAI NSEKQSSI YVQ AKPAGLSLELIFDATMPGNKTPIEEQLMQLKQLCSVDATSNETRFLQVKWGKMRWESRGYF AGRAKSLSVNYTLFDRDATPLRVRVILALVADESLVLQETEQNLQSPAKIALRIQDGVSLALM

[0452] AASTASTLSGGVDYLTLAWQNGLDNLNGFVPGEILQATRGDES

[0453] SEQ ID NO: 8 (PVC8 / PVC-Pnf8-Spike tip(VgrG)

[0454] MSHQLKIIADGKALSLLAAVDVDTCYRVNSIPSATLKLSVPDRPLSSFSQTDVQTELAHCQVG

[0455] KTLRLELIDGSKKWVLFNGLITRKALRIKNKQLLLTLWKHRLQLMVDTQHSQLFKDKSEKAIL

[0456] STLLNQTGINARFGKIAALDQKHEQMVQFRCSDWHFLLCRLSATGAWLLPAIEDVQFVQPD

[0457] ALKSNSAYTLKSRGDENKDIWKDAYWQFDNQINPALLEVSGWDISKQQVQSGGRYGKIAL

[0458] GKAALSPDGLASLNKTGWDICYSSPLTTQESGYLAQGLLLNQRISGVTGEFLLKGDGRYQL

[0459] GDNIQLTGFGSQLDGTASITEVRHRLNRRIDWETTVSIGLQHEYLPILPDAPELHIATVAKYQ QDSAVLNRIPIILPVLNRPNEFLWARLGKPYASHESGFCFYPEPGDEVIIGFFENDPRYPVILG AMHNPKNKAPFEPTQDNREKVLIVKKGEAQQQLVIDGKEKMIRINAGENQIMLQQDKDISLS

[0460] TKKELTLKAQTM NATM DKSLAMSGKNSVEI KGAKI N LTQ

[0461] SEQ ID NO: 9 (PVC9 / PVC-Pnf9-Baseplate wedge)

[0462] MENQILTQLYGRGWAFPPVFSLEKGVEMAEGAEDVRQSLQILFSTEPGERLMRENYGCGL NDFMFENIRNELIAEIESHIHDNVLRYEPRADMTDIQVRQSPGMGNTLQVQVMYRLRGSDIN

[0463] QQIQGVLALSEGRVTEVV

[0464] SEQ ID NO: 10 (PVC10 / PVC-Pnf10)

[0465] MSEAIWDGDVLQFDPNFGNRQVTVPSPGKISGTGHAQVSGKKVCILGDEKQVRVSATYITT

[0466] THTTPGTGTITISALDAGQQALQCTSGAALIIKGQQFTAMFTPELPAMNNTVTPPQPDVTTPS SGKGRFITQQNFATVN

[0467] SEQ ID NO: 11 (PVC11 / PVC-Pnf11 )

[0468] MELNELTNKLSNLVPMTDFKLDNRASLQLLKYIEAYTKIIPFNSGDKYWNDFFFMSGNTPEKL

[0469] AKLYQKEIEPNGELLPQQAFLLAVLRLLETPISLLNVLPAAHRELYYRELLGLSSHAAQPDQV

[0470] ALSMELNSTVMEQLLPEGTLFEAGQDEQGNALQYALDASLLANRGYISDLRWLRNDGEKQ

[0471] WVTSAPWDLQAQVSLPSDGIRLFGKTNSDQQVFGGVLITSSLLAMEAGIRKIIVTFEQEMNT QELVAQVSSGNQWLTLTSEVNKKEVTLTLSDKEPAISAPEDLDNLFFTQPVLRLQGKDSQAL PEVTGISVSEKDDTKDTSFEMYHLTPFGYSSDIEPLEENPALYLGFTDVKPGQTLALYWKLK

[0472] SPQQPTVSWYYLDQHNQWAELDSWVSDGTQNLYQDGTWHVELPVDASNQAEQMPVGRY

[0473] WLRAVVEVPAHEGALGKAPWLYGLIYNAMTATLVNVDSISDSHFLTPLPASSIQRPVEPIIVL

[0474] ASVNQPWASWGGRIPESYSAFFERIAQNLSHRNRSLTWGNMVTLLKERYVSIFDVKYPGND

[0475] ELTRVPALEQQQLTVIPANRYNDSDDSLRPVLNPARLQEMADWLQQKDSPWASIEVRNPEY

[0476] LDVKIHYEVIFKPDVNEDFGYRQLQQQLCEVYMPWSIDEQRPWLNNSINYFQLLATIQQQP

[0477] LVERVTRLTLHRADSSDESDGTASVEAKDNEVLILVWEEDDNLQYRGNDYE

[0478] SEQ ID NO: 12 (PVC12Z PVC-Pnf12-GGDEF)

[0479] MSNQDALFHSVKDDIHFDTLLEQAHQVIEKQAEKLWSDTAEHDPGITFLQGISYGVSDLAYR

[0480] HTLPLKDLLTPAPDEQQQEGIFPAEFGPHNTLTCGPVTADDYRKALLDLHSSDSLDGTQQD

[0481] EGDFLFRSVQLVREPEKQRYTYWYDATKREYSFVNSEGAKEFTLRGNYWLYLEPTRWTQG

[0482] NIAAATRQLTEFLTKNRNIGESVSNIIWLQPVDLPLLLDVELDDDVGAQDVPGIFAAVYSTAE

[0483] QYLMPGAQRYRTEVLQNAGMSNDQIFEGPLLEHGWIPELPAARDYTQRLTLNLSRLVNSLL

[0484] EIEGIKHVNRLRLDDSFDKTAIEPVKGDTWSWSIKEGYYPRLWGEDPLNQLAQQNGPLRVIA

[0485] KGGISVSVSKEQIQASLPSQSLIQNEPVILAYGQHRDVGSYYPVSDTLPPCYGLQHSLSESE

[0486] HLLPLHQFMLPFEQLLACGCQQIAMLPRLLAFQREGYEVWGDQWPFKSGSVNDDAHQDYA

[0487] PALKDLLGQIALDSDHELDIINYLLGYFGTQRAPRTFTTQLDDFRAVQQGYLAQQPTLTYHRS

[0488] NIRIDQVSSLQKRIAARMGLGGELFKPQPDLSQLPFYLIEHRALLPVKPNSQFDKEQKPASVT

[0489] EEGGSQTGQHYWIEQKGIDGKLTQGQVINLILYEGEQGETQFTIRGQMVFKTEGDKFWLD

[0490] VNNSAQLEYNLARVMTAAKASKLFWQNSPVWMEDMGYRLAYASDQSSLPVNQRRLTRTV

[0491] QTPFPPMWVGSEITLLKQVGIVNLKKAESEKLYAKVVSFDRIEGTLIIERLGNSTLAFPTSEE

[0492] AWRYSWYFSGEKYERTDRFSFVISWVNSDLIKLPGVDPYKLEEWVKETILTEFPAHISMIIH

[0493] WMDREAFLNFANTYQRWQNNGTPLGDAAYSILESLTLGKLPSALKGVGTMRIATSSQREEV

[0494] VGSNGDQWNTDGITQNELFYVPKES

[0495] SEQ ID NO: 13 (PVC13 / PVC-Pnf13)

[0496] MNETRYNATVQEQQTLSNPKAVGPDIDKLKDKFKEGSIPLQTDFNELIDIADIGRKACGQAP

[0497] QQNGPGEGLKLADDGTLNLKIGTFSNKDFSPLILKDDVLSVDLGSGLTNETNGICVGQGDGI

[0498] TVNTSNVAVKQGNGISVTSSGGVAVKVSANKGLSVDSSGVAVKVNTDKGISVDGNGVAVKV

[0499] NTSKGISVDNTGVAVIANASKGISVDGSGVAVIANTSKGISVDGSGVAVIANTSKGISVDNTG

[0500] VAVIANASKGISVDGSGVAVIANTSKGISVDGSGVAVIANTSKGISVDSSGVAVKVKANGGIK

[0501] VDANGVAIDPNNVLPKGVIVMFSGSTAPTGWALCDGNNGTPNLIDRFILGGKGTDINGVSTN

[0502] TASGTKNSKLFDFSSDEATLTIDGKTLGRALSLQQIPNHAHFSGIIMDTEKVNYYGSKKITTNV

[0503] WGVTTGDNTSVRYIYKSSGVLDSNNNVSNSTLGGNSLQTHDHDIKITGTGKHSHKNKVTVP

[0504] YYILAFIIKL SEQ ID NO: 14 (PVC14Z PVC-Pnf14)

[0505] MTSEPNLLNRITITIEANNQQVARKVLHGSLLNQANINKLFNSYFNEYEINRGVYLETLILNLGT

[0506] INFHDFNSLFPTLLKAALNKEFSQYQINNHREEMLFNETISNQATDKSYIFGDNKLIDAENFIH

[0507] FLYQKHSTLNLVEAMGNNGIEKLTNQLTQIENKFALLLAKSCLSEEGLKRLLAIKQPDLLIAINR

[0508] RLSERINRPQYQEKLVSCGQLIFSALGYIQQYNIQEIPKPDEKVIARITTELNNNGLLNTIPIITL FRQSGINDSSLNDWLKKIWQVRSISQLCRKYLSAKEYQYLSEHFVSKSVDKNRYDEEPVNQ

[0509] SILSRLNNNSIKEGNNHSQLCTLSRLYSEPVVLPEQTILRQVSNTVDQSILSRLNNASIKEGNN

[0510] QSQLRTLSRLYSEPVALPEQTI PRQVSNTGI LI LWPM LPTLFNQLGLLEKKKFI H RQAQFN AV DFLDYLIWGTEDVKVERKVLNNVLCGLMADEITEPMPIEPEKQWIIIQWLDAIISQLSGWKKLS

[0511] RNDVRQLFLQRPGELLINEQEIKITIQQQPFDALLTDWPWPMNMACFSWLSQPLTITWL

[0512] SEQ ID NO: 15 (PVC15 / PVC-Pnf15-ATPase)

[0513] MNISPVFYDSLNQDNDRDLSFLFSELERIDLALQHHFYCVESQRSELLDEFLLTEAEVVTRLD KPLGKPHWINDDYLAISQKGNVSLMAASRLMDLIERFELTDFERDVLLLGLLPHFDSRYYRLF

[0514] SLIQGGQQGRLPSFALALELFCHSALEKQVQQASFLHRAPLMGCQLLSIDTSQKTLAWLQTP FITDSGVYHFLLGHHYIMPALEHCAEWLTPTGIGCYPEGLKQVLGNVLLSDNDNIRPIVLLRG

[0515] MAGSARAYTITNMMASEGKQTLLVDISKLADSDEKNIILQIKHILRETRMHGACLLLRNFCLLV EQNKQLLDSLSELLNQPELRIVCLIEPYSPLVWLKKIPVLLIEMPLLTPAEKARLLIASLPDNCS

[0516] EDIDTITLSQRYTFNPETLPLILQEAQLYQQQRDPLDILQQCDIRQALNLRAQQNFGQLAQRII PKRSLKDLLVSDEIAQQLREILIAIKYREQVLAGGFKDKIAYGTGISALFYGDSGTGKTMAAEV

[0517] IADHIGVDLIKVDLSTVVNKYIGETEKNLSRIFDLAEQDAGVLFFDEADALFGKRSETKDSQDR HANIEVSYLLQRLENYPGLVILSTNNRGHLDSAFNRRFTFITRFTYPDEKIRKKMWQEIWPRN

[0518] IKISEDIDFNELAQRTSVTGANIRNIALLSSFFASEQGNDEVSNENIEIALKRELAKVGRLTF

[0519] SEQ ID NO: 16 (PVC16 / PVC-Pnf16) - includes FLAG tag at C-terminus

[0520] MLNTQTIIDVNKAMDAMLRAYLNQDIAIRFDLPELDTMQSDAMVSIFLYDIHEDLQLRSAESR GFDVYAGRLLPGWVNIKCNYLITYWEASKPATDASSPDSQPDNQAIQVMSQVLNALINNRQ

[0521] LAGIPGAYTQVVPPKESLNSLGNFWQSLGNRPRLSLNYSVTVPVSLNDGQDSATPVTAVSS TVEQTASLSQEVVSHALRELLITELGGGEDNRLVLSKVELSAVKETMTQDSPAQMIILLSVSG

[0522] ITRQEYLKEIDNIFDRWVNNAEVITTIDDCGIRIESITKDNLVGIDYKDDDDK

[0523] SEQ ID NO: 17 (Photorhabdus asymbiotica strain ATCC43949 PVCPnf operon, pyc1 - pyc16; e.g. corresponding to genes PAU 03353 to PAU 03338 of the sequence of GenBank accession no. FM162591.1)

[0524] ATGTCTACAAGTACATCTCAAATTGCGGTTGAATATCCTATTCCTGTCTATCGCTTTATTGTTTCTGTCGGAGA

[0525] TGAGAAAATTCCATTTAATAGTGTTTCAGGATTAGATATTAGTTATGACACCATTGAATACCGAGATGGTGTTG GTAATTGGTTCAAAATGCCGGGTCAGAGTCAGAGCACTAATATCACCTTGCGTAAAGGCGTTTTCCCGGGGA AAACAGAACTGTTTGATTGGATTAACTCTATTCAGCTTAATCAGGTAGAGAAAAAGGATATTACCATCAGTTTA ACTAATGATGCAGGTACCGAATTATTAATGACCTGGAATGTTTCTAATGCTTTTCCCACTTCATTGACTTCACC TTCATTTGATGCCACCAGTAATGATATTGCAGTACAGGAAATTACGCTGATGGCAGATCGGGTGATTATGCAG GCTGTTTGAAGCATTGATATTTAATCATCTCATATAAGGGAACTTTTATGACAACCGTTACCAGTTATCCTGGC GTTTATATTGAAGAATTAAATAGCCTGGCCTTGTCAGTTTCAAATAGCGCCACAGCGGTTCCTGTTTTTGCTGT GGACGAACAAAACCAATATATTAGTGAAGATAATGCAATCCGTATTAATTCGTGGATGGATTATCTTAATCTGA TTGGCAATTTTAATAATGAAGACAAATTAGATGTTTCTGTGCGTGCTTATTTTGCCAATGGAGGTGGATATTGT TATCTCGTCAAAACAACGAGTTTAGAAAAAATTATTCCAACCTTGGATGATGTAACCTTATTGGTTGCTGCGG GCGAAGATATTAAAACGACAGTAGATGTTTTATGTCAGCCAGGAAAAGGGTTATTCGCAGTCTTTGATGGCCC TGAAACAGAGTTGACTATCAACGGTGCGGAAGAGGCAAAACAAGCCTATACCGCCACACCATTCGCTGCGGT TTATTATCCTTGGTTGAAAGCGGATTGGGCTAACATAGATATTCCACCCAGTGCAGTGATGGCGGGAGTTTAT GCATCGGTGGATTTATCCCGTGGTGTATGGAAAGCGCCTGCCAATGTTGCGTTGAAAGGGGGCCTGGAACC TAAATTTTTAGTCACGGATGAATTGCAGGGTGAATATAACACTGGCCGCGCTATCAATATGATTCGTAATTTCA GTAACACAGGTACTACGGTTTGGGGTGCAAGAACCCTGGAAGATAAAGACAATTGGCGTTATGTTCCAGTGC GACGCTTGTTTAATTCTGTGGAGCGGGATATCAAGCGTGCCATGAGCTTTGCTATGTTCGAGCCTAATAATCA GCCTACTTGGGAGCGGGTACGGGCGGCGATTAGCAACTACCTTTATAGCCTGTGGCAACAGGGGGGATTAG CTGGCAGCAAAGAAGAAGACGCTTATTTTGTGCAAATTGGTAAAGGTATAACGATGACACAGGAGCAGATTG ATGCAGGGCAAATGATTGTTAAAGTCGGTTTGGCTGCTGTACGGCCTGCGGAATTTATCATTCTCCAGTTTAC GCAAGATGTAGAACAGCGTTAATCATATGATTATGAGGAGTTATCATGTCTGCTATTCTGAAAGCGCCTGGCG TTTATATTGAAGAAGACGCTTCCCTAGCGTTGTCTGTCAGTAACAGCGCGACTGCCGTGCCTGTTTTTATCGG AAAATTTACTCCGACAGTGGTTGATTCAATCCAAGTCTGTACCCGTATCAGCAACTGGCTTGAATTCACTTCC TCTTTTTCCCTAGCTCCAACAGTTGAGATTGTTGTCCAATCTAACACTGAATCTGAATCTGAATCTGAAACTTA CCACTATATTGAGACAATCAATTTATCTCCAGCTGTGGAAGCATTGCGACTCTATTTTCAAAATGGCGGAGGA GCTTGCTATATCTACCCATTAAATGATGCTGAAGATGAATTGGTTCTGGCGGCCATACCAGAAGTCATTGAAC AGAAAGGTGATATTACTCTGTTGGTTTGCCCGGAACTCGATCTGGATTACAAAACTAAGATCTATGGCGCAGT GAGCTCACTGTTGAATGATAACAAAGTGGGCTATTTCCTGATTGCGGATAGCAATGATGGAGAATCTGTGTCA GGAGTATGGAATAGTGCTAAGGCCGCCGCCTATTATCCCCAGTTGGAAACTAACCTAAAATTTTCCACGTTGC CTGGGGATAAGGACATTCGTATCAGCGGTTATCAGGATGATGATGAAACACATAAACCGAAAAACTTGGATG AGCTCAGGACAATCAACGAGGCGTTGGCACAGGATATTGATGCAAGATTGCTCGAGGAGAAACAACGTGCT GTCATCATTCCGCCAAGTGCTGCCATTGCGGGCATTTATTGCCAAACGGATAATCGTCGCGGTGTTTGGAAA GCGCCAGCCAACGTTGCGCTCACAGGGATCGGGAGTTTGCTTGATAAGGTAGACGATGAACGGCAGGGAGA GATGAATGACAAGGGAATCAATGTCATCCGTTCATTTACCGACCGTGGTTTTATGGTCTGGGGAGCCCGTAC TTGTGTGGACGCTGCCAACATCAGCTGGCGTTATATTCCTGTTCGTCGCCTGTTCAATTCCGTTGAACGAGAT ATCCGCCAGGCGCTGCGCGCTGTGTTGTTTGAAACTAATAGTCAGCCTACCTGGGTACGTGCTAAGGCTGC CGTTGATCAATATCTTTATACCCTTTGGCAGAAAAATGCATTGATGGGTGCTCGCCCGGAAGAAGCTTATTTT GTGCAAATTGGTCAGGATATCACCATGTCCGAGGCTGATATTAAACAGGGTAAGATGATCATGACTGTTGGTT TGGCAGCAGTGCGGCCAGCTGAGTTCATCATTCTGCAATTTACGCAGGATGTTGTTCAGTAATCTCCATGACT AAACGCCAGGCACTGTATTGACAGTGCCTACTCTAACCATCTTGGAGGAGGTGATGATGATGGAGAGACTCC AACCGGGTGTGACTTTAACAGAAAGTATAATCACGATGGGTCAGCAAGAGATACCCAGTGCTGTGCCGGTGT TTATTGGTTACACCGTTCGTTATCCGGAACAATCGGAAGCATCAGTCCGTATCGACAGTTTGGCCGAGTATAC CAGCCTGTTTGGTGACGACCATGTGATGATGTTTGCTGTCAGGCACTATTTTGATAATGGCGGGCAACAGGC ATTTGTTTTACCCCTGAAGGACAATATGCCATCAGTGGAGATGACCACAGCTGAAGCGGAAAATCTGATAGC CGCATTGCGCTCTGCTACGGTTAGCGAAGCCATTGGTGGGCATAGTCAGATTACACTGATTTTGGTACCGGA TATGGCTCGGCTTAATGACAGTGATATTGATGACTCCTCAACCCAGGTAAGCCTGTGGTCCCAAGGCTGGGA GGCGCTGCTGCAATTGAGTCAGGTTAGGCCCAACCTCTTTGTGCTGTTAGATGCGCCGGATAATGTTGAACA GGCGCAGAAGTGTATGACAACGCTATCGTCAGATTATCGTCAATGGGGGGCAGCATATTGGCCTCGTCTGG AAACTACCTATCAGAAAGAAATATCTGGCAAGGACAATGAATCTCAGGGAATTTTCCAGGGGACTGTTCTGTC ACCCACAGCCGCGGTCGCAGCGGTAATTCAACGCACGGATAACGACGCGGGTGTTTGGAAAGCACCGGCC AATATTGCCTTATCCCAGGTTATTCGACCTGTTAAATCTTATCTTCAGGGAAGTGTACTGTTTAACAGCAGCG GCACTTCGCTCAATGTGATCCGCAGTTTCCCAGGTAAGGGCATACGGGTATGGGGATGCCGCACTCTGGAA AACACGGATAATACGCAGTGGCGCTATCTGCAAACACGTCGGCTGGTTTCCTATGTAACAGCGCATTTGACC CAATTGGCTCGCATGTATGTCTTTGAGCCAAATAATGAACTTACCTGGATGAAGTTAAAAGGACAAAGTTACA ACTGGTTACGGCAATTATGGTTGCAGGGTGGCTTGTATGGTTCACAGGAGGATGAGGCATTTAACATTCTGT TAGGCGTAAACGAGACGATGACTGAGGATGATGTTCGTGCAGGAAAAATGATCATGAAAGTTGAGTTGGCTG TGTTGTTTCCTGCCGAATTTATTGAGATCAGTTTGGTGTTTAATACCCAAACAGAGGCGCTGTCTTAAGAAGG AAAAAGTACGATGAACGATTATTACACACCCGTGGTATCCCATCGTTTTATGGCGAGTTTTATTTTTAACCGCA TTCCCGATCCGCTGGATATTCGTTTTCAGCGTATCTCTGGCCTTAGTCGGGAACTACAGGTGACTCAGTACA GTGAGGGAGGAGAAAATGCCCGTAATAACTATTTAGCTGAGAAAATCCAACACGGTACGTTGACTTTGGAAC GGGGCGTGATGACAGTCTCGCCATTGACCTGGATGTTTGATCGGGTATTGAGTGGTGAAAAAATCGCTTATG CCGATGTGGTGGTGATGCTACTGAATGAAAATTCACTGCCATTGTCCAGTTGGACGTTGAGCAATGCGCTGC CGGTACGCTGGCAAACCAGCGACTTTGACGCTAACAGCAATGCCATATTGGTGAATACCCTTGAATTGCGTT ACCAGGATATGCGCTGGCTTGGAGTCAAAATATGACAGTAGAAATCAGAGAGTTACTTATCCAGGCAAAGGT AGTGCCATCAACACGACCGACTGAATCAGAACGGCAAAACCATTCTTTGATACAGGAAAGTCTGGATGAGGC GACTTGGGTGGAAACGATAAAACGCGAAGTGTTGGCCGCATTACGCGATGAGGAAGGGTGGCGTCCATGAG TCTGATTGAACGTGGTTTAGCTAAGCTGACAATTAATGCTTATAAGGATAGGGAAGGGAAGATACGGGCAGG AACGTTGCAGGCCATGTATAACCCTGACTCCTTGCAACTGGATTACCAAACGGATTATCAGCAATCCCAAGC

[0526] GATTAATAGCGAAAAGCAAAGTAGCATTTATGTACAGGCCAAGCCCGCAGGGTTATCACTTGAATTAATTTTT

[0527] GATGCCACGATGCCGGGTAACAAAACCCCCATTGAAGAGCAGCTCATGCAGCTCAAGCAACTGTGCAGTGT

[0528] GGATGCAACCAGTAACGAGACGCGATTCCTGCAAGTTAAATGGGGCAAAATGCGTTGGGAAAGTCGGGGTT

[0529] ACTTTGCTGGCAGGGCCAAGAGTTTGTCTGTGAATTACACTTTGTTTGATCGTGATGCGACTCCCTTGAGGGT

[0530] ACGGGTAATATTGGCATTAGTGGCTGATGAAAGTCTGGTGTTGCAGGAGACTGAACAAAATCTGCAATCTCC

[0531] GGCAAAAATCGCATTACGCATACAGGATGGGGTATCTCTGGCTCTGATGGCAGCCAGTACGGCATCAACATT

[0532] GTCAGGCGGTGTGGATTATCTGACGCTGGCCTGGCAAAACGGTCTGGATAATCTCAATGGGTTCGTTCCGG

[0533] GTGAAATATTGCAGGCCACCAGGGGAGACGAATCATGAGCCACCAACTGAAAATTATTGCAGATGGTAAGGC

[0534] ACTGTCACTTTTGGCCGCGGTAGATGTGGACACCTGTTATCGGGTTAACAGTATACCTTCTGCGACATTGAAA

[0535] CTGAGCGTACCGGATAGGCCACTCTCTTCTTTCAGTCAGACGGATGTTCAGACAGAACTGGCCCACTGTCAG

[0536] GTAGGGAAAACCCTGCGTCTGGAATTGATTGATGGTAGCAAAAAATGGGTGCTGTTTAATGGTCTTATTACCC

[0537] GTAAGGCTCTGAGAATTAAGAATAAGCAATTATTGCTCACTCTGGTTGTCAAGCATCGGTTGCAACTGATGGT

[0538] GGATACCCAGCATTCACAGCTGTTTAAAGACAAAAGCGAAAAAGCGATCTTAAGCACGCTATTGAATCAGACC

[0539] GGAATCAATGCTCGCTTCGGAAAGATAGCGGCGTTAGATCAAAAGCATGAACAGATGGTGCAATTTCGTTGT

[0540] TCAGACTGGCATTTTCTGTTGTGCCGACTGTCGGCAACCGGTGCATGGTTGTTACCTGCCATAGAAGACGTT

[0541] CAGTTTGTTCAACCTGATGCTCTGAAATCAAACTCAGCCTATACCTTGAAGAGCAGGGGGGATGAGAACAAA

[0542] GACATCGTTGTCAAGGATGCTTACTGGCAGTTTGACAATCAAATCAACCCCGCTTTGCTGGAAGTCAGTGGC

[0543] TGGGATATCAGTAAGCAGCAGGTACAATCAGGCGGTCGCTACGGAAAAATCGCGTTGGGTAAGGCGGCACT

[0544] CTCTCCTGATGGATTGGCATCCCTTAATAAAACGGGTTGGGACATTTGTTATAGCAGTCCGTTAACAACCCAG

[0545] GAAAGCGGTTATCTGGCACAGGGATTATTGCTTAACCAGCGCATTTCTGGGGTGACAGGAGAATTTTTGCTC

[0546] AAAGGAGATGGGCGTTACCAGTTGGGAGACAACATTCAGCTGACTGGATTTGGTTCACAGTTAGATGGTACG

[0547] GCAAGCATTACTGAGGTTCGCCACCGTCTTAATCGGCGAATTGATTGGGAAACCACGGTGAGCATTGGTTTA

[0548] CAACATGAATATTTGCCGATATTACCTGATGCTCCCGAACTACATATTGCGACAGTAGCGAAATATCAGCAGG

[0549] ACAGTGCGGTGTTAAACCGTATCCCCATTATTCTGCCGGTACTGAATCGTCCCAATGAATTTTTGTGGGCCAG

[0550] ATTGGGGAAACCTTATGCTAGCCATGAAAGCGGTTTCTGTTTTTACCCAGAGCCAGGTGACGAAGTTATTATT

[0551] GGTTTTTTTGAAAATGATCCGCGTTATCCAGTTATTTTAGGTGCTATGCATAATCCGAAAAATAAGGCCCCTTT

[0552] TGAACCAACCCAAGATAATAGGGAAAAAGTATTGATCGTTAAAAAAGGTGAAGCGCAACAACAATTAGTCATT

[0553] GATGGCAAAGAGAAAATGATCCGAATTAATGCGGGTGAAAATCAAATAATGCTTCAGCAAGATAAAGACATTT

[0554] CTCTGTCAACGAAAAAAGAATTAACACTGAAAGCGCAGACAATGAATGCCACGATGGATAAATCATTGGCAAT

[0555] GTCCGGGAAAAACAGTGTTGAAATCAAAGGCGCAAAAATTAATCTTACCCAATGAAAGGTGACGATGAATGG

[0556] AAAATCAAATACTGACACAACTCTATGGTCGTGGTTGGGCTTTTCCTCCGGTCTTTTCCCTTGAAAAGGGGGT

[0557] AGAGATGGCTGAAGGGGCGGAAGATGTGAGACAAAGTTTGCAGATTCTGTTTAGTACTGAGCCGGGGGAAC

[0558] GTCTTATGCGTGAAAATTATGGCTGCGGATTAAATGATTTTATGTTTGAAAATATCCGCAATGAACTTATTGCT

[0559] GAAATTGAATCCCATATCCATGACAACGTATTACGATATGAACCCCGGGCTGATATGACTGATATTCAGGTTC

[0560] GTCAATCCCCTGGCATGGGGAATACTTTGCAAGTGCAGGTCATGTATCGCCTGAGAGGGAGTGATATCAATC

[0561] AACAAATCCAGGGAGTACTTGCACTGAGTGAAGGCCGGGTGACGGAGGTAGTATGAGTGAAGCGATTGTGG

[0562] TGGATGGTGACGTGTTACAGTTTGATCCCAACTTTGGCAATCGGCAGGTGACGGTTCCCAGCCCAGGAAAAA

[0563] TTAGCGGCACAGGACATGCGCAGGTAAGTGGAAAAAAAGTGTGTATTCTGGGGGATGAGAAACAGGTCAGG

[0564] GTTTCTGCAACCTATATTACAACAACACATACTACGCCGGGAACAGGAACCATTACTATCAGTGCTCTGGATG

[0565] CTGGCCAGCAGGCCCTTCAGTGTACCAGTGGGGCGGCTTTAATTATCAAGGGGCAGCAATTTACGGCGATG

[0566] TTTACGCCTGAATTGCCAGCCATGAATAATACAGTGACTCCGCCACAACCGGATGTTACGACACCTTCATCAG

[0567] GAAAAGGACGTTTTATCACTCAACAAAATTTTGCTACCGTAAATTAGAGTATTGACTGAATTAAATAGAATTAA

[0568] CGAAGGTGTAAATAATTATTTATTTGCTGACGAATCGCTGTGACAAATAAACACAGGTGATGTTATGGAATTAA

[0569] ATGAGTTAACTAACAAATTGTCAAATTTGGTGCCAATGACCGATTTTAAATTAGATAATCGAGCCAGTTTGCAA

[0570] TTGCTTAAATATATTGAAGCGTATACGAAGATAATACCCTTTAATTCTGGCGATAAATATTGGAATGACTTTTTC

[0571] TTTATGTCAGGAAATACGCCAGAGAAACTTGCAAAATTATATCAGAAAGAAATAGAACCCAATGGGGAGTTAT

[0572] TACCTCAGCAGGCTTTTTTGTTGGCGGTTTTGCGTTTATTGGAAACACCAATATCCTTATTAAATGTATTACCT

[0573] GCTGCTCATCGTGAGCTCTATTATCGGGAGCTTTTAGGCTTGTCTTCCCATGCGGCACAGCCTGATCAGGTT

[0574] GCTTTATCTATGGAACTGAATTCGACAGTGATGGAACAGCTGCTCCCTGAAGGAACCCTGTTTGAGGCTGGT

[0575] CAGGATGAACAAGGCAATGCATTGCAATATGCCCTGGATGCCAGTTTGCTGGCTAATCGTGGATATATCAGT

[0576] GACTTGCGCTGGTTACGGAATGACGGGGAAAAGCAATGGGTTACTTCTGCTCCATGGGATTTACAGGCACAG

[0577] GTGTCACTGCCGTCTGATGGGATACGATTATTTGGTAAGACAAATAGTGATCAGCAGGTATTTGGTGGGGTG

[0578] TTGATAACGTCATCACTTCTGGCGATGGAAGCGGGGATAAGGAAGATCATTGTTACTTTTGAGCAGGAGATG

[0579] AACACCCAAGAACTGGTGGCACAGGTCAGCAGTGGAAATCAATGGCTAACATTGACGTCTGAGGTAAATAAG

[0580] AAAGAGGTCACACTGACACTGTCAGACAAAGAACCGGCAATCAGTGCGCCAGAGGATCTGGATAATCTCTTT

[0581] TTCACGCAACCGGTACTCAGGCTACAGGGAAAGGATAGTCAGGCACTGCCGGAGGTGACGGGTATCAGCGT

[0582] TTCGGAAAAGGATGATACTAAGGATACCTCTTTTGAGATGTATCACTTAACACCATTTGGTTATAGCAGTGATA

[0583] TAGAGCCATTGGAGGAAAATCCAGCGTTATATTTAGGCTTTACTGATGTAAAGCCAGGGCAAACACTGGCGC

[0584] TGTATTGGAAATTAAAATCCCCGCAGCAACCAACCGTTTCCTGGTATTACCTGGATCAACATAATCAATGGGC

[0585] TGAATTGGATTCATGGGTCAGTGATGGAACCCAGAATCTGTATCAGGATGGTACTTGGCACGTTGAGTTGCC

[0586] TGTGGATGCATCCAATCAGGCAGAGCAGATGCCAGTTGGACGCTATTGGTTGCGGGCAGTGGTGGAGGTAC

[0587] CCGCTCATGAGGGGGCGTTGGGGAAGGCTCCTTGGCTATATGGTCTAATCTATAACGCCATGACGGCAACC

[0588] TTGGTTAATGTAGATAGCATCAGTGACAGCCATTTCTTAACCCCTTTGCCTGCCAGCAGCATACAGCGGCCC GTTGAACCCATCATTGTGTTGGCATCGGTCAACCAGCCTTGGGCATCATGGGGTGGACGTATACCTGAATCC

[0589] TACAGTGCCTTTTTTGAACGGATAGCTCAAAACCTGTCTCATCGAAACCGGTCCTTAACCTGGGGAAATATGG TGACATTACTCAAAGAGCGTTATGTCAGCATCTTTGATGTTAAGTATCCAGGTAATGATGAACTCACCAGAGT GCCAGCATTGGAGCAGCAGCAACTAACAGTGATTCCAGCAAACCGGTACAACGATAGCGATGATTCTCTGCG TCCGGTACTGAATCCTGCTCGTCTGCAAGAGATGGCTGATTGGTTGCAGCAGAAAGACTCTCCCTGGGCCTC TATTGAGGTCAGGAATCCAGAATACTTGGATGTGAAAATCCATTACGAGGTGATTTTTAAACCTGATGTGAAC GAAGATTTTGGCTATCGCCAGCTACAGCAGCAACTGTGTGAGGTGTATATGCCTTGGAGCATAGATGAGCAG CGGCCCGTTGTATTGAATAACAGCATTAATTATTTCCAGTTGTTAGCCACTATTCAACAGCAACCGCTGGTTG AGCGAGTCACTCGTCTGACACTACATCGGGCTGATTCTTCTGATGAGAGTGATGGTACAGCATCTGTGGAAG CCAAAGATAATGAAGTGCTTATTTTAGTCTGGGAAGAGGACGATAATCTGCAATACCGAGGAAATGACTATGA GTAATCAGGATGCACTGTTTCATAGCGTTAAAGACGATATTCACTTTGATACCTTGCTGGAACAAGCTCATCA GGTGATTGAAAAACAGGCTGAAAAACTGTGGAGTGATACGGCAGAGCATGATCCGGGTATCACATTTTTGCA GGGAATCAGTTACGGTGTGTCAGATTTGGCTTACCGACATACATTACCCCTGAAAGATTTACTGACTCCGGC GCCGGATGAGCAGCAGCAAGAGGGAATTTTTCCTGCCGAATTTGGCCCGCATAATACACTGACTTGTGGGC CGGTGACAGCGGATGATTATCGCAAGGCATTGTTAGATCTACACAGCAGCGACAGCCTGGATGGTACTCAG CAGGATGAGGGGGATTTTCTGTTCCGGAGTGTGCAACTGGTGCGTGAACCGGAAAAACAGCGTTATACCTAT TGGTATGATGCAACCAAGAGGGAATATAGCTTTGTCAACAGTGAAGGGGCTAAAGAGTTTACCTTGCGGGGG AATTACTGGTTGTATCTGGAACCAACCCGTTGGACTCAGGGTAATATTGCCGCTGCTACCAGACAACTGACA GAATTTTTGACTAAAAATCGCAATATTGGTGAATCTGTCAGCAACATTATCTGGCTACAACCGGTTGATCTGC CACTGTTGCTGGATGTTGAACTGGATGATGATGTAGGTGCACAGGATGTCCCCGGTATTTTTGCGGCGGTGT ATAGCACCGCAGAGCAGTATCTGATGCCTGGAGCACAGCGTTACCGTACGGAAGTACTGCAAAATGCTGGG ATGAGCAATGATCAAATCTTCGAAGGTCCATTATTGGAACATGGCTGGATACCAGAGCTGCCGGCAGCCCGT GATTATACTCAAAGGCTCACTCTCAATCTTAGCCGGTTGGTAAATAGTCTGCTTGAGATTGAGGGCATTAAAC ATGTGAATCGTCTTCGTCTGGATGATAGCTTCGATAAAACTGCTATTGAACCCGTTAAGGGGGATACCTGGTC GTGGTCGATCAAAGAGGGCTATTATCCACGTCTTTGGGGAGAAGACCCACTTAACCAATTGGCGCAACAAAA TGGCCCGCTTAGGGTGATAGCCAAAGGAGGGATTAGCGTCAGTGTGAGTAAAGAGCAAATCCAGGCCAGTT TACCCAGTCAATCACTGATTCAAAATGAGCCGGTAATATTGGCTTACGGCCAGCACCGTGACGTTGGCAGCT ATTATCCCGTCAGTGATACTTTGCCGCCTTGCTATGGACTACAACATTCTTTGTCTGAAAGTGAACACTTATTG CCACTTCATCAATTTATGTTGCCATTTGAACAATTATTGGCCTGTGGTTGTCAACAGATAGCCATGCTCCCGC GGTTACTGGCTTTTCAGCGCGAAGGTTATGAGGTTTGGGGTGATCAGTGGCCCTTTAAGTCAGGCTCAGTGA ATGATGACGCCCATCAAGATTATGCCCCTGCATTAAAGGATTTGTTAGGACAGATTGCGCTGGATAGTGATCA TGAATTGGATATTATTAATTACTTGCTGGGTTACTTTGGCACACAGCGGGCACCGCGTACCTTTACGACACAA CTCGATGATTTTCGTGCGGTCCAACAGGGTTATCTGGCCCAGCAACCGACATTGACTTACCACCGCTCCAAT ATTCGTATCGATCAGGTATCGTCGCTACAAAAACGTATTGCTGCTCGCATGGGGCTGGGCGGTGAGTTGTTT AAACCTCAACCGGATCTGAGCCAACTGCCTTTTTATTTGATTGAACATCGAGCGTTGCTGCCAGTCAAACCCA ATAGTCAGTTTGATAAGGAACAGAAACCAGCCTCGGTGACAGAGGAGGGGGGCAGCCAAACAGGTCAACAT TATGTGGTCATTGAACAGAAGGGCATTGATGGCAAGCTGACACAGGGGCAAGTGATCAATTTAATTCTGTAT GAAGGAGAGCAGGGAGAAACCCAATTTACGATACGCGGTCAGATGGTATTCAAAACCGAGGGGGATAAGTT TTGGTTGGATGTGAATAATAGTGCGCAACTGGAATATAATCTGGCGCGGGTAATGACAGCAGCCAAGGCGAG TAAACTCTTTTGGCAAAACAGCCCGGTATGGATGGAGGATATGGGCTATCGTCTGGCCTATGCTAGTGACCA ATCCTCATTGCCTGTGAATCAACGGCGCTTGACCCGCACAGTGCAAACTCCATTCCCGCCGATGGTTGTTGT

[0590] AGGTAGCGAAATCACCCTGTTAAAGCAGGTGGGGATAGTCAATTTAAAAAAAGCGGAGTCAGAAAAACTTTAT GCAAAAGTTGTTAGCTTTGATCGCATTGAAGGGACCTTGATTATTGAGCGTTTGGGTAATTCCACTCTGGCTT TTCCTACCTCGGAAGAGGCGTGGCGGTATAGTTGGTATTTTTCGGGGGAGAAATATGAAAGGACTGACCGCT TTTCATTTGTGATTAGCGTAGTAGTGAACAGTGACTTAATTAAATTGCCCGGTGTTGATCCCTATAAATTGGAA GAATGGGTGAAAGAAACGATTCTTACCGAATTTCCAGCTCATATTTCTATGATTATCCATTGGATGGATCGGG AAGCCTTTTTAAATTTCGCCAATACCTATCAGCGTTGGCAAAATAATGGTACGCCACTGGGGGATGCGGCTTA TTCCATTCTAGAAAGTTTGACACTTGGTAAATTGCCATCTGCCTTAAAAGGTGTTGGCACAATGCGTATTGCC ACATCTAGTCAAAGAGAAGAAGTGGTGGGTAGTAATGGTGATCAATGGAATACAGATGGAATAACCCAGAAT GAATTATTCTATGTTCCTAAAGAGAGCTAGGAAAAATAAATATCTGCCACTAATGATGTTGAATTAAATATGTTT TCTGGAGTTAATCATGAACGAAACTCGTTATAATGCAACTGTACAAGAACAACAAACATTATCTAATCCAAAAG CTGTTGGACCTGACATCGATAAATTAAAGGATAAATTTAAAGAGGGCAGTATTCCCCTGCAAACCGATTTCAA TGAGTTAATTGATATTGCCGATATTGGACGTAAAGCCTGTGGTCAAGCGCCACAACAAAATGGCCCAGGAGA AGGATTGAAATTGGCTGATGACGGTACGCTTAATTTAAAAATAGGCACTTTTTCCAATAAAGACTTTTCTCCAT TAATATTAAAAGATGATGTTTTATCTGTAGATCTTGGTAGTGGTCTGACTAATGAAACCAATGGAATCTGTGTC GGTCAGGGCGATGGTATTACAGTTAACACTAGCAATGTAGCTGTAAAACAAGGTAACGGAATTAGCGTTACTA GTAGTGGTGGTGTTGCCGTTAAAGTTAGTGCTAATAAGGGACTTAGCGTTGATAGTAGTGGTGTTGCAGTTAA AGTTAATACTGATAAGGGAATTAGCGTTGATGGTAATGGTGTTGCAGTTAAAGTTAATACTAGTAAAGGAATTA GCGTTGATAATACAGGTGTTGCAGTTATAGCTAATGCTAGTAAGGGAATTAGCGTTGATGGTAGTGGTGTTGC AGTTATAGCTAATACTAGTAAAGGAATTAGCGTTGATGGTAGTGGTGTTGCAGTTATAGCTAATACTAGTAAA GGAATTAGCGTTGATAATACAGGTGTTGCAGTTATAGCTAATGCTAGTAAGGGAATTAGCGTTGATGGTAGTG GTGTTGCAGTTATAGCTAATACTAGTAAAGGAATTAGCGTTGATGGTAGTGGTGTTGCAGTTATAGCTAATAC TAGTAAAGGAATTAGCGTTGATAGTAGTGGTGTTGCAGTTAAAGTTAAAGCTAATGGCGGAATTAAAGTAGAT GCTAATGGTGTTGCAATTGATCCTAATAATGTACTCCCCAAGGGAGTGATTGTAATGTTCTCTGGCAGTACTG CACCAACTGGTTGGGCGTTATGTGATGGCAATAATGGTACACCAAATTTAATCGATCGATTTATTTTAGGTGG GAAAGGGACTGATATTAATGGAGTGAGTACTAATACAGCTTCAGGTACTAAAAATAGTAAGTTATTCGATTTCA GTTCTGATGAAGCTACATTAACTATTGATGGTAAAACACTGGGGAGAGCATTATCGTTACAGCAAATACCTAA TCATGCACACTTTAGTGGAATAATTATGGATACAGAGAAAGTTAATTATTATGGAAGTAAAAAAATCACAACAA ATGTGTGGGGTGTAACAACAGGAGATAATACTTCAGTACGATATATTTATAAGTCATCAGGTGTACTTGACTC TAACAATAATGTCTCCAACAGTACCTTAGGCGGAAACAGTCTGCAGACGCACGATCATGATATTAAGATAACG GGCACAGGAAAACATTCTCACAAAAACAAAGTAACAGTCCCTTATTATATTCTGGCTTTCATCATAAAGCTTTA ATATATATGAAAAATTGAAAATATAAATTATCCATTAATAATAAAGAGGATATTAGCATGACTTCGGAGCCAAAT CTGTTAAACCGGATTACAATTACTATTGAAGCTAATAATCAACAAGTAGCTAGAAAAGTATTGCATGGCTCCTT GCTTAATCAAGCTAATATAAATAAATTATTTAATTCATACTTTAATGAATATGAAATTAATAGGGGTGTTTATTTA GAAACATTAATCCTGAATCTTGGTACGATAAATTTCCATGATTTTAATTCATTGTTTCCTACTCTCCTAAAAGCT GCATTGAATAAAGAATTCAGTCAATATCAGATAAACAACCATAGGGAAGAAATGCTATTTAATGAGACAATATC AAATCAAGCTACTGATAAGTCTTACATATTTGGCGATAACAAATTAATTGATGCAGAGAATTTCATTCACTTTTT ATATCAAAAGCATTCCACATTAAATCTAGTAGAAGCAATGGGAAATAATGGTATTGAAAAATTAACAAATCAGT TAACACAAATAGAAAATAAATTTGCGTTATTATTGGCAAAAAGTTGTTTGTCTGAGGAAGGCTTAAAACGACTC TTGGCTATCAAACAACCCGATTTATTAATCGCTATCAATCGCAGATTATCTGAAAGAATAAATAGACCACAATA TCAGGAGAAGCTTGTTTCCTGCGGACAACTGATATTTAGTGCTCTGGGATATATACAACAGTACAATATACAG GAAATTCCTAAACCGGATGAAAAAGTTATTGCACGCATAACAACTGAACTTAATAATAATGGTTTGCTTAATAC AATACCTATTATTACACTATTTCGTCAGAGTGGGATTAACGATTCATCACTAAATGATTGGCTAAAGAAAATCT GGCAGGTGAGATCAATTTCACAGTTATGCAGAAAGTATCTTTCTGCTAAGGAATACCAATATCTGTCAGAACA TTTTGTTTCAAAGAGCGTCGATAAAAATAGATATGATGAAGAGCCCGTAAATCAGAGCATATTATCAAGGTTG AATAATAATTCCATTAAAGAAGGAAATAATCACAGTCAACTCTGTACTCTCAGTAGACTATATTCTGAACCCGT TGTATTACCTGAACAAACCATTCTACGTCAGGTTAGTAATACAGTAGATCAGAGCATATTATCAAGGTTGAATA ATGCCTCCATTAAAGAAGGAAATAACCAAAGTCAACTTCGCACTCTCAGTAGACTATATTCTGAGCCCGTTGC ATTACCTGAACAAACCATTCCACGTCAGGTTAGTAATACAGGTATATTAATTCTATGGCCAATGCTACCTACAC TATTTAACCAGCTTGGTCTACTTGAGAAAAAGAAATTTATCCATCGTCAGGCCCAGTTTAATGCCGTTGATTTT CTTGATTACCTGATTTGGGGAACCGAAGATGTGAAAGTGGAACGAAAGGTTTTGAATAATGTTCTATGTGGGT TAATGGCTGATGAAATTACTGAACCAATGCCTATTGAACCAGAAAAACAATGGATAATAATTCAATGGCTGGA CGCTATTATCTCCCAACTTTCTGGCTGGAAAAAGTTAAGTCGTAATGACGTCCGTCAATTATTTCTACAACGAC CAGGAGAATTACTGATCAATGAACAGGAAATTAAAATCACAATACAGCAACAACCATTTGATGCTCTGTTAACT GATTGGCCGTGGCCAATGAATATGGCTTGTTTTAGCTGGTTGAGTCAACCATTAACCATTACGTGGTTATAAC CATTGACCACAATGACTTAGTCTGAGTAAAAAATATGAATATATCGCCTGTTTTTTATGATTCATTGAATCAGG ATAACGACCGTGATCTATCGTTTTTATTTAGCGAACTGGAACGAATAGATCTCGCTCTTCAACACCATTTTTAT TGTGTAGAAAGTCAGCGAAGTGAGCTCCTGGATGAGTTTCTGCTCACTGAGGCGGAAGTGGTGACCAGGCT GGATAAGCCACTTGGTAAACCTCATTGGATAAATGATGATTATCTGGCGATATCGCAAAAGGGCAATGTAAGC CTAATGGCAGCGTCCAGATTAATGGATCTGATCGAACGCTTTGAACTGACTGATTTTGAGCGCGATGTTTTAC TATTAGGCTTATTGCCCCATTTTGATAGCCGCTATTATCGACTGTTTTCGCTGATTCAAGGGGGACAACAGGG TCGATTACCTTCTTTTGCGCTGGCATTGGAACTGTTTTGCCACTCGGCGCTGGAGAAACAGGTACAGCAAGC GAGTTTTCTGCACCGGGCACCTTTGATGGGTTGCCAGCTATTATCCATCGATACTAGTCAAAAAACGCTGGC CTGGCTCCAGACTCCCTTTATTACTGACAGCGGGGTATATCACTTTTTACTGGGGCATCACTACATTATGCCG GCTTTAGAACATTGTGCTGAGTGGTTAACACCGACAGGGATTGGCTGTTATCCTGAAGGATTAAAACAAGTAC TGGGTAACGTATTGTTATCTGACAACGATAATATTAGACCGATTGTCTTATTACGGGGAATGGCCGGCAGTGC CAGAGCTTATACCATTACTAATATGATGGCTTCAGAAGGGAAGCAAACACTGCTGGTAGATATATCCAAACTT GCTGATAGCGATGAAAAAAACATTATTCTTCAGATAAAGCATATTTTGCGGGAAACCCGCATGCATGGAGCAT GTTTATTATTACGGAATTTTTGCTTGTTAGTGGAACAGAATAAACAACTATTGGACTCCCTGTCAGAGTTATTG AATCAACCTGAATTAAGAATTGTTTGCCTGATTGAGCCTTATTCCCCATTGGTATGGCTGAAAAAGATACCGG TATTACTGATTGAGATGCCACTTTTAACGCCTGCGGAAAAAGCCAGATTGTTAATTGCCAGCTTACCGGATAA TTGTTCCGAGGATATTGATACGATAACTTTAAGCCAGCGTTACACTTTTAACCCAGAAACCCTGCCATTGATTT TGCAAGAGGCCCAGCTTTATCAACAGCAGCGAGATCCGCTGGATATCTTGCAGCAATGCGATATACGCCAGG CATTAAATTTGCGTGCTCAACAAAATTTCGGTCAATTGGCACAGCGGATTATTCCTAAGCGCTCATTAAAGGA TTTATTGGTATCCGATGAGATTGCTCAGCAGTTACGGGAAATACTCATAGCAATTAAGTATCGGGAACAGGTT CTGGCGGGAGGGTTTAAAGATAAAATTGCCTATGGCACTGGTATCAGCGCCCTGTTTTATGGTGATTCAGGC ACTGGAAAAACCATGGCAGCAGAAGTGATTGCTGACCACATTGGCGTTGACTTAATAAAAGTGGATTTATCTA CAGTAGTGAATAAATACATCGGTGAAACAGAAAAAAACTTATCCCGTATTTTCGATTTGGCGGAACAGGATGC AGGGGTATTATTCTTTGATGAAGCTGACGCACTGTTTGGTAAACGCAGTGAAACTAAAGATTCCCAGGACAGA CATGCCAATATTGAAGTTTCTTACTTATTACAGCGCCTGGAGAATTACCCGGGTCTGGTCATTTTATCCACCA ATAATCGTGGTCATTTAGACAGTGCTTTTAATCGTCGTTTTACTTTCATTACCCGTTTTACTTACCCGGATGAA AAAATCCGTAAAAAAATGTGGCAGGAAATTTGGCCTAGAAATATAAAAATATCGGAAGATATCGATTTTAACGA ATTAGCTCAACGAACAAGCGTGACTGGCGCGAATATCCGCAATATTGCTTTATTGTCTTCATTCTTTGCTTCA GAGCAGGGGAATGATGAAGTCAGTAATGAAAATATTGAAATTGCATTGAAGCGTGAATTAGCTAAAGTCGGA CGATTAACATTTTAAAAGTTATCACAATGAAAGTATTGAAATATTAAATAAATTTATTACCAAAAAGTTATCACG ATATAATTTAAGAGAGGTTTTTTATGTTAAACACGCAAACTATTATTGATGTCAATAAGGCAATGGATGCCATG CTGCGCGCATATCTGAATCAAGATATTGCCATTCGTTTTGATCTACCTGAATTGGATACTATGCAATCTGATGC GATGGTAAGTATCTTTCTTTATGACATTCATGAAGATTTACAGCTTCGCTCGGCAGAATCAAGAGGGTTTGAT GTTTATGCCGGGAGGTTATTGCCTGGTTGGGTAAATATTAAATGTAACTATCTGATTACCTATTGGGAAGCTT CTAAGCCAGCGACTGATGCCAGCAGTCCGGATAGCCAACCTGATAACCAGGCAATACAAGTGATGTCACAAG TATTAAATGCCTTGATTAATAATCGTCAATTGGCAGGTATTCCTGGTGCTTATACTCAGGTTGTACCGCCTAAA GAGAGTTTAAATAGCCTGGGGAATTTCTGGCAATCACTGGGTAATCGCCCACGGCTTTCTCTCAATTATTCAG TGACAGTACCTGTTAGCCTAAACGATGGTCAGGATAGCGCGACTCCGGTTACCGCGGTTTCTTCTACAGTGG AACAAACGGCATCGCTCAGTCAAGAAGTGGTTAGTCATGCTTTACGCGAATTACTCATTACGGAATTAGGAG GAGGAGAGGATAACCGGTTGGTACTGAGTAAAGTTGAATTATCCGCAGTGAAAGAGACGATGACTCAAGACA

[0591] GTCCGGCTCAGATGATTATATTGTTGTCTGTTTCAGGCATTACACGACAGGAATATTTGAAGGAAATTGATAAT

[0592] ATCTTTGATCGTTGGGTAAATAATGCTGAAGTTATTACCACTATTGATGATTGTGGGATTAGAATTGAAAGTAT

[0593] AACGAAAGATAATCTTGTAGGAATTTAA

[0594] SEQ ID NO: 18 (Photorhabdus asymbiotica strain ATCC43949 PVCIopT operon, pyc1 - pyc16-, e.g. corresponding to genes PAU 02112 to PAU 02099 of the sequence of GenBank accession no. FM162591.1)

[0595] ATGGCCACAACCACAGTTGACTATCCAATACCGGCTTATCGATTTGTTGTCTCCGTTGGTGATGAACAAATCC CTTTTAACAGCGTTTCGGGGCTGGATATTACTTATGATGTCATCGAGTATAAAGATGGCACCGGTAATTATTAT AAAATGCCGGGTCAACGTCAGTTAATCAATATTACACTGCGTAAAGGGGTATTCCCTGGCGACACTAAACTTT

[0596] TTGATTGGCTTAATTCCATTCAGCTTAATCAGGTTGAGAAAAAAGATGTTTCAATTAGCTTGACCAACGAAGTT GGAACTGAAATTTTAATGACCTGGAGCGTAGCCAATGCATTCCCAACCTCATTAACATCTCCTTCTTTTGATG CCACCAGCAATGATATCGCTGTTCAAGAAATAAAACTGACTGCCGATCGAGTCACTATTCAGGCAGCTTAAAG

[0597] CATCACGATGATTGATATATCAGACGGGACAAAATGATCCTCAAAATTTGGCACAACGGCTACCCGTCCAACT AAATTTACCCTCTTACAGTTCACGCAAAATATCGCACAATACAATTGGAGGCAATATGCCAACAACAACTTATC CCGGCGTTTATATTGAAGAAGACGCCTCACTGTCACTTTCCGTTCGCTCAAGTGCAACGGCGGTGCCCGTTT

[0598] TTACCGTTGAAGATGACAGTCAACTTCATACTCCTACCAGAGTGAATAGTTGGTTAGAATATCTGACAAAAAAA GCAGATAAAAAATTCAATTCTACCGACAAACTTGATATCGCATTGCGCGCTTATTTTATTAACGGCGGCGGAT ATGGTTATCTCGTCAAAGCGGGTGAATTAACAAATCAAATTCCAAAACTTAACGATGTCACATTACTGGTCGC

[0599] GGCTGGAGAAAATATCAAAGATGCTGTGAGTACACTTTGTCAACCGGGCAAAGGCTTATTTGCCATTCTGGAT GGCCCAACCGAAGAGTTAAAGTCTGATGGCAAATCCAGAGATCCGTATGATCAAAGCCCTTTTGCCGCCGTT TATTACCCCTGGCTAGTTGCTGATTGGGCAGACAATATTCCGCCAAGCGCGGCCATTGCCGGTATCTATTGT

[0600] TCAGTTGACCGTACCCGCGGTGTCTGGAAAGCCCCAGCAAATGTCATATTACAAGGCGGGGTGAAACCGAA GTTTAAAGTCACCGATGACTTACAAGGTATTTACAACACCGGTAAAGCCATCAATATGATCCGTGAATTTCCG AATACCGGTGTCACCATCTGGGGCGCCCGCACACTTAAGGACGAAGATAACTGGCGTTACATCCCAGTTCG

[0601] CCGCCTGTTTAACAGTGCAGAGCGAGACATTAAAAATGCCATGAGTTTCGCGGTCTTTGAACCTAACAGCCA ACCCACCTGGAAAGCTGTACACCGAGCTATTGATAATTATCTCTATGCCCTTTGGCAACAAGGAGGGCTAGC AGGAAACAAAGCTGAACAAGCTTACTTTGTGCAAATTGGTAAAGGGATAACCATGACCGATGATGATATCAAG

[0602] CAAGGGAAAATGATTGTTAAAGTGGGTATGGCCGCAGTGCGCCCGGCTGAATTTATCATCCTTCAATTTTCAC AAAATGTAGCACAGTAACCGTACTGAGGCGCGGTTTAACACCGCGTCCATTCAGTCTATTGAATGGAGGAGA CAATAATGATAACGGAGATAAAACAGCCGGGCGTCACCATCACGGAAAATTCGATATCCCCGAAATCAGATA

[0603] ATGAATTTATCGGCGTCCCCGTTTTTATTGGCCATACCGAAAAAAATTCAAGCCATAAAACGGCTGTTAAACTA AATAGCCTGATGGACTTTACCCAAGCTTTCGGTGCATCAGGATTAACCTATTATTCAGTACGCCACTTTTTTGA AAATGGTGGACAGCAAGCTTATATCTTGTCACTGGGGATTAATCAACAGCTAAAAGATTTTCAATCATTGATTA CCGCCCTGCAATGGAACTGGGTAAAACAAGCCATTGCCGCAGAAAACGAAATCACATTGATTGTTGTGCCTG

[0604] ATATTACCCGTTTTAATGATCTCAGCGCTCAAAAAAGCCTTTGGCTACAACTCTGGCAATCAATACTTGAACTG TGTAAAAGTCGGCGTGGCATCATGGGATTACTGGACGCGCCTGATGATCCAACATTAGCAACTGAGTGTTTA AAACAATTCTCTTCCACTGATCGCCAATGGGGCGCCGTATACTGGCCAAGGCTAAAAAGTACCTACCAAGAA

[0605] AACGGTACATACATTGTACTTTCACCTACTGCTGCGGTCGCCGCCGTTATGCAACGCAATGACAGTCAGAAA GGCATATGGACTGCTCCCGCCAATGTGGCTTTAGCCAACGTCATCGGTCCGGTACGTTCTTACATTGAAGCT GGAACCTTGCTGAATCAAGAAGGCACTTCGTTGAATCTGGTGCGTAGCTTCCCCGGCAAAGGCATTAAAATC

[0606] TGGGGCTGCCGCACTCTGGATAACATACCTCATTCTCCCTGGCGTTATATCCAAATTCGCCGTTTGGTTTCCT ATATCGAAGCTCATATAACCCAACTTGGCCGCGCCTTTGTCTTTGAACCCAACAACGCCATCACCTGGATGAA ATTTAAAGGTCAGGCCCACAACTGGCTACGTCAATTATGGCTAAAAGGTGGATTACGGGGCACTCAGGAAGA

[0607] TCAAGCATTTGAGGTGTTACTGGGTGTTAATGAATCCATGAGTGAAACGGATATCTTGGCCGGAAAAATGATC ATGAAAATCAGGCTGGCGCTGTTAATTCCGGCAGAATTTATTGAGCTGAGTCTGACGTTTGATATCCGTAACA ATACCGTACCTAGCTAATCTAAACAGGGGAAAAACATGTACAACTTATACACCCCGTCAGTATCTCACCGTTT

[0608] TATCGCCAGTTTTCTGTTTAACAACATTCCCAGCCCACTTGATATCGCCTTTCAGCGTATATCTGGCCTGAGC CGAGAACTGCAAACCACCCAACATAGCCAAGGTGGAGAAAACGCCAGAAACGTCTGGTTATCCGAGAAGAT CCAACATGGCAGCCTGGTGCTGGAGCGCGGTGTTATGACCATCACTCCCCTCACCTTGGTTTTTGATCGCGT

[0609] GCTGCGCGGTGAAAAAGCCGTGTATGCCGATGTTGTCATCATGCTACTGAATGAAAATGCGTTACCCGTGGC GAGCTGGACAGTCAGTAACGCGCTACCGGTTCGTTGGTCCACCAGCGACTTTGATGCTAATAGCAACACCGT ACTGGTGAGTTCTCTGGAATTACGTTATCAGGATATGCGCTGGTTAGGAGTAAAAGCATGACGGTAGAAATTA

[0610] AAGAACTGATTATTCAGGCTAAAGTCACCGATTCTACGAGTGATCAACTCGCCCCAAGAACATTAGCCCAAGA AAAGCTGGATAACGCCCGTTTGATTGACATAGTGAAACGGGAAGTGTTAGAGGCATTACGTGAAGGAGGCCA TCATGAGTTTAATTGAACGTGGTTTATCCAGACTCACCCTAACCGCTTTTAAAGACCGAGAAGGTAAAGTTTC

[0611] CGTGGGTCGCTTACAAGCCATGTATAACCCCGATACGATCCAGCTTGACTACCAAACCCGCTACCAACAGGA TGAAAGTGTTAATCGTGCCAGCCAAAGCAGCCGTTATGTATTATCCCAACCCGCCGGATTATCCTTAGTTCTG CTGTTTGATGCCTCGATGCCCGATAATAACATGCCGATAGAAACCCAGCTTGCGACCCTGAAATCCCTGTGT

[0612] GCGATTGATGCCAGCACCAAAGTACCCCACTTCCTTAAAATCAAATGGGGCAAAATGCGCTGGGAAAACAAA GGTTATTTCGCCTGCCGAGCCAGTAGCCTGGCCGTCAACTATACCCTGTTTGACCGGGATGCCACACCATTG CGGGCCAGCGCCACTCTATCTCTGGTAGCGGACGAAAGCTTTATTATTCAAGCTACCGAACGGCAGTTAAAA TCACCGCCGGCCACTGCGGTTAGCGTAACTGATATGCTCTCCCTGCCTTTGATTGCTTTAGATGCTGGAGCG

[0613] TCTCTGGCTGGTGGCATTGATTATCTCTCGCTGGCCTGGCAAAACGGTCTGGATAATCTTGATGACTTTACCC

[0614] CCGGACAAACACTGCAAGCGCGGGGGGATGCATGAAGATACCCATGATAACCCTCAAAATAGGTGGCAAAA

[0615] CGCTCAATCAATTGACTGTCATCAGTCTGACAATAAACCATCAAATCAATGGCATTCCCTCGACCAACATCAC

[0616] CTTGGGGATCGCTGGCGATGCGAGCCATATTTTCGACACCAAAGCCCAAGCTGAACTGGCAAGTTGTCGCC

[0617] CCAATAATGAACTCACCCTACAGATCCAAAAAACCGTGGTGTTTAAAGGGAGCATCGTTCGACAAGCACTTGA

[0618] ACTGAAAGGTCAAGACAGCATCATTACCCTGACAGCAAAACATCCACTACAAAAGTTAACTCATAGCCTCCAT

[0619] TCACAATTATTCAGTCAACAGAGTGATGAAGCGATTATCAGGAAATTATTCAATCAGGCGGGTATCCAAACAA

[0620] CGATAAAGCAGGCTCCTCAACTTAAAACCGTTCATGAACAAATGGTGCAATTTCGTTGCAATGACTGGGCATT

[0621] CCTAAAAAGCCGATTGATTGCCACTAATACCTGGCTGTTGCCCGGCAATGAATCGGTTACTTTGATAACACCT

[0622] AAGGCCCTGAATCAATCGACAGTGCATACTCTTCATCGACAGGCCAGTGCTGAAGATATTGTGTTATTTGCAG

[0623] CGGATCTCCAATGGAATAACCAATATAGCCCTAAAACGGTGAGTGTACGTGCCTGGGATATTGCTCAACAAA

[0624] AGCTTTCCCCAGCAATTAATACCCAAAACAGTCAGCTTGGCAGTCATAAATTGGCCGTGGACAGTATCGCCG

[0625] CACTGGCTGATAAAGAGTGGCAATGGGCTTACAGCTATCCATTAGATAATGAACAAGCCAAACACCTTGCTCA

[0626] AGGCATTATGAATAACCTGCGAAGCCATAATATATCTGGCAGTTTTGAAATCGAAGGTAATCACCGTTATCAA

[0627] CCGGGGGATGTCTTGGCGTTAAATGGTTTTGGTCAGGGGATGGACGGTCAAGGGATTATCACCGGAGTCAG

[0628] TCAGATAATTAATCAGCGGCAAGGCTGGCACACCCTATTAACCTTAGGCATGTTACCCGATGTAGAACCGCC

[0629] GGTGCCTCAGGTGAAAGAGTTGCATATCGGTATCGTGGAAAAATACCAGCAAGACCGCCAATCACTAAGCCG

[0630] TATCCCAGTCAGAATACCCGCATTAAACTTGACCAAAGGTGTCCTTTTTGCCCGGCTAGGTAAACCTTATGCC

[0631] AGTCATGAAAGCGGATTTTGCTTTTATCCCGAACCGGGAGATGAAGTGATTATCGGATTCTTTGAATGTGATC

[0632] CTCGTTTTCCAGTGATATTAGGTTCCATGCATAATCCGAAAAATAAACCACCGTTAGAACCCAGTGAAAAAAAT

[0633] CCGGTGAAAACTTTAGTTATCAAGCAAGGGGATAAACAACAAGCATTAATATTCGATAATAAAGAAAACACGG

[0634] TGGCACTTAATAGCGGCGAAAATAAAGTCTCTCTGCAACAGGATAAAAACATTACGCTCAATTCAACTAAAAA

[0635] TCTCATCACTCAGGCCCAAGAAATTAATATACAAGCGGAAAAATCTCTGTCAGCCACAGGAAAATCTGGCGTC

[0636] GATATTAAGGGCGCGAAAATTAACTTAACCCAGTAATGAGGTATTGAAATGACAAGCCAAATATTAGCCAATA

[0637] TTTACGGTTGCGGCTGGAAATTTCCGCCACAGTTTTCTATTGAAACTGGCGTAGAAATGGCCGAAGGTGCCG

[0638] AAAACGTTCGCCAAAGTATGAAAATCCTTTTTTTAACTGAACCCGGTGAACGAATTATGCGTGAAGATTATGG

[0639] TTGTGGTCTGAATGATTACATGTTTGAAAATATCAGTGATGAATTATTATCGGAGATTCAAACCCGCATTGAAG

[0640] AACGAGTATTGCGCTATGAACCCCGTGCTGAAATCACAGATATCCAAGTAACTCAGAAAACAGACTCACCGA

[0641] ATACTTTACATATTCAAGTGACCTATGCCCTGAGAGGCAGCCAAATCAGTCAACAGCTTGAAGGGGTTCTTGA

[0642] GATCAACGAAGGTCAGGCAAAGGTGAGTCTATGAGCAAACAACTCATTATTGATGGCGACAGCCTGCTATTC

[0643] GAGCCATTATTCGGCAACCGGCAGGTCACTATTTTGATGCCAGCGACCATCAGAGGCAGCGGACACGCGCA

[0644] AATCCAAGGCAGAAAGATAGCGATTGTCGGCGATGAAAAAAAGGTACAACTTCAAGCGCAATACATTACCCC

[0645] AAGCCACCCGGTACCTGGCATAGGCACAGTTACCATTGCTCAATTAGATACCAGCCAGCAAGTCAACTTTTG

[0646] CCACAGCCCTGCCACAGTGATAGTTGTCGGGCAGCAATTTACCGCTCGATTTACCCCATCACAGCCGGCAAT

[0647] TAATCCGTCAACCGGGCCAGATGTCACAACACCCAGTATGGGCAAAGGCCGTTTTATTGCCAGTCAACATAC

[0648] TATCAACGCCGGATAAATAACTCTGCAAAATCATTATTCAATAACGTTCCTATTCTGCAATAGCTATCAGCAAT

[0649] ATATTCAAATAACAGGTGGTATAATATGGGACTCACCGAATTAAAAAATAAACTCTCTGCTATCGTACTCGATA

[0650] CGGATTTTAAACTTGATGAAAGAAGTACACTGGATATTTTAAACTGGCTACAAGAATATGCTAAAAAAATCCCT

[0651] TTCAATCAAGAGAAAAAACAGTTCTGGGATAGTTTCTATTTTATTCAGGAAAATAGTCCTGAGAAATTAGCCGA

[0652] TCTTTACCAAAACGTTAATAAAACGAATGGCCATTTACCGGCCCATCAAGCTTTTGTTTTAGCCTTTTTAAAAC

[0653] TTTTAGAAACCACCAAAGTATTATTTAATACTTTTCCGGCACGACATCGTGATCTTTATTACCGGGAATTATTA

[0654] GGTCTAAAACCCAGAAATGCCCAAGCAGATAGTGTTGCTTTAGGCATTACCTTAAATACAGATAACACAGAAC

[0655] ATCTTATTCCTAAAGGAACCTTGTTCGATGCCGGGCAGGACAGGGCCGGAAATCCGCTACAATACGCATCAA

[0656] ATGCAGATTTACTGGCGAATCAAGGAAAATTGAGCGATCTGCGTTGGTGTCGAAAAGATAATGATAGCTGGC

[0657] AATCTGCAATACTACTGAACCACTCAGATAATATTGAATTACCTGAAAACAGTATTCGACTTTTTAGTCCAACG

[0658] CCGGATGATATTCCCGTTTTATCCGGTTATTTGATAACTTCGTCTTTATTTGCTATGCCAACGGGGGAACGCA

[0659] GTATTACATTGACTTTAGCAGATAATTGGCATGGTGATATTAAGCACATCACCGCTAAAATCAGTTCGGGAGA

[0660] TCACTGGCTTTCACTATCAGTAAAAAAAGAACAAGACAATAGTATTCACTATCTTAAACTTTATTTATCAACCAA

[0661] TGATGACCCCATCGGTCCTCCTGATGCTTTGGATAATATAGCGTTTGATGTACCGGTATTAAAGCTGGGCACT

[0662] GTTCAGGGACCTATACTACCCAAGATTACGGGTATTGAAATTAGCATTAACGGCAACAGTAATGTACATTATT

[0663] CCTCTGATAACGGTATTGAAAAAATAGATGCAGCTAGTTTTCCCTTTGGACAATCACCGTCACCAGGTTCCGG

[0664] TTTTAATCTGATTGCCCCTGAATGGTATGGTACAGAAAGCGCCAAAATTACTCTTACTCCTCAATGGACTGGA

[0665] TTACCCAAAGAGGGGTTTAAAGAGTGGTATCAAGGATATAGTTCTACCCCCGAAAATAATGCATTTAAAGTAC

[0666] AGGCTTATTTAATCACACCTCAAAAGAGAGAAAAATTTAATGAAGCTCAGTCATTATTTAATGAAAGTAAAGAC

[0667] AAGAAACCACAAGGAAAAAGCCTAACTTTTACCTTACCTGCAATGGATTATTCCTTTGCAAACAGCCCATCAT

[0668] CTAATAACTGGCCCGCATCAATACGCATAGAACTAACCGAACAGGATTTTATGCATGCCCAATATTGGCAAAA

[0669] TCCTACGGGTAAAAAACAGCCCTATACCCCCAAAATGAACACATTACAAATTCAGTTCAGTGCCAAAGTTAAA

[0670] CCCGAACAATTTTCCGTTTATTCTCTCACGCCTTTTGGTTGGGGAAAAACAGGAGAAAATAGAACATCATTAA

[0671] CCCATGATACATTCTATTTAGGTTTTACCGATGTATTACCAGGACAAACTTTATCCCTGTACTGGCAGTTAGAA

[0672] GGTATTAAAAAGCTCCCTTTATCCTGGTCTTATCTGAATCAAGAAAATACCTGGAGTCCATTGGATAATCAGGT

[0673] GCATGACCAAACCCACAACCTATTTGATCGAGGAATCTGGCGTACCTCATTGCCACATGATGCTTCAAACCAA

[0674] GCCTCTCAAATGCCAAAAGGACAATATTGGGTGAAGGCACACATTTTACAAACGAATCAAGCAACCCTGACT

[0675] GATCTGTATTGGTATCGAAAAGATAATGATGTCTGGAAATCCGCAACACCTCTTAGCCTTTCAAATAACATGAA

[0676] ATTACCCGCAAACGGTATTCAGATTTTTAGCCCAACATCTCATGATGTTCCAGTTCGATACGGCTACCTAATTA CTTCATCTTTATTCTCATTCCTCAAGAAAGGACGCAATATCACATTAATTTTAGCAGGAGATAGCTGGGAGGG TAATCCTGAAAACATCACCGCTAAAATCAGTTCAGGAAATCACTGGTTAACACTATCCGTCGAATATCTGAGT AATACTAATAGTCTTAAGTTGCAATTATCAGATAATAATAATGATCCCATCAGCCCCCCTAATGCTCTGGATAA TATGACGTTTGACACGCCATTGTTAAAACTAGAAGCCACTCAGGATTTCACTTTGCCCTGGATTTATAAGGTAT GCGTTAATAGCAACAATATACTCTCTACCTCTGACAGCTCAGATGCAGCGATTACTCGTTTCCCCTTTGGCCA ATCACCATCGTTGGGTTCCAGCTTTAGTCCGAAAATCGTTTTCCCGGAATGGTTTGAATCTGAATACGCATCA GACACCACGATCACGATTACCCCTCAATGGGTTAACCTGCCCACAGAAAACTTTTCATCGTGGTATGACGGA TATATTAATAAACCTGCCGATAATAGCGTATTTAAAATAGAGGGTTATTTACTTACTCATTATCAGGGAAAAATC AAACTCACAGAAGCTGAGACAGGAAGCGAAACCCAAGCATTATTCAATGGAAACAATGCACCACAAGGAAAA AGCCTGACTTTCACTTTACCTAATAGGTATAACTTCTATCCGCGCAACCATCAGTCAATGAAGATAGAAATAAA ACTCGTTAAACAAGACTTTATGCACACTCAACATAAGAGCAATCCCACAGGCAAAAAACCACCCTATACCCCG CAAATCAGTGCCTTACAGGTGGAATTCAATGCTACAGCTTTCCATCGAAAATTCTCCGTTTATCCTCTCACGC CTTTTGGCTGGGGCAAAACAGGAGAAAATAGCACACCATTAATTCATGATACATTTTATTTAGGCTTGACCGA TATATCACCAGAGCAAACTTTTTCTCTGTATTGGCAGCTAAAGGGCCTTAAAGAGCTACCTTTGTCTTGGTTTT ATCTAAGTGAAGAAAATAGCTGGAAATCATTAAATAGATCAACTTACAACCAAACCCACAACCTGTTTGAATCA GCAGAACAAAGTATCCTATTACCACGGGATGCTTCAAACCAAGCCTCTCAAATGCCATTAGGACGGTATTGG CTGAAAGCACAGATAGAACAGGAGAAAAAACAGATAAAGATAGCGCTTCCTGATTATTATCCAAGAATCAGG GGGCTGTTGTATAACGCTACCATCGCCACTTTAATCAACGCTGAAGCTGTTGAGCAATCTCACCTTATCAACG GATTGGCTGCTAACAACATTAAACAACCGGTTAACTCATCCGTTGCCATCAACGAAGTTATTCAACCCTGGAC ATCCTGGAACGGTCGCCCAAAAGAAACCGAGTCAGCATTCCTGGCACGAGTTCCTGCCCGGCTCTCTCATC GTAACCGAGTGCTAAGCTGGGGTAACATTGCCACTTTATTAAAAGAGAATTTTAGTAGCTTATTCGATGTCAA ATACCCTTCTGTCAGTGAATTAACCAAAATTCCAGCGCCAGAAAAGCGACAATTAACCATCATCCCCGACAAC CGCTATAAAGATAATGATGATTCACTACGCCCAGTATTGAACCAAGCCAGACTGACCGAGATGGTCGAATGG TTAGATCGATTAAGTAGCCCTTGGACAACTATTGAAATTAAAAATCCCACATATGTTAACGTTCTGATCCACTA TGAACTGATATTTACCTCGGATGTTAACCCCGATTATGGCCTCCATCAGCTACAACAAGAACTCAGTCGAAAA TATATGCCGTGGGGAGAAAATGCAGCTATTGGCGTAACACCCGGTAATCGTATTGACTACTTCCAGTTATTAG CCTCAATTCAACAATCACCGCTGGTTGAACGGGTCACCAACTTAACGTTAAAAAAAGGCAGCCAGCCTACCG TAAGTGAAAGTATAGAAGCCGCCGATGATGAAGTACTGATTTTAGTCTGGTCATAAAAACTTCCCCAACCTAA GGAATTAACAAATGAATAATCGAGATATGCTATTTCCTATCATTAAAGACGATATTACCTTTGATTCTTTATTCG CCCAGGCAAAAGCCGTTATTGAACAACAATCGGGGCAGCTCTGGAATAATACAGGTGAAAATGATCCCGGCA TTACTTTATTAGAAGCCTGTTGTTATGGCGCATCCGATCTGGCCTATCGCCACACATTGCCACTGCGAGATTT GCTTACTCCTCAAGAAAATGAACGAATAGATGATGGCATTTTTCCCAAAGAATTTGGTCCACAACAAATACTG ACCTGCGGCCCAATTACCGCGGAAGATTACCGTCGAGCTTTGTTAGATTTGCGTAGTGATAACACCGTTGAA GGTTATTTTTTCTTTAATGATGCACAGCTCATTCGTGAACCGGAAAATCAACGCTATTCATATTGGTATAACAA AGAAAAACGCGAATACAGTTTTACTCAAGACCAATACAGCGAACAATTACAGTTAACACTGAGAGGAAACTAT TGGCTCTATTTACTTCCCAGTCGGAAAACCCAGCTCGATAACACCCTGGCTGAAGAAAGACTCAACATTTTTC TGAAAGATAACCGAAACTTAGGAGAATCGGTCAGTAAAATTATTTGGCTAGAACCCATTAAACTGTCATTGAA AATTGATATTCAGCTTGATGATGACGCCAAAGATATTGCTGATATATTTGCTAAAGTTTATATGATTGCAGAAC AAATGGTGCTTGAAAAACCATTACGTTATACCACTCAAGCGATGAAAGAACTGGGTTACAGTCAGGAACAAAT ATTTGAAGGCCCTTATTTACACCACGGTTGGATACCGAAATTACCTCAAACCAAAGATTATACTCACCCTACC GTATTAAATCTCAGTCCTTTAATTAATCAGTTACTGGCTATCAAAGGGGTGAAACATATTACCCAATTTACATT GGATAAGCCTGATAAAAAAATTTCTAAGTTACCAAATGATAATTGGTCTTGGGAAATCGCTCCGGGATATTAC CCAAAACTATGGGGAGATACTCCATTAGAATTAATTACCTCACCAACAAGCCCACTCACCATCACGGCAAAAG GGGGAATTAAAATTGCTATTACTAAACAACAGATAGAAAAAAACATAATGACAGAACCACTAATTAATACACAG CCAGAATTATTGAACTGGGGTAAACATCGCAAAGTCCTGGATTACTATCCGATAAGCAATAAATTACCCGCTT GCTATGGATTACAAACTAATACCCAACAACAGCTACAGTTGCATCAATTTATGCTGCCTTTTGAACAAATGCTA GCGAATAACTGCGCTGAACTTGCTTTATTGCCAAGACTATTAGCTTTTAAACAACGAGGAAATACGGTACATG GCATTCAATGGCCTTTTAAAGAAAATACGGTTGGTCAACATGTTCATAAGGACATAGTATCTAATTTAAACAAT AATGCTACGAAAATCGATAATAATGCCGATGACTACGACAAGGAACTCGTTATTCTAGATTATTTGTTAAGATA TTTTGGGGCTCAATGTGCAATCCCACGACTATCACCAGACCCACCACAATCATCATTAACAGAACCTCAGACT AAAAAAGATTTTCTATCTACTCAGCGCGAATATCTGGCTCAACAGCCAAAACTGACTTATCAGCGTAACAATAT TCGGATTGATAAAGTATCAGCACTGCAAAAACGTATCGCTGCCCGATTAGGTCTGGGAGGAGAATGTTTCAA AGCAGAGCCTGACTTAGCTCACCTTCCTTTCTACCTCATTGAACATCGTAGGCTCTTACCAGTAAAACCTGAT ATAAAATTCTATATTGAGCAACAACCTAATTCTCTGGAAATTGAAAATGATAAATTAAAAATCACACAGAAAGAT TCAGCGGGTCGGTTACTGCAAGGTCAAGTTATTAACCTGGAATTTCGTGAGGGCTATGATGAATTTACATTGC TAAACTTAATGATAACTGAAGTGACAAGAGATACATTCACCATTAGCATTAATAATAGCCGTGATCTCAGAGAC AATCTGGACAAAGTGCAACACGCGTTTGAACAAACGAATAATCTGAGCTGGCACAATAGCTTAATATGGATGG AAGATATGGATTATCAATTGGTTTATGCCAATGGAGAACAACTGGAAAAAGCGGAAAATGAACGATGGATTAC CATTAACAATCAAAGTGCTTTCCCTGCTATGATCGGAGAGAATGATGAAATCACACTAAAAATTCAATCCGATT ATGAACTTAAAACCAAAGTCGTGCGGCTTGATTATAACAACAAAAAAATTCTGATTATAAAAGATGCGACATCA ATAAATAATTTTCCGCCAAAAAGAGAAGCATCATATTATTCTTGCTCTTCTCTAAAAGACAATGGGTACGGATA TTCGGATGAATATAAATATGAACTTACTTATATTGATACAGATTCTACAAAAGAAAATGAGTGCTGGATTACTAT CAGCGATCCAAATAATTTGTTTTCTCCTGATATCATCGCAGAGAATGACGAAATTATATTGAAAGCTAACCCTA ATTATGAGTTTAAAACGCACGTAGTAAAATTTGATCGTATTAATAGACAAATATTACTTAGGAAAAATACAGAC CTGGAAAATAATTTTCCATCAGAAAACAACACATCGCACTATCGCTGGCATTTCTCTGGTGAAAAATATGCCC AAACTGACCATTTTTCATTTGTTGTCAGTGCAGTACTGAATCGAGAATTAATTGAGAGGGGCACAGTCGATCT CTATAAATTAGAGTCTTGGGTAAAAACTGAGATTTTATCTGAATTACCCGCGCATATCTCACTCGTTATTCATT GGCTATCATCGGAAGAATTCGAAAAATTTGCCAGTACTTATAAAGTTTGGCAAAATAATGGCGCTCCTTTAGG

[0677] TGATCACGCATATAAAATTCTAGAAACATTAACACTTGGGAAAAAACCTTCTACTTCAGCAAGAAGGTCCAGC

[0678] AGCTATATAGAAGCACAGTAATAATTCTTACAGAACATTAACCCATATTTATCTTATAATATCAAACATCATAAA

[0679] AACAATCTTCAGCTCATTATAATGACATATTTCATACTCAGGTTTCTTCATATCTGTTAATTACAAAGAGAATAT

[0680] TAATATGATCTCAGCACCAAATCTGTTAAATCGGATTATCATTACTATTGAAGCGAATAACGCACAGGCAGCTA

[0681] AAAAAGTATTGCATGGCTCCCTGCTTAATCAATCCAGTATAAACAAACTCTTTGATTCATACTTTAACCAATAT

[0682] GTTGTTAATCAGACTATCTACCTGAAGACACTCACCCTGAATCTTGGCGAAATACGATTAAATAGTTTTAATTC

[0683] ACAGTTTGTTATTCGGCTTAATACTATTCTGAGTCAAGCATTGAGCCAATATCAGGTAAATAATCAAACTGATA

[0684] TTGAGAAATTTATTTATTACTTATATCGAAAAGATTCTATATTAAACCCAATAGAGGAAATCAATAATCGTGAAA

[0685] TTACTGACATCAATATTAAGCAATTAATTAACCAATTACCCCAGATACAAAACAATTGGACACTATTATTGGCA

[0686] AAAAGCTGTTTATCCACACATAGCCTGAAAAAACTCCTGGCTATCAAAAAAACAGCTTTATTAACCGCCATTAA

[0687] TCGTAAATTATCTGAAAAGATCAATATATCACCCTATCAGCAGGAATCGGTTTCCACCTGGCAATTGATACTGA

[0688] ATGCGCTGAAATATATACAGCGACATAATACACAGGAAATACCTGAACCCGATGCGAAAGTCATATCACTCAT

[0689] TACAACGGAACTCAATGACAATGCCATTAATACAGCACCAATTATTGCATTATTTCGCCAAGTTATAACCAACC

[0690] ATTCCCCACTGAATAAGTGGCTGGAACAACTGTGGCAAACAAAGCGAATTTCACAGTTATGTAAAAAACAGCT

[0691] GTCAATTGAAGAATACCAACATCTATCGGAGCGCTTTATTGCCAAACACGGGAATAAAAATAAATCTGATAAA

[0692] AAATCATCCATGACTTCCGAACCGCTGTTATTACCTGAACACCCTCCACCACGTCAGGTCAATAATGCTGGAA

[0693] TATTAGTTCTGTGGCCGATGTTACCTACTCTATTTAACCAATTCGGCCTGTTTGAAAAACAAAAATTTATTCATC

[0694] GTCAAGCTCAATTTAGGGCTGTTAATCTACTTGATTATCTCATTTGGGGAAACGAAGAAACACAGACAGAACG

[0695] AAAAATATTGAATTGCGTTCTGTGTGGGTTAATTGCCGATGAGGACACGGAATCAATCCCTATTGAGCCAGAA

[0696] AAACAACAGGTAATAGAACAATGGTTAGATGCAGTTATCAGTCAACTTCCTGCCTGGAAAAAATTAAGCCGCA

[0697] ATGATAGCCGCCAATTGTTTTTACAACGCCCGGGGGAATTGCTGACAAATGAGCAGGAAATCAAAATTACGG

[0698] TACAACCTCAACCATTCGATGCACTGTTAAATAACTGGCCCTGGCCGTTAAATATCGCCAAACTTCCCTGGCT

[0699] GGATCGCCCTTTATTAATCAACTGGTAAAACATTGACAAGGTTTATATGAAAGAACATCAATATAGAATAGTCG

[0700] ATCTACGCTGGATTTATTCCCATTTGGAGCGCATCGATCTGCTGTTACAACGTCACTATTACCAAAAGAGAGA

[0701] CAAATACGATTCATTGCCAGAAAGTTTTTTGCTTGAAGAAGATGAATTAGAACAACGTCTAGCAAAACCGTTG

[0702] GGTATTCCTCATTGGCTAACAGCAAATACCGGCGCTGGTGATACAGAAACAGAAAATCATTCTGCTTCCGGC

[0703] ACATTATCACTGCTAGTCACGCGTTTTAAACTCACTGAATTTGAACGTGATGTGTTATTGCTAGGTTTATTACC

[0704] GCATTTTGACAACCGCTATCATGCGTTATTTGCTACTCTGCACGGTAACAGTAAAAAACAGTGGCCCAGTTTT

[0705] GATTTAGCGATTGAATTATTTAGCCAACATCAAAGTAACTGGCAATTATTTCAACACCACTTTTTACCGCAAGC

[0706] TCCATTAATCAATCACCATTTATTACGACTCAATAACCAAGAGGAACCCATTTGGCTACAAACTCAATTTTTAA

[0707] CTCACAATGCAGTCTGGTCTTTTTTATCCGGTCAGCGCGTCATTTTACCTCCCTTAATATCCTGCGCTTACTG

[0708] GCATATTCCAACCTCACAGACTTGGTATCCACCAATCCTTGGTCATGCATTTGAAAAAATATTGCTGAATGAAA

[0709] CGGACGAAATACGCCCGCTGGTGGTTCTTAAAGGAAAACAGGACAGCGCCAGAGAACTGGCAGTCAGTAAT

[0710] ATTATGGGAATTCACGGCATTAACACTTTAACGTTCGATTTATTTCACCTGCCAGATGAAGAGTGCACCACCT

[0711] CAATACTCAATCTGCTAATAGATGCAATACGAGAAACCCGGCTACATAATGCCTGTTTATTAATCCGTAACTTT

[0712] TCTTTGCTGGCAGAGGAAAAGAGAATATCGCATAGAGAATTATCAGCTCTACTGAATCAACCCAAATTACGTG

[0713] TGGTTTGTCTGGCAGAGTCAGAAGAATCATTAGCATGGGTTAAACACCTGCCGATAGTGCAAATTAATATGCC

[0714] ACCGGCGACGCTGGCAGATAAAAAAACGATGCTGGAAGCCAGTTTGCCAGATAATGTCACTAAAGGAATTAA

[0715] TATAACTCAATTATGTCAACGTTTTTCATTTACAGCAGAAACATTACCGTTAATTATCAAGGAAGCTCATCAATA

[0716] CCAAATCCTCCGACAACCGGAAGATCAATTGAAAGAATCTGATCTACGTAAGGCATTAAATTGCCGCGCCCA

[0717] ACAAAATTTCGGTAAATTAGCCCAGCGTATGACACCAAAACGAAGTTTTAATGATTTGGTTATTTCCGCTGACT

[0718] TAACTCAACAGTTGAAAGAAATCATCGCAGCAATTAATTACCGTGACCAAATTCTGGGCGCAGGTTTTCGGGA

[0719] AAAAATCAGCTATGGTACTGGTATTAGCGCCCTATTTTACGGTGAATCCGGGACGGGGAAAACCATGGCCGC

[0720] AGAAGTGATTGCCAGCTATCTTGGTGTTGATCTGATTAAGGTAGATCTTTCTACCGTGGTGAATAAATACATC

[0721] GGTGAAACCGAAAAAAATATCTCCCGTATTTTCGATCTGGCCGAAGCGGATTCCGGGGTGCTGTTTTTCGAT

[0722] GAAGCCGATGCCTTATTCGGTAAACGCAGTGAAACCAAAGATGCCCAAGATAGACATGCCAATATTGAAGTTT

[0723] CTTATTTATTACAGCGACTAGAAAATTATCCGGGATTAGTGATTTTAGCGACTAACAATCGCAACCATTTGGAT

[0724] AGTGCGTTTAATCGCCGCTTTACCTTTATTACCCGCTTTACTTATCCCGATGAAGCATTACGCAAAGCAATGT

[0725] GGCAGGCAATTTGGCCTGAACAACTTAAGTTATCAGATCAACTTGATTTTGAGCATTTGGCTAAACAGGCAAA

[0726] TCTGACCGGTGCTAATATCAGAAATATTGCCTTATTATCATCAATATTAGCTACAGATAATAATAGTGATCAAAT

[0727] TGAAAATAAACATATAGCGCGAGCATTGATACTTGAATTAAATAAAACGGGCCGATTGATTTTTTAATCATTTA

[0728] TACCCAATAAATTTCGAGTTGCAGCGCGGCGGCAAGTGAACGAATCCCCAGGAGCATAGATAACTATGTGAC

[0729] TGGGGTGAGTGAAAGCAGCCAACAAAGCAGCAACTTGAAGGATGAAGGGTATATAGAATTGGAGTGAATATG

[0730] ACAAATATAATTAACCCTAATAATGCGATTCTTGAAGTTAATAACGCATTAAATGATATTTTATCTCAGTATTTA

[0731] ACTAATATTGATATCCGCTTTGATCTACCAGAAATAAATTCAATCCCATCAACCCCTACAGTGAGTATATTTCTT

[0732] TATGATATACATGAAGACCTACAATTACGTTCTGCTGAACCAAGAAGTTATCATCCTACCACCAGCTCATTATT

[0733] GCCGGGATGGGTAAATATTAATTATAACTATTTAATTACTTACTGGCATTCAAGTAATCCATCAAGCGACAGTT

[0734] CTACCCCTGATAGTCAACCCAATAATCAAGCGGCACAAGTCATGACTGCTATTTTAAATGCATTGGTTAACAA

[0735] CCGACAATTACCTAAAATTCCTGGCGCATATACCAGAGTCATTCCACCTCAAGAAAATCTAAATAGCTTAGGT

[0736] AACTTTTGGCAAGCGCTTGGCAATCGCCCTCGCCTTTCTTTATTATATTCAATTACCGCACCGGTAAAACTGC

[0737] AAAATATTAAAGATGTCATAAAGCCCATTAGCCAAATTTCCACTTCTGTGGATCAAAAATCAAATCTGGATAAT

[0738] TCGCAAATCAACCAAGCCTTATTTAGCAAATTGGGTGCCGATTTAGGTGGCACACAAGATGTTCGTCTTGCTC

[0739] TTGCGAAAGTGAATCTGACAACCAAACCTGCTAAAGAAAATAATGAAAATCAAAATAATAAAAATGTAATTATT GAAGTTTCTGGCATTACCCATTTGGATTATTTACCCAGAATAAAAGGTATTCTTTCAACATGGGTAAATAGTCA TAGTGCTGTTGTTAGGATAAATGATATTGGTATTATTGTTTCAGAATATAAATATGATAAATTAACAGGCGTTTA A

[0740] SEQ ID NO: 19 (Photorhabdus asymbiotica strain ATCC43949 PVCPaTox operon, pyc1 - pyc16)

[0741] ATGAATACAGCTCAAGAAATTATTAACCGTTTATCGGGGAGAGCCGTTACGCTTGGTTGGGATGTTGTTATTG CTTATGACCGAAAAAAAATTAACACTCTGTTAGAGCAACAATATGTTGAAAAGGTAAAAAACGGGGAGAACTT CCCGCTTATCAACTGGGAGAACCAGAGAAAAACACTTCAATTTAAAGATCTTCAATTAGGTGTTCCACTTATTT CTTTTGAGAATTCAACACTGGAAAATTCAAGGGCGCTTGCCACGATAGAATTTATTTCAGGAGCTATTATTGAA TTTAGTGACTCCGGGCAAATAATCAACTATAAGAAGATTGAACCTAGTCATGGTTATGGCATGGTGCTGACTA TCGATCTCATGGCTGGTACAGGTTCAGTAGAAGAACAAGGTCGGGTGATAATAAATCTTAACGAAGGCGCCA TACTCGATTTGCATGTTATCCAACAACCGCCAGCAGAAGTGGTAGAATTTTTCCGCACTTGGTTGATGGCTAA TAAAATGACTTATGAATTAGGTAAGCTGGATCTGAGTAGTCAAGCTGGTCTAGTGCCTCGTTCTTTTCGTATTC GTACTCAGCGGGCGCCTGAAAAAATTCGTAAAGCGACGAGCGATGAAGGAAATGGCGCTGTTTTGTTGTTTG TTGCCACTAACTATAACCCTACAAGTGGAACTTTACCTGCCAAGGATTATCCGTGGCTAATCCCTGAGGAATA TTCAGGCGCATTGCTTATCGGTAATAAATGCTTATTTAAAGACATTCTGAAACCGAATCTGGATCAGTTGTTTG ATAAAGGGGAATGGACATTAAAAGTTCAGCAAACGGATTCTGATCAACTGCTGCATTATCTGGAGGCAAACTC TGCATATATAACAGATAAGCCTTATATGGCAGACTTTGAAGGAACTCAGGATGGAGTCTGGACAGGACGTTAT AAATTTGAGACTGGCCGGGGACATTATGGGGTGTATGAAAATGTACGCTTTCCTATCAATGGAATGTTGATGA AACCGGCTAAAACTGGATTACAGTTATCAATAGATTCACCACAAAGCCATCAATTTAATGTTGATTTCGGAATG AAGTGGTTCCATTGTGCTAATATAATGTGTGGTTATTCCTGGTTTAACGAGACTTACCCATTTTATCTTGATGG AAAATCATTTTATCAAGTTCATATTGACCCTGATAAAGAGGTGATTTATTTTACTGGGCCAGATGAAGATATTA ATATTGTAGGAAATTACAGCCCGCCTGCGTGGTGGCAATCTAAATGGCAAAAACATATCAGTGATGATTTTAC GGATATTTCCTCGGAAAAATTTAAGCGACTCAGTCAAATAAAATTGCCAGAAATATGCATGTTTGCCGTGAAC CATTTATTATTTCCTGGTCATAATACTTTGCTGTTGAAAGACGTTTATTTACCGGGTGATATGGTGATTTTCGG TGATATTAACCCATCACTTACCGCTTTTCGGGTTACGCCATTAAAAGCAACAGTGGTGGCAAAGGGAACCCAA CAATTTAAAGCCATAGAAACTAATTGATGATTATACCCTTCATCCTTCAAGTTGCTGCTTTGTTGGCTACGTTC ACTCACCCCAGTCACATAGTTAGCTATGCTCCCGGGGATTCGCTCCCTGGCCGTCGCGATGCATCTTGAAAT CCATAGGGTATATATTTAATTGGATAAGTCTTTTTTATTTTAACATTATAACCTGATTCTTTTTGGATAAAATTAA AGGATTATTAACATGTCTATTACACAAGAACAAATCGCTGCTGAATATCCTATTCCTAGTTACCGTTTTATGGT TTCTATAGGAGATGTGCAAGTCCCTTTTAATAGTGTTTCGGGATTAGATAGGAAATATGAGGTTATTGAATATA AAGATGGCATTGGTAATTATTATAAAATGCCAGGACAAATACAGAGGGTTGATATTACACTTCGGAAAGGCAT ATTCTCTGGGAAAAATGATTTATTTAATTGGATTAATTCCATTGAACTCAATCGGGTAGAAAAAAAGGATATTA CAATTAGTTTAACTAATGATACTGGCAGTAAAGTCTTAATGAGTTGGGTTGTTTCGAACGCCTTTCCGAGCTC ACTGACGGCCCCTTCATTTGATGCTTCAAGTAATGAAATTGCAGTACAAGAAATTTCATTAGTTGCTGATCGG GTAACAATTCAGGTTCCCTGATAACTAAAAACTTTAAGGAAAAATAATGTCTGTACAAACAACTTATCCCGGAA TTTATATTGAAGAAGATGCATCATTGTCTCTATCTATCAATAATAGTCCAACAGCAATCCCTGTTTTTATCGGTA AATTTTACAACTTGGATGGTTCCTTACCTAAAGTGGGAACATGTTCTAGAATTACCAGTTGGTTAGATTTCACT AAAAAATTTTCGGTAGCTCCTCCTCAAACCATTTCATTGATCGCGTCGCCAATTGCTGACACACAAGAAAGTG TACCCAAAGCAGTTCAATATACTTATAAGGCCGAGTTTGAAACCTCAGAAAATCTGGCAAATGGTGCCTATGC GGTACAACATTATTTCCAGAATGGCGGTGGTATTTGCTATATCATACCTTTAGTTAGCGTGAAAAAAGAGGAT GCTGCGATTGAGTTAACAAAATTACCTGAATTAATTGAAAGACAACAAGAGATTACGTTAATCGTCTGCCCGG AGGACGATAAGACGCTCACTGTTGATAGCAGTAAAAAATCGGATGTTTATAACAGCATCAATACATTATTGAG TAATAAGGTAGGTTATTTTCTCATTGCAGATTCAGATGATGGCAAAGCAGTTCCTGATACGTTGCCGGAAAAA ACTGCGGTCTATTATCCTGGTTTACTAACTTCTTTTACACAACGCTATGCCCGACCTGCCGATTCTGCTATCAA AGTGACCGGTATTACAAATATATCAACTCTGGCTGATATTCACACCAACTTGGCCGATGACTACTCAACAGCA AGTCAGGTTATTAATGATGTTTTGGAAAAAAATAATAAGCTCGCATCGTCTCCCATTATTTTACCTCCCAGCGC CGCTGTTGCTGGTGCTTATGCCGCTGTTGATGTGAGTCGTGGTGTTTGGAAAGCACCTGCGAATGTGATGTT AAGTAATGCCACGCCAATCATTAGTATTTCCGATGCGGAACAAGGTGTGATGAACCCATTAGGTATTAATGCT ATTCGTAGTTTTACTGGTAGAGGTACTTTGATTTGGGGAGCTCGTACTCTGGATAAAACGGATAACTGGCGCT ATGTTCCTGTACGTCGTTTATTCAATAGCGCAGAGCGAGATATTAAGTTAGCAATGCGTTTTGCAGTTTTTGA GCCTAACTCCCAACCAATTTGGGAAAAGGTCAAGGCTGCTATCAATAGCTATTTGCAGTCACTTTGGCAGCAA GGTGCACTGCAAGGCAATAAACCCGATGAAGCCTGGTTTGTACAAATTGGTAAAGGCGTGACCATGACAGAT GATGATATTAAGAATGGGAGAATGATTATCAAAATCGGCATGGCGGCAGTACGTCCGGCAGAATTCATTATTT TACAGTTTACGCAGAATATCGCCCAGTAACTTAGGTCTATACCCTATAGATTTCAAGATGCATCGCGGCGGCA AGGGAGCGAATCCCCGGGAGCATATACCCAATAGATTTCAAGTTGCAGTGCGGCGGCAAGTGAACGCATCC CCAGGAGCATAGATAACTATGTGACTGGGGTAAGTGAACGCAGCCAACAAAGCAGCAGCTTGAAAGATGAA GGGTATAGATAACGATGTGACCGGGGTGAGTGAGTGCAGCCAACAAAGAGGCAACTTGAAAGATAACGGGT ATATTTAATATGGGCGATTTATTGCCCATTTTTGTGAAAGGAAATGAGTTATGTCGCCAACGCTACCCGGTGT

[0742] AACGATGACTCAGGCGCAGATAACAGCGTTCGGTGTCAGTACATTAAATATGCCCGTATTCATAGGGTATTGT ACGAGATTGCCTGCCTTTTCAGCGCCTGTAAAAGTAAACAGTTTAGCTGAAACAGAACAAATAATAGGGAAAG AAGGGCGTTTGTATGCTCTATTGCGCCACTTTTTCGATAACGATGGGATACAAGCTTTTATTCTGTCGTTAGG CGCACCTGCTGGGGAAAATGCTAATAGTTGGCTTGAGGCATTACAACAGCCCGATTTGTATGCGGCTGTTGC AGCAGAGCCGCTAATTACACTTTTAGCCGTCGTTGAGGCAAGTGAACTGAACCAAAAAGAAGGTAATGAGGC TGTGGAAGCTTGGCGACAGTACTGGAAAGCAGTATTAGCGTTATGTCAGGCACGCAGTGACTTGTTTGCCAT ATTGGAGGCACCAGATGATACCGCATTAATCAAGCGTAGTTTGCAGGATTTTCATCATAAGGCACGTCAGTTT GGCGCTCTCTACTGGCCAAGGCTAGAAACATCTTATCAATCCTCTCAGTTAAAAATTTTGTCTCCTATTGGTG CAGTAGCAGCGGTTATTCAAAGTAATGATGTCCGGCGAGGGGTAGGACATGCACCTGCCAATATAGCGTTAA AACAGACGATTCGCCCGATAAAGTCCCGCCTGGAATTAGAAGAGTTGTATGAAGAATCGGATGGTTCACTGA ATCTGATTTGTAGTTTTCCAGCTCGTGGTACTCGTATTTGGGGATGTCGTACGTTGGCGGGTATTGATTCACC TTGGCGTTATATTCAAACCCGATTATTGACTTCACACGTGGAAAGGCAACTCAGCCAGTTAGGGTGCATGTTG ATGTTTGAACCTAATAACGCAGTCACTTGGATGAAGTTTAAAGGCCATGCTGGGAATCTATTAAGGCAGCTTT GGTTACAAGGGGTGCTGTATGGGCAGCGTGAAGATGAAGCCTTTTCCGTTGAAATAGATGAAAACGAAACGA TGACTCGCCAGGATATTGATGAAGGCAGAATGATTGCTCGTATTCATTTGGCATTGTTAGCACCGGCAGAGTT TATCGCTGTGACTTTGAATTTTGATACTCGCTCAGGCATTGCGACGAGTACATAATAAATCGGAATATCTCCAT GACACTACCAGCAGAGCTTTATACCCCAGCGGTTTCACATCGTTTTATTGTTAATTTTCTTTTTAAAGGTTTAC TTCCTTCTCCCGTAGATATTCGATTTCAACGTGTTTCTGGTTTAGGGCGTGAGTTACAGGTTGAACAGCGCCA TCAGGGGGGAGAAAACGCACGGAATCATTGGTTGGCTGAACGTATACAGCATAATAGCTTGATATTAGAAAG AGGGGTTATGGTCGTTACCCCTTTAACACTGATGTTTGATCAGGTGATGCGGGGGGAAACTCTCAATTGGGC AGATGTGGTAATTATTCTTCTCGATCAGGCTCAACGTCCGATAACAAGTTGGACCTTGAGTCATGCGCTACCG GTTCGCTGGCAAACAGGAGATTTAGATGCCAACAGTAACCAAGTGCTGATTAACACCTTAGAGCTGCGTTAT GAAGATATGCGCATTATAGGGGTAAAATTATGACTATCGAAATCCGTGAACTCATTGTTCAAGCCCGTGTTGT CGGGACTGATACCAAAACAACACGAACCGTTCCTTTATCTATTGTGCAAATGGAAACACTTATAGAACAACGT CTGGTTGAAAAAGTGAAGCGGGAGATATTAGACGTACTCCGGGAAGAACAAGGTGGTGGGTTATGAGCTTG CTTGAACGAGGTCTGGCTAAACTCACGATTACGGGTTGGAAGGAGCGTGAGCGTAAACATCAGATTGGTAAA CTAGAAGCAATGTATAACCCGGAAACACTTCAACTGGATTATCAAACTGATTATCTCCCTGATGTTAGCAATAA TCAGGTAACAGTGAGTAACCGCTACGTTTTGTCAAAGCCCGCAGGGTTAACACTATCCTTGTTATTTGATGCC AATATGGCTGGTCTTACGACAACCGTCGAGTCCCAAATCACTACCCTCAAATCGCTTTGTTTAGTTAATGCAA GTACTGATGAACCCAATTTTTTGGAAATTAATTGGGGGGCAATGCGTTGGGAAAATAAAAATTATTTTGTTGGT CGGGCTAGTGGATTGTCTCTGACTTATTTGCGCTTTGATCGTAACGCAACACCATTGCGTGTGAGTGCGCAG CTCACATTAGTCGCAGATGAAAGCTTTGTGCTCCAGGATAACCAAGCCAAGTTAGATGCGCCGCCGGTATCA GTAGTTAATGTCCCGGATCTGACTTCATTACCTGCACTGGCGAATATCGCTAGCGTAACCACTATGTTGGGA GTGGATTATTTAATGTTAGCCCGCACCAATGATATGGATAATTTGGATGATATGCAGCCAGGTCAGACATTGC GAACACCGGAGGCATCATGAGTTTTTTAGATAACAGTAACTTCAAGCCATCAGATATCAAACTGTTCGTTAAC ATTCAGGGAGTGGAGAAGGAACTCAACGAACTGATAGTAAGCGAATTGAAAATCTCCCGACGTATCAATGCC ATTCCGCAGGCAGTTGTAAAGCTAAGAGCGAAAGAGAGTGAAAGTGGTGTATATCAGTCTGATGTACAGCGG ATGTTGAAGAGTTGCCGTCCGGGAGTAAAGGCAGAGCTTCGTATTTTGAATACCCGGCTATTCAGTGGCGAT ATTGTGCAGCAAAAAACAGAGTTAGTGTATGCGAAAACACACACTATCAAATTGGTGCTACGCCATGACTTAC AGCGCATCACCGGTAATTTTCGTACCAGAGTGTTTGCGAATACCCGTGATCGTAAAGTGATAGCCGATCTATT GAATACCGCAACATTAAAGCCGGCATTTTCGGGGACATCACATTGGGATATAGATCATGAGCAACTGGTTCA GTATCGTTGCAGTGATTGGCAATTTTTGTTGCAACGGCTCTATGCTACGAATAGCTGGTTGTTAGCTGAAGAA GATAAAGATAACACTCAGGGGAAAGTGACCATTATTGCTCCAAATTCTTTGCCCCTGAATGAGCGTTGGACAC TGCAACATCAGGCTGATCATCAGGCTATCCGGCTTTACAGCACGGAGCTGATGCTGGATAACCGGTTTGATA CAGCGGAGGCTGTTGTTAGTGCTTGGGATATTGATGATCAGGCATTACTCGTGGCGTGGAAAGAAACCCTTA GTCAAGTTGGGAAAGATGCGTTAGCGTCAGATAATTTTAGCCAGACAAATAAAGATTCGAGTGAACTGTTATT AAGTTGTCCGCTCTCTACAAAAGAAGTTCAATTTTTAACGCGTAGCCAATTAGTCATGCGGCGCTTGACGGCC GTTCGTGGTTCACTGAAGGTTGAAGGCAGTACTAAGTACCGTTTAGGGCATGAACTGATGTTGTCAGGTTTT GGTGAAAATATGGATGGCTCACAAATACTGACGGGAGTGGATCATCGAATAACGGCAGAAGAAAGTTGGAAA ACAACCTTACATGTGGGATTAGAACTGCCGTTAAAGGCAGAGTATGTCACTCAGGTTAACGGTGTTCATATCG GCAAGGTTGCTGATTATCAATCAGATAGCAAAAAATGGGATCGTATTCCTGTTTTGATCCCTGCATTTGGAAC GAATATTCCCTTGTTTGCCCGATTGGGAAAACCCTACGCCAGCCACCAAAGTGGATTTTGTTTCTATCCTGAA ACGGGTGATGAAGTCATTCTCAGTTTTTTGGAAGGGGACCCTCGTTATCCTGTCATTATTGATTCCCTGCATA ATCCTAAACAACAGACTCCATTGCAAATCAGCAAAGAGAATAATCTCAAAATGTTGATGATTAAGCAGAGCGA TAAAGATGAGCAACAATTGTTATTTGATAGCCAGCAACAAACAGTCGCGTTAATCGGTAAGAAAAATATCGAG

[0743] GTTAAAGGTGAGTATATCAACCTGACTAAATCAAAGGGGACTCGATAATGGCAAATACGCTTATTGGCCAGGT ATATGGTCAAGGATGGGCTTTTCCCATTAAATTTATTCCTGATAATAAAGAAACCGCAGATCAAACAGCCGGT ATTGTTATGGCTCAAGGGATTGAAGATGTCAGTCAATCGCTGGAAATATTATTTCTTACCGAGCCTGGCGAAC GAATTATGCGTGAAGATTTTGGTTGTGGTTTACAAGATTTTGTTTTTGAAAATATTAGTGATACGCTAATTTCTG CCATCAAAAATCGTATTCAGCAAGCAATATTACGTTATGAACCTCGCGCATATTTATTGAACGTTGATATTCAA ACCAAAGAAAACCAACCTGGACATCTGCTCATTCAGATTAATTGGAAATTACGTGGTAGTGATATATCTCAGC GTTTAGACGGAGTGCTTAGACTCCATTCAGGTCAAGCATTGGAACTGTTATGACCAATTATATTATTATCGAC GGGGATCTCATTCAAATAAATCCCAAATTTGAGGGTGATCGAACTCTTACGATTAATGGTATTCCTAAAATAAG CGGGAATGGAGATGCGCAAATTGAAGGAAAAAATATTTGTGTGTCAGGTGATCACTTAACTGTCTCAATTCCA GCCATTTATATAACCTCCAGACATCCTGTTGCAGGTAGTGGAAAAGTGAAAATTACAAATTTATCTGACGACC AACTAGCAGAATTTTGTGTTAGTGGGGATGTTGTGATTATTGAAGGCAGTCAGTTTGAAGCTCAGTTTACACC GGATAAGCCGGCCACTAATCCAAGTAACCAAGATGCAGATAATCCTGCGCCTTCGAATGGGAGTGGGAGATT TATACACTCACAGAACTTCGTTAAGGCAGAAAAATAAAAAATTTTGCCGAAGCGGTTAATAAGTATGAATAAG CGGGGCGGATAAAAACATGGATCTTGCTGAATTAAATAATACGTTGATGAATGACTTACCAACGACCAATTTT AAGTTAGAAACAAAGGACCCATTAACGCAATTAAAGTGGTTACAACGTTATACAGAAAATATTCGTTTTTATGC GAATGATGATTATTTCTGGCATCAATTCTGGTTCTTAAAAAATCACACACCAGAAGCGCTCTTTGCTCGTTTGC AAGGTGAAACGTTGGCTGATGGAGAATTGCCTCCTCATCAAGCGCTATTGCTGGCCTTTTTACAACAGCTTAA GACGCCAGGAATCATGCTTGATACTTTTTCAGCCCGTCATCGGCAATTGTACTATCAGGAATTGCTAGGGATA ACGCAGAAAGATGCACAACCTGATCATGTGGCGCTTGGCGTGGTATTAAGTACTGGTATTGCAGAATATTTAT TACCGACAGGCACATTAGTGGATGGTGGACAAGACAGCAGCGGAAATTCACTGCAATATGCGTTGGATACCG

[0744] ATTTATTGGTTAATCCAGGGCAATTAACAGATGTTCGCTACAGCTATTTGGATCATAAGACCTATAAAATCTTC

[0745] ATCTTGCAAGATGATAAAGCGAATATCAGTTGGCCCTCTTCAGGCGCTCGTTTATTTGTAGCACCTGAGGGCA

[0746] ACGGACAGGAAAAGGCACCTGAACAAAAGTTGGCACTTTACCTGGGATTTGATGATATACAGCCAGGGCAAA CTCTTTCTTTATTTTGGCAATTCATTGCATCAACTCCCCTGACATTAAAATGGTTTTATCTGAACGAGATAAATA ACTGGGTGAAGCTAGATAGTGTCAGAGATAACACGGATGGCTTTTTTATCAGTGGATTATGGCAAGCGATATT ACCTGATGATGCGGTGAAAATGTATTTTCCAGAGACAACTTCTGTAAAACGCTACTGGATTAAAGCTGAGGTG

[0747] GAATCGCTTACTGAATCTGGCGATTTGTGGCAACCGCTATTAGAAGGCATCTTGTATAACGCTCAAACAGCAA

[0748] CGCTGGTTGATGCAGACAACACAGATGAAAAGCACTTTCATGATGGGCTGATGCCTTTTAGCGTGCAGCATT

[0749] TGGTCAACACCGTTTCAGAGGTAAAAAAAATTGAGCAGCCCTGGTCTTCTTGGGGGGGAACGCCACAGGAA

[0750] GACACTACTGATTTCTTCCATCGAGCGGCAACACGTCTTCAGCATCGCCAGCGTGCGTTAACTTGGGATAAC CAAATTGCCATGTTGAAGGCTGAATTTCCGCGGATTTATGATGTCATCTCACCAAATATCACGTGGATGAACC

[0751] AACTTCAGACATCAAATACGCAAACGCTGATCGTTATTCCTGATGTGAACTACAGCGACAACAAGGATCGCTT ACGGCCACAATTCAGCCCTGCCAGCTTGCGACAAATGAGTGACTGGTTACAGATTCACACTAGCGCATGGGC GAATCCACAAGTGGAAAATCCAATTTATATTGATGTCTCTGTGACCTATGAGGTGCAATTTAGTGCGGGTGTG AATCCTGATTATGCCCTCCGGCAATTACAACAATGGTTGAGTTCAATTTATATGCCATGGTATCACGCAGATA

[0752] AAAAAGGTGTTGCCGCTGGCGATCAAATCGATTTTTACCAACTGTTTGCAGATATTCAGCGAGTACCTTACGT

[0753] GGAGCATGTCAAAACATTGACATTGACCACAAAAGACACCTCATTAACCAATGGCGGGGTTATTAAGGCACA

[0754] GCAAAATGAAGTGCTGGTGTTGGTATGGCAACAAGGAGAACAAATTAGGCAGGGAGAATCGAAATGAGGCA

[0755] GCATAATGAGTTATTTCCTGTAGTAAAAGACGCGATAAGCTTTGAAAACCTGCAAGCTCAGGGTGAGAAGGTT

[0756] ATTAGTGATCAGTCCGGTAACATATGGAGCGATAAAGATAAACATGATCCTGGTATAACATTACTAGACTCTTT

[0757] AAGTTACGGTGTTTCGGATTTAGCGTATCGGCACTCATTACCTTTAACCGATTTATTAACCATTGCTGGAAAAG

[0758] ATACGCTTTTTCCAGCCGAATTCGGGCCACAGCAGACGCTAACTTGTGGCCCTATAACACTGGATGATTACC

[0759] GGCGTGCGTTACTTGATTTACATGGTAATGATGCATTTAAAATATCAGCTAGTGACCCCAGAGACTTTTTGTTT

[0760] CAGGATATACAGTTAATTTGTGAGCCAAAAAGTAAGCGTTATAAATACTATTTCAATCCCGAAACGCTTGAATA

[0761] TACATTCACGCCACCTTCAGGGGATAAATTTAAAACTTTAACACTACGAGGGAATTATTGGCTTTATTGGATAC

[0762] CAACCCGTTGGGCAGGTAAATCAGCTAATTTGCCGTTAGTTAAGCGGGTGATGGAAGATTTTCTCCGTGAAA

[0763] ATCGAAATTTGGGGGAAAATGTTGTTCAAGTGACACGGGTGATATCAACGCCTATTTATCCTGAGCTGGTCAT

[0764] TGAGCTGGCGGATGATATTACAGATGCGGCATCAGTATTAGCATCAATCTATATGCTATTAGAACAGTGGGC GATGCCGATGCCTGCTCGCTTTACTACCGAAGCATTACAGGCCAAGGGATTAACAAACGAAGAGATCTTTGA

[0765] TGGGCCGTGGTTGCGTCATGGTTGGATACCTCAGTTACCGACCTCTCAAAACTACCATACAGGCATGGTTCT

[0766] GAAGATGAATCATCTGATTAACCAATTGCTGGCGGTTGAAGGTATAAAGCGCGTAGTTAGCCTGACGTTGCC

[0767] AGAAACAGAATATTTGCATCAGATAAAAGATGATAATTGGTCCTGGCAATTAGATGTTGGTTATTATCCATTAT

[0768] TATGGGGAGCTAATCCACTAGAGGTAATTACAGAGAAAAATAACAATTATGTCAAATTGTTCGCAAAAGGTGG

[0769] GGTACGATTACAACCTGATCAGAAAAGTGTTGAGCGGTTATTATCACAGGAATCACTCATTAATAATGCTGCA

[0770] TCCACGTTACCGGCTGGTAAGGTGCGTGATCTCAAAGCCTATACACCTATAAGCCGCAGGTTGCCTGCCTGT

[0771] TATGGTTTGCAGAATACTTTGCAAAAGTTAAAACCTGAACAACGACACTTATATCAGTTCCTATTACCATTGGA GCAAATGCTTGCTGATGGATGTGCGCGGCTTGCATTTTTGCCACATTTGTTAGCATTTAGGGACCGAAGCGG AAATATCAGTGATACACTCTGGCCTTTCAAGAATACAGAGGACACAATTGCCCAACAGGTTCATCAGGAATAT

[0772] GCCGGTACATTAAAAGCCTTTCAACAGCAGGAAATTAGCCTGTTTGATGATAAAAATAGACCGCATCATGGCA ATATCAATCGGGAATTAGATATTCTTGATTATCTGCTAGGGTATTTTGGTACACAACGTGCAAAGCGTCCATTA ACGCAGGATATTCATGATTTTCTGCAAACCCAGCGAGGTTATTTGGCACAGCAGCCGGAGTTGGGTTATCAG CGTGATAATATCCGTATTGATCGAGTTTCAGCTTTACAAAAACGTATAGCAGCCCGAATTGGGCTAGATGGTA

[0773] CTATTTTCAAAGAATCGGTTGATTTAAGTAAGTTACCTTTTTATTTGATTGAACATCGTCAGCTTTTACCAAATT

[0774] TACCCCATCTTGACTTTCAACATGATCAAACTCCCCAATCTTTTGTGATTTCCGACAACATTGTTAAAGTGAAA

[0775] CAAGCGGGAATAGCAGATAAAATCGTTCGTGGACAGCTTATTGATTTTATAGATATTGAAAGCAAATTTACCG

[0776] TTCGTGCCCAAATGATTGTCGCTGTAGAGGGAAATGAATTTTCTCTGGATACAAAAAATAGTATTCAACTTGAA

[0777] AAGAATCTGCAGTTATTACAATCAGCGTCTGAGAAAAACAATTTACGATGGAGAAATAGCACGGCGTGGTTAG

[0778] AGGATATGACGTATCGTATCAATTATACTGACGATCAGGTTATAGACGATAAAACAAAACAATGTCGTTTACAA

[0779] AGTAATACTAAATCGCCTTTTCCAGCCTTAATTGCACCAAAAAATAAGATTACGATTATTAAGCAATCTTCTCC

[0780] ACTCTCCAGTATTGCTGAATTTACTGATGAACCAGAATTCAAATTAGTTGCAACGGTGACAGAGATTGATCGG ATTGAAGGGATATTGACTATCGAACGGGATGACAACCAACTCCCTTTCCCGACTAAAGAAGAGAGTAATCAAT ATATATGGTACATATCTGATGAAAACTATATTTCAAGTGATCGTTTCTCTTTTGTGGTGAGCGTCGTGCTGAAT CGCGGTTTGGTTGAAAGGGAAGATATTGATCAATATAAGCTAGAGGAATGGATAGAGCGTGAAACACTTGCA

[0781] GAGTTTCCTGCACATATTTCGTTAATTACTCATTGGCTGGCATCTGAAAATTTCGATGATTTTGCGAAGACATA

[0782] TCAACGTTGGCAAAACAATGGGGCGCAGTTAGGGGATGAATCCTACACCATTTTGGAAAAACTGACATTAGG

[0783] GCATTTACCAACAGGACTTACTGGCATTAGTAATATGTTTATTGCTACAGAAGCTCAGCGTCTAGAAGTTGTT

[0784] GGCGAGAGTGGTAATGAGTGGAATACCCAGGCAATTATTAACAACGAACTATTCTATGTTCCCTCACAGAATA GTTAATACCGAGTGTTGTGATCAACTTTTATTATAAGCCGGAGGATAAATGGACAACAAAAATAACAAACCTAC TGATCAAGAGATTCTAAAAACATCACGGGCTGTCGGAGAAATTCCTTCAGCGGATAATTTAAAAAATCGTTTTA AAGCTCGTTCGATTCCATTAGAGACGGATTTTACTAATCTCATTGACCTTGCTGAAGTTGGACGATTGGCTAT

[0785] CGGCCAGTCACCATCGCAGCAAAGTAAAACGCCTGGCACCGGAATGGAATTAACTTCGGATGGTAAATTACA AGTCAAGGCTGGGGCAGGTGTTGATATCGATAATAATAATCGTATTACTATTAAGTCTGGTCATGGAATTAAG GTTGATGGAAACGGCATTTCCGTTAAACCAGGTTCGGGTATTAAGGTTGATAGTAATGGTGTAAATGTCAATA

[0786] TTGATGATTTTTGGGAGGAAATACGCAATAAAATTATGCCTAAAGGAACCATGCTGCCTATTTATGGCACACC

[0787] TAACCCCTCTGCGCTGCCAACAGGATGGGAATGGTGTGATGGTAAAGATGGCAGACCTAATTTAAAAAAAGG

[0788] GAAATATAACTTACTATCAGGTCAGTCTTCAGGTACTGATACTTTTTGGGCAGATAATAAGAATGGAGATACA

[0789] GAGATCAACGTGTTATTTGTTTACTATATGATTAAGGTTGTGTAATATCTTAAGTAATATGCATTACTCTAAAAT

[0790] GAATGATTTATATTTAAGTAACATAATAATTAAGTTGTGTTGTAGGGCTGTTTTTATGAGAAATATAAAAACGGA

[0791] GGTAATAATTGGCTTCAAAATATCAGTGATGAAATAGAGTTATTTCGCTTTATAAAAATTTTGTTTTATTTCTTTT

[0792] AATAATTATTTATAGAAGGTAATGATATGTGCACACAAAAAAACGTGTTAGATAGACTGAAAGATAGAAATATT

[0793] ACATTGGGTTGGGATGTTGTTGTTGCATATAACCAAGAAAGTGTTAATAAGTTATTGAAGCAACAATATGTTGA

[0794] AAAAGTTTACTCAAATGAACATTTTGTTTTTAAAGATTGGCATGATGATAATAAAACGAAATTTATTGAGGGATT

[0795] AACAGTAGGCGCTCCACTAGTTTCATTTGAGGAGGCGTCTTTATCCGATGCTAATGTAAAAGTGACACTTAAC

[0796] TTTCTTTCTGGTAGATGGAGAGTTATACAAGCAAATACCGGCACACCAATTGAATGGAAAGAAATTGTTCCTG

[0797] GCAGTGGCTATAAAGCAGAATTAGTTGTTCCGCTTAAATCAATAACTGGTAGTGTAAGTAAAAAAGATATCATA

[0798] TTAAAATTCAAAGATGCTGTCGTAAAAAAAATAAATTTATTTGACAATCAAGAGCCTGATTTTATTAATTATTTC

[0799] AAGCAATCGATCAGTGAGGGAAATTATACTTTAGGGCAACTGGTGACAGACAGCACACCGGGATTAATTCCT GCTGAATTTCATATTCGTACTCAACCCCATCCAAAAACACGTGAGCGTGGTTCTCAATATGTAGGAAATGGTG

[0800] CGGTACTGTTGTTTATTAAAACGCAATATGGCGGAAGTGGAACATTGCCTGTAAATGATTTTGATTGGTTAATT

[0801] CCTGATGATCATACTAGCGCATTAGTCATTTCGAGTAAGACCATGATGGGGCAAATATTGCCAAAACAATACA

[0802] AAGATAAATTGCCTGGTGATCCTCAGTTTAGCCCACCAAAAAGAGTCAATGATAAACAAGACTCTGCTTATTAT

[0803] ATTACGATTACCGATGGTGGATTTGATGGTAATAGCCCTATAGAGAAGTCATGGTTACGTTCTGATTATAGCA

[0804] ATGGGATTTGGACTGGTGAACGTGGTAATGCTATTATTGGTGAAAAAGGAAAGCGGATACCACCACGTTTTC

[0805] CATACCAAAATTTTGTTATTAAACCTCATGGTGAATCGTTATTTCAAGGATGGGAGAATAAGATAAATTACACT

[0806] CAAAAGTGTGCAAGATATTTCCGACATCATAGTAATAGTATAACTTTCGAAGATACTGCATTAATGGATCTCAG

[0807] TATTGGTGGACAAGGTAGTATCAATTGCCAGATTGATGGTGAACATTTCTATTTAAAATCAGATGATTTTTCCC

[0808] CCAATGTCAGCTATGAACCAACTTCATTCTGGGATAAATTTATCGGTGGGGTGGATGCAAATGTGAAAGATGA

[0809] ATTCAGAGATGAATTAGCACAACAGGCAGAAGCAAAGTTAAAACAGGTATTTAATATTGAATTGCCTGAAATC

[0810] AGTCTGTTTTCTATTAAACATCTGCTCTTTCCTGGCATGGATGTTATGCAACTTAAACAGGGTTATTTCCCAGG

[0811] AGATTTGATTATCTTTGGGGATATTTCACCTAAATTGACCACAATTCAGGTGGCTCCTTTGGAAGCCATGGTT GCCCTTAAAGAAAATCAAAAATTCACTGTCGTACCTGAAAATAAAAATGTTAGTTGGAAGTTGGATCATAATAG TGAGGCTATCAATGATCCGGGAAATATTGATGATAAAGGTATTTATACGGCACCGGGCAGAATCAGATCTGG

[0812] TTCTGAAGTCATTAAAGTCACTGCAACTGACGGCGATGGAAATCAGGCATCGGCGGCGCTGACGTTGGTTCC

[0813] TTCTTCTGTTGCATTAACACCTTCTTTTGCTTTTATCTCTGAAGCAGATAAGAAACCTATATTATTATTGGCGAA

[0814] TGTCCTAGACGGAAAAGCAGTAACATGGAATGTGGAAAGCTGTACAGGCAGCCAATGTGGTTCTGTTGATCA

[0815] GAATGGGCTTTATACTCCACCAGCAGGGCGTTTTAACGATGGATTTACTTTTGCATCCATCACCGCAACTGCA

[0816] AAAGATGGTAGTCAAGCACGAACCATTATTTGTCTAATGGCATCAATGCCAGGACATGGTTTTTACAAGGTTG

[0817] AACCTAATTTACGTTTGAATGTGAAAGTAGGGGAAGAAATTATCTTTAAAGCGCAGGCAGATAGCTATAATGG

[0818] TGATCCTGATACTTGGGAAATTTTCCCTCCTCGCGGAAAATTAAGTGAACCTGAGTTTGAACCCAATAATGAT

[0819] CCTGAAACTAATGATACAATTTTTGGTCATTATAAGGTGACCTATACCGCGCCGACTAATGTTACCTCACCTG

[0820] AATTGCTTGTTGTCCATGTATGGGAGAAAAATAGGCATAATGAGAAAAACAAAGGTAAGGCAGGATATGCACT

[0821] TATTGAAATTATCCCAGATGATAAATAGAAAATTTATTTAAATAAAAATCACAGCGGGTTTATCTCGCTGTGATT

[0822] AAAGTCATCTTTTTTTATAGATTGTTTATCTCTAATAATAATTTTATTTTATAATATAAAGGAAATTAAAATGAATA

[0823] ATGAATATAAAAATAACACCGTGAATTGGCGTATTTCACCTGATACGGTAGGAAGTATTGATAATAACGGTTTA

[0824] TATACAGCACCTAATCGGGTAAAGAATATCGAATTTGTCCAAGTAATGGCAAGCGATGCTAATAATAATCAAT

[0825] CTTCTGCGATTATTACTGTTATTCCCTCTTCTGTTGCGTTAACGCCATCGTTTACTTTTATCTCTGAGGCAAAA

[0826] AAAACATCAGTCACTTTTAAAGCGACAGAACTTGAAGGGAAAAAAGTGACATGGAGTATAAATAATTATACCA

[0827] GTAATCAGTATGGTTCCATCGATCAAAATGGTATCTACACACCACCGGAAAGTCGTTTTAACGATGGATATAC

[0828] TTTTGTATCTATTACAGCAAAAGCGGAAAATGGCGCTGAAGCGCAAGCGCTTATTTGCTTGATGGCCAAAATT

[0829] CCAGGGCATGCCTTTTTCGATGTTCAGCCTAATATATGTTTAAGTGTGAAGCCTGGAGAAGAAATCATTTTTA

[0830] GAGCTAACGCAGATCGTTATAATGGTGATCCTGATTCCTGGGAAATTTTCCCGTCTCTTGGTAAATTGGGTGA

[0831] GCCTGAGTATATAAAAAATAACGATCCAGAAATTCCTATTTATGGATATTATCAAGTGAAATATATTGCGCCAA

[0832] CCAATATAAATTCTTCCCAAATACTCGTTGTGCGTACTTGGGAATATGACAAACATGATGAGCATAATCAAGGT

[0833] AAAGCAGGATATGCATTCATTGAAATTGTGCCAGAAAATGAGCTTTAATATATATACCCAATAGATTTCGAGCC

[0834] GCAGCGCGGCGGCAAGTGAATGAATCCCCAGGAGCATAGATAACGATGTGACTGGGGTGAGTGAACGCAG

[0835] CCAACAAAGAGGTAACTTGAAAGATAATGAGTATAAATGACTTTAGTAAGAGAAATTATGGCTTCATTCAGAAC

[0836] TATTTATTAGAGTAATTAACTTTATAAAGACATTTAATGGAAAATATAATAGAAAAATTTAATATTAATATTGAAG

[0837] TCTCATCTGAAATTATTGGAGAGAGTTTATTAAACTCCCCTTTATTGATGAGTAGAGAAATCAGCAATCAATTA

[0838] TCTGAAATATTATTAGATTATAAAGAATATAATATTGCATTGGATAAGTTAGTGTTAAATATAGGAGAAATACCC

[0839] TATGAAATATTTGAACAACAATTCTATGGTCGTTTGGGAAAATTATTAAATGAAAAGTTAACAATAATAATAAAT

[0840] GATAAATTATTGGTAAAAAACATATCAACCTCGTTATTTCCTGAATGTTTTAGTGAAAAAAGAAACCCATTATTA

[0841] AATAGAGTCATAAAAAATTTACCTTCTAATTTGGTTTTTGAAGTTCATTCAATGGTAAAAATAGAATCAGTAAAT

[0842] AACAAAAAACAAGCTAATATATTGACATCTTATCTGGCTTATTCTTTTTTTAATAAAAGCAAATTACAACAACAT

[0843] TTATTTTCCACTAGTAATAATAAATTAATTGAGAGCTTATACGCACTTTTTCTAACGGATCAGAATCGAATACCT

[0844] ACTGCTCATAAAATAGGAAAAGGTGCACTTATACTATCTGCCCTTATTTGGCTTTATTCTAATTCCAATGATTAT

[0845] CTGCCCAAACCAGAAAGCACTCTGTTGTTACAAATAGAACAGGATATAAAACAAGGATATTTGCCTTTAACGT TGTTAATCACTTTCTTCCAGAACAGAAATGGCGGGCGTGTTTTTTGCGATTGGCAGTATGCGTTATGGCAAAT

[0846] CGATATCATCAAAAATCACTTAGGCATTAAAATAACATCGAAAGAACCCCATTTACGGGAGAAAATAATGTTAC AACCAGTTAATGCTTCTGATCGATCCTCTGTGCTGATATCAGACGAAAAATTGACAATACCGTTAACAATTACA GGTGCGGGATTAGTGCTTCTCTGGCCACTATTAACTCCACTATTTTCGTCTTTTGATTTGTTAGATAAGAAAAG TTTTTCAGACAATTTGGCACAGGAAATAGCATTTAATTTATTGGAATGGTTAGTCTGGGGAGATGAGATGCTG TTACATCAGGAATCATCATTATCTTTATTACTCTGCGGAATAGATCACCAAACAATACTGGAGCGCCAGGTTCT TATTCCTGAGCACAAGGAAAAATTAAATAACTGGTTGCAAGGTATTTGTACTCAACTTTTCTCTTGGAAAAAGC TAGGGATCGATGATATGCGCCAACTTTTTTTGCAGCGTCAGGCTGCACTTTATTATGAAGATGATGGCCGTTG GTTATTAACGGTGCAGCGTGAAGCTTATGATGTATTACTGACTCAAATGCCTTGGCCGTGGCCATTGAATATT GTGACATTACCTTGGCTAGCTGAGCCGATTAGTATCACTTGGGAAGGTATCTCTGAACCAACGGATTTGTCAT TTTGGTAATCCAATATCTCATTAGGAACTCTATGCATGTACGATTTATCTGATGATCTTGCCAGACAGAATATT TCACCGGAATATGAATTGACGGTTTTGCTGTCTCAGACTGCTATATTGGATAAACGAATTCGTTTACGAATTCA GGAATTAATGCAACAGCAAACACTATTGGGAGAAAGTGGACAGACGTCTTTTGATGATATTTCATTTTCATTC GTTTCGAGTGAACAACAAAAATCATCTTATTTGGTGTCACCGCATCAAAATTGGACGAAAGAGGATTTTCCTC CTGAGCCGATCCCATCTCGTAGCCGTCTAGGACAATTAGTTGAACGGTTTGACTTAACTCAATTTGAAATTGA TTTGATTTTATTGTGCCTGTTGCCTCATCTTGACAGACGTTATCTAACGTTATTTTCTCTTGTTCCGGTAAGTG GAGGTAATAACAGCAAAAAGCAGATGTTAACGTTGGGATTGGCTTTGGAGTTGCTTTGTCCGAGTGTAGTAG AGCGCAATGCGCAACGTGCCAGTTTATTACCACAGGCACCGCTTTGGGATTATCGTTTATTTCAGTTGCGCG GTGATATGTCTGTTTCCTACGATGAAATACCGTTAGCAATCGATAATTCTCTTATGCATTGGTTATTGGGGCAT GATGCTCTCCCGATTTCTCTTCTCTCCCGGGCTCATTGGCTTCCTGTTCCTGAAGTGCCTGATATTTTGCCTG ATTTCACCAACCAATTGATAGAACTCTGCCAAATGGAACAAGAGGGGATGCTGACAATAATCGCCGGCGGAG CCGGAAGTGGCAGCAAAACAAGTGTTGCACGCGCAGCATCACAAGTAGGGCGCTCTGTATTGTTGTTATCGT TAGCATCAGTGACACTGAGTGAACATGAAACTATTACACTGATAACACTGGCATTACGTGAAGCACAACTAAG AAATGCCTGTCTTATGTTTGAAGCTTTGGATGAGTTTTGTGAAGCACGCCCCGCTTTGCAGCTCTGGCTAGGA AATCGACTGGCTCGTTGTTCGATTCCGCTGTTTTGTCAATTACCTAAGCAAGCATCATTATTGCCATTGGATG CAATTTCACAAGTTGTATTGTCTATGCCAATGCCTTCTTTAATGGTGAAGGCTGCAGCATTAGCTTCAATGATG ACGAATTATTTTCCAGACAATTCATTGGATGTTGAAAGTTTAGTGACATGTTTCCATCCTTCTCCATTGATATTG AAAAAGGCCCTTAGTGAAGCAGAAATTTATCGCCGACTACGGGGGGAAACGGCTAGTTTGAGATTAGATGAT GTGCAAATGTCCCTGCGTTTTCGGTTACAGCAGAATTTTGGACGTTTAGCACAGAGAATTACACCACAACGAA CCTTTGATGATTTGATCATCAGTGAATCTCAACAGCAACAATTACAAGAAATCCTGGCGGCTATTCGGCAACG AGATAGGATGCTAGAGCAAGGATTTGCTCGTAAAGTGAGCTATGGGACGGGTATCAGCACGCTATTTTTTGG TGAATCTGGCACAGGAAAAACGATGGTAGCAGAAGTGTTAGCTGGTGTTTTAGGTGTGGATTTGATCAAGGT AGATTTGTCCACTGTGGTTAACAAATATATTGGTGAAACTGAAAAAAATCTGGCTCGTGTTTTTGATTATGCCC AAGAAGACGCCGGGGTATTGTTCTTTGATGAGGCAGATGCATTGTTTGGCAAACGAAGTGAAACTAAAGATG CAAAAGATCGTCATGCTAATATTGAAGTTTCCTACCTATTGCAACGCCTTGAAAGTTATCCAGGGCTGGTGAT ATTAGCCACCAATTACCGTAATCATTTAGACTCAGCATTTAGTCGTCGCCTGACTTTTTCGGTACGATTCTCTT TTCCAGATGTTTCCTTACGGGAACGGATGTGGCGGATTATCTGGCCATCGGGAATTCAATTAGCCGACGACA TCAGTTTTTCAGCGTTGGCAAAACGGGCTGAATTAACGGGGGCGAATATCCGTAATATTGCGCTACTCGCTA GTTGGCTGGCAGTAGATGAAGGAAATGAAAAAATTACTATGGCTCATATTGAATGCGCATTACGACGTGAACT GAGTAAAGTTGGGCGCATTGATTTACCTTAATTTTTCTTTGTAATCGGGAGACAACTATGGTTAAAAATATCAA ATCAGATGAAACCTTACTGATATTAAATAGTAAAATAGAAGATGCATTAAAAGCGTATTTACCGGGCGAAGAT GTCGTTATTCGGTTCGATATGTTTGGTAAAAATGAAAATCCAGATTCTCCTACCGTGTGCGTTTTTCTTTATGA TATTCAGGAAGATCTGCAATTACGCGTGGGAGAAGGGCGGCAATACCTGCCTGCGACAGGAAATTTTGTCCC GGGATGTGTCAATGTTCGTTGTAATTATCTTATTTCCTACTGGGAGCCGGAACAGAGCGGAGGGCAGGGATC GCCAACCATACGTTCTAATAGTCAATCAATGAAGATAATGAACTGTGTATTGAATGCATTAATTAATCATCGTT CATTTCCTGGTTTACCCAGAACTTATACGAGAGTTCTTCCTCCTAATGAACAATTAAATAGCTTAGGAAACTTT TGGCAATCATTAGATAATAAGCCTCGACTATGTTTAAGTTATATGGTGACTATTCCTATTCAACTTACCCCGCC GACAGAGAAGGTATCTCCTGTCATTACCTCAAAAACTGATATTACTCGAAAACCATCGCTTAACTTTTATCTTG AGGCAGATGAAATTATCCGTCAGGCATTAGTTGATGCCTTAATATCTCAAACAACAGAATCTATGGATACGAT AACTAGCTGGCTGGCAAAAGTTGTTATTATTTGTCGACCACCAGAAATAATGAATAAACAAATGATTGAACAAA CTGTGAAATTAATTATTGCTGGAATTACAGAAGAGGGATTAGCTGGAAATATAAAGACAATCACTCAAAAGTG GGTGGAAGAGAAGACGATTATTGGTGAAATCGACGATGTTTCTCTAGTTATTTCCCAAGTTGACACGACAGC GTTGTCTGCTGTAACAATACCGACATCTGTTTAA

[0847] SEQ ID NO: 20 (PVCpromF)

[0848] TATCATATGTCTACAACTCCAGAACAAATTGCTG

[0849] SEQ ID NO: 21 (PVCpromR)

[0850] ATCTCTAGAACAGATATTCCAGCCAGC

[0851] SEQ ID NO: 22 (ParaINF)

[0852] GGCGTCACACTTTGCTATG

[0853] SEQ ID NO: 23 (ParaINF)

[0854] TCGGTGGCAGTAAATTGTCC SEQ ID NO: 24 (F1 primer)

[0855] ATGTCTACAAGTACATCTCAAATTGCG

[0856] SEQ ID NO: 25 (F2 primer)

[0857] GACTCCCTTGAGGGTACGG

[0858] SEQ ID NO: 26 (F3 primer)

[0859] TTCTGATGAGAGTGATGGTAC

[0860] SEQ ID NO: 27 (F4 primer)

[0861] TGAATAAAGAATTCAGTCAATATC

[0862] SEQ ID NO: 28 (R1 primer)

[0863] TAGTGGCTGATGAAAGTCTG

[0864] SEQ ID NO: 29 (R2 primer)

[0865] GGAAGCCAAAGATAATGAAGTG

[0866] SEQ ID NO: 30 (R3 primer)

[0867] CATTTCTTCCCTATGGTTG

[0868] SEQ ID NO: 31 (R4 primer)

[0869] TTAAATTCCTACAAGATTATCTTT

[0870] SEQ ID NO: 32 (PVC16Z PVC-Pnf16) - absent FLAG tag at C-terminus

[0871] MLNTQTIIDVNKAMDAMLRAYLNQDIAIRFDLPELDTMQSDAMVSIFLYDIHEDLQLRSAESR

[0872] GFDVYAGRLLPGWVNIKCNYLITYWEASKPATDASSPDSQPDNQAIQVMSQVLNALINNRQ

[0873] LAGIPGAYTQVVPPKESLNSLGNFWQSLGNRPRLSLNYSVTVPVSLNDGQDSATPVTAVSS

[0874] TVEQTASLSQEVVSHALRELLITELGGGEDNRLVLSKVELSAVKETMTQDSPAQMIILLSVSG

[0875] ITRQEYLKEIDNIFDRWVNNAEVITTIDDCGIRIESITKDNLVGI

Claims

CLAIMS1. A chromatographic method for separating pre-contraction state PVC Nanosyringes from post-contraction state PVC Nanosyringes, the method comprising: a. providing a sample containing both pre- and post-contraction state PVC Nanosyringes; b. loading the sample on a monolith anion exchange column having (preferably quaternary amine) binding moieties, under conditions (e.g. pH 7-8, temperature 19-25°C) wherein pre- and post-contraction state PVC Nanosyringes become bound to the column with differing affinities; c. applying a gradient of ionic strength to the column that provides for differential elution of the pre- and post-contraction state PVC Nanosyringes, wherein the gradient is generated by incrementally (e.g. linearly) increasing the salt concentration of a mobile phase elution buffer that is applied to the column; and d. eluting the pre- and post-contraction state PVC Nanosyringes into separate elution fractions under the gradient based on their differing affinities to the anion exchange column, and collecting fractions having the pre-contraction state PVC Nanosyringes separated from the post-contraction state PVC Nanosyringes.

2. The method according to claim 1 , the method comprising monitoring eluate for ultraviolet absorbance at a wavelength of about 280 nm (A280) and collecting fractions that elute under a base peak of A280 (e.g. having highest A280 value, the highest A280 being relative to any other A280 value detected across the eluate profile), the collected fractions having the pre-contraction state PVC Nanosyringes separated from the postcontraction state PVC Nanosyringes.

3. The method according to claim 1 or claim 2, comprising collecting fractions that elute when the ionic strength of the mobile phase elution buffer in the column is equivalent to that of a buffer comprising (or consisting of) about 0.28M-0.47M NaCI (and optionally also about 2.7 mM KCI, about 10 mM Na2HPC>4, and about 1.9 mM KH2PO4);, optionally when at a pH of 7-8 (preferably a pH of about 7.4), optionally when at a temperature of 19-25°C (preferably 20-23°C), wherein the ionic strength is calculatable using the following formula:where q is the molar concentration of ion i (M, mol / L), Zj is the charge number of that ion i, and I is taken over all ions in the solution.

4. The method according to claim 3, comprising collecting fractions that elute under an ionic strength equivalent to that of a buffer comprising (or consisting of) a concentration of about 0.37-0.47M NaCI (and optionally also about 2.7 mM KCI, about 10 mM Na2HPC>4, and about 1.9 mM KH2PO4); optionally when at a pH of 7-8 (preferably a pH of about 7.4), optionally when at a temperature of 19-25°C (preferably 20-23°C).

5. The method according to any one of the preceding claims, comprising collecting fractions that elute when the conductivity of the mobile phase elution buffer in the column is between 34 mS / cm and 48 mS / cm, more preferably between 40 mS / cm and 48 mS / cm; preferably when the elution is conducted under pH conditions of pH 7-8, more preferably about pH 7.4; preferably wherein the elution is conducted at a temperature of 19-25°C (more preferably 20-23°C); preferably wherein the conductivity values correspond to conductivity measured at 20-23°C in a 1 cm path cell.

6. The method according to any one of the preceding claims, wherein in step b the sample is comprised within a loading buffer such that the loading step occurs under a pH of 7-8 (preferably about 7.4), preferably wherein the loading step is carried out at a temperature of 19-25°C (preferably 20-23°C).

7. The method according to any one of the preceding claims, wherein the pH of the mobile phase elution buffer is maintained at pH 7-8 during elution e.g. wherein the elution is conducted under pH conditions of pH 7-8, preferably about pH 7.4.

8. The method according to claim 7, wherein the elution is carried out at a temperature of 20-23°C.

9. The method according to any one of the preceding claims, wherein the pH is maintained at a stable value (e.g. of pH 7-8, preferably about pH 7.4) throughout steps a- d of the separation process.

10. The method according to any one of the preceding claims, wherein the temperature is maintained at a stable value (e.g. of 19-25°C, preferably 20-23°C) throughout steps a-d of the separation process.11 . The method according to any one of the preceding claims, wherein the ionic strength gradient is a linear gradient.

12. The method according to any one of the preceding claims, wherein the concentration of salt (e.g. NaCI) in the mobile phase elution buffer at the beginning of the gradient is at least two times lower than the salt concentration that causes elution of pre-contraction state PVC Nanosyringes.

13. The method according to any one of the preceding claims, wherein the ionic strength gradient is established by increasing the amount of an input elution buffer (having a salt concentration that is higher than that at which the pre- and post-contraction state Nanosyringes elute from the column) from 0-30% (e.g. the remainder provided by running buffer) over 8-12 (e.g. 10) column volumes (e.g. for a column volume of 1 ml or 8 ml).

14. The method according to claim 13, comprising collecting fractions that elute when the amount of input elution buffer is at 14%-24%, preferably 18%-24% (e.g. the remainder provided by running buffer).

15. The method according to any one of the preceding claims wherein the salt concentration in the mobile phase elution buffer at the start of the gradient is that having an ionic strength equivalent to 137mM NaCI (optionally equivalent to a buffer having an ionic strength of 10 mM Na2HPC>4, 1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, e.g. at pH 7.4).

16. The method according to any one of the preceding claims, wherein the ionic strength gradient is provided by an increasing concentration of salt (preferably NaCI) in the mobile phase elution buffer, preferably starting at a NaCI salt concentration of about 120-150 mM (e.g. about 137mM).

17. The method according to any one of the preceding claims, wherein the ionic strength gradient is provided by an increasing NaCI concentration in the mobile phase elution buffer, and the method comprises collecting fractions that elute under a NaCI concentration of about 0.28M-0.47M (preferably about 0.37M-0.47M); preferably at pHconditions of pH 7-8 (more preferably about pH 7.4), preferably at a temperature of 19- 25°C (more preferably 20-23°C).

18. The method according to any one of the preceding claims, wherein the loading flow rate in step b is 1 mL / min for a 1 ml column; or 4 mL / min for a 4 ml column; or 8 ml / min for an 8ml column.

19. The method according to any one of the preceding claims, wherein the elution flow rate is 1 mL / min for a 1 ml column; or 4 mL / min for a 4 ml column, or 8 ml / min for an 8ml column.

20. The method according to any one of the preceding claims, wherein the column has been equilibrated with the same buffer as a loading buffer in which the sample is present while loading the column.

21. The method according to any one of the preceding claims, wherein the loading buffer comprises or consists of 9-11mM (preferably about 10 mM) Na2HPO4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 130-150mM (preferably about 137 mM) NaCI; 2.5- 3mM (preferably about 2.7 mM) KCI, and a pH of 7-8 (preferably a pH of 7.4).

22. The method according to any one of claims 1-20, wherein the loading buffer comprises or consists of 18-22mM Tris-buffered saline (preferably about 20 mM Tris Trisbuffered saline), 130-150mM (preferably about 137 mM) NaCI; and 7-9 mM MgSC (preferably about 8 mM MgSC ); and a pH of 7-8 (preferably a pH of 7.4).

23. The method according to any one of the preceding claims, wherein the input elution buffer (e.g. at its highest salt / NaCI concentration) comprises or consists of 9-11mM (preferably about 10 mM) Na2HPC>4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 1.5- 2.5M (preferably about 2M) NaCI; 2.5-3mM (preferably about 2.7 mM) KCI, and a pH 7-8 (preferably a pH of 7.4); or comprises or consists of a buffer having an ionic strength equivalent thereto.

24. The method according to any one of the preceding claims, wherein prior to loading the sample, the column is equilibrated with a buffer (e.g. loading buffer as described above) of pH 7-8 (preferably pH 7.4) .

25. The method according to any one of the preceding claims, wherein prior to loading the sample, the column is equilibrated with a loading buffer comprising (or consisting of) 10 mM Na2HPO4, 1.9 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI, pH 7.4.

26. The method according to any one of claims 1-24, wherein prior to loading the sample, the column is equilibrated with a loading buffer comprising (or consisting of) 18-22mM Tris-buffered saline (preferably about 20 mM Tris-buffered saline), 130-150mM (preferably about 137 mM) NaCI; and 7-9 mM MgSO4(preferably about 8 mM MgSO4); and a pH of 7-8 (preferably a pH of 7.4).

27. The method according to any one of claims 24-26, wherein prior to equilibrating the column, the column is washed with a suitable preparation of water (e.g. endotoxin free water), preferably with 8-12 (e.g. 10) column volumes of said water.

28. The method according to any one of the preceding claims, wherein prior to the elution step, unbound material is washed from the column using loading (aka running) buffer, preferably using 8-12 (e.g. 10) column volumes of loading buffer; preferably wherein the loading buffer for washing has an equivalent ionic strength (e.g. salt concentration) and an equivalent pH to a loading buffer used to load the sample; preferably wherein both loading buffers are the same.

29. The method according to any one of claims 1-27, wherein prior to the elution step, unbound material is washed from the column using a first wash buffer comprising or consisting of 9-11mM (preferably about 10 mM) Na2HPO4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 130-150mM (preferably about 137 mM) NaCI; and preferably also 7-9 mM (preferably about 8 mM) MgSO4.

30. The method according to any one of the preceding claims, wherein a wash step is conducted prior to the elution step (and optionally subsequent to claim 28 or 29) to remove endotoxin from the column using a wash buffer comprising an endotoxin removal agent (e.g. a detergent such as CHAPS); preferably wherein the wash buffer has an equivalent ionic strength (e.g. salt concentration) and an equivalent pH to a loading buffer used to load the sample; more preferably wherein the wash buffer additionally comprises said endotoxin removal agent but is otherwise equivalent to the loading buffer e.g. thereby providing a washed monolith column; preferably wherein the wash step comprises washing the column with 20-40 (e.g. 30) column volumes of said wash buffer.

31. The method according to claim 30, wherein the wash buffer (e.g. a second wash buffer for the wash step conducted prior to the elution step) comprises or consists of 9- 11mM (preferably about 10 mM) Na2HPO4; 1.7-2.2mM (preferably about 1.9 mM) KH2PO4; 130-150mM (preferably about 137 mM) NaCI; 2.5-3mM (preferably about 2.7 mM) KCI; 15-25 mM (e.g. about 20 mM) CHAPS; and preferably 18-22 mM EDTA (more preferably about 20 mM EDTA); and preferably also 7-9 mM (preferably about 8 mM) MgSC ; and a pH of 7-8 (preferably a pH of about 7.4); and / or32. The method according to claim 30 or 31 , wherein the wash buffer is PBS containing a detergent that acts as an endotoxin removal agent (e.g. wherein the wash buffer comprises or consists of 10 mM Na2HPO4, 1.9 mM KH2PO4 phosphate, 137 mM NaCI, 2.7 mM KCI, 20 mM CHAPS, 20 mM EDTA, and preferably also 8 mM MgSC , pH 7.4).

33. The method according to any one of the preceding claims, wherein subsequent to step d), the eluate (e.g. collected fractions) is passed through an endotoxin removal column.

34. The method according to any one of the preceding claims, wherein the monolith column comprises channels of at least 1 pM in diameter, preferably about 2 pM in diameter.

35. The method according to any one of the preceding claims, wherein each fraction of eluate comprises a volume of 0.3-0.6 ml (preferably about 0.5 ml).

36. The method according to any one of the preceding claims, wherein in step a) said sample is a sample that was previously processed to remove expression (e.g. host) cell protein / debris following lysis of the expression cells.

37. The method according to any one of the preceding claims, wherein the following steps are carried out prior to step a): i. culturing prokaryotic host cells (preferably E. coli) comprising a nucleotide sequence encoding PVC structural proteins (PVC 1-16), wherein the nucleotide sequence is operably linked to an inducible promoter;ii. inducing expression of the PVC structural proteins (e.g. with arabinose) by the host cells and allowing the PVC proteins to assemble into Nanosyringes in the host cells; iii. lysing the cells (e.g. by homogenisation) thereby providing a lysate comprising said pre- and post-contraction state Nanosyringes.

38. The method according to any one of the preceding claims, wherein prior to step (a), a (the) lysate comprising the Nanosyringes is subjected to one or more clarifying purification step(s) to remove (e.g. non-Nanosyringe) host cell material, thereby providing a purified sample containing both pre- and post-contraction state PVC Nanosyringes (e.g. said purified sample providing the sample in step (a)).

39. The method according to claim 38, wherein said one or more clarifying purification step(s) include PEG precipitation, flocculation, centrifugation, affinity chromatography, membrane filtration (e.g. tangential flow filtration) and / or diafiltration, to remove (e.g. non- Nanosyringe) host cell material, thereby providing a purified sample containing both pre- and post-contraction state PVC Nanosyringes (e.g. said purified sample providing the sample in step (a)).

40. The method according to claim 38 or 39, wherein the clarifying purification step comprises binding pre- and post-contraction state PVC Nanosyringes (in embodiments wherein an affinity tag such as a FLAG tag is associated with at least one of the PVC proteins) to a resin (or column) having a moiety that binds the affinity tag, washing through unbound material, and subsequently eluting the Nanosyringes thereby providing a purified sample containing both pre- and post-contraction state PVC Nanosyringes (e.g. said purified sample providing the sample in step (a)); optionally wherein the affinity tag is fused to the c-terminus of PVC16; optionally wherein purified sample containing both pre- and post-contraction state PVC Nanosyringes is referred to as a “pre-anion exchange chromatography purified sample”.

41. The method according to any preceding claim, where the collected fractions (e.g. enriched for the pre-contraction state PVC Nanosyringes) are subjected to a volume reduction step (e.g. concentration), preferably by using centrifugal filter or tangential flow filtration (e.g. with hollow fibres).

42. The method according to any one of claims 37-41 , wherein the host cells are cultured to an optical density at 600 nm (ODeoo) of 5.5-8.5; preferably wherein the host cells are cultured to an optical density of 600 nm (ODeoo) of 6-8.

43. The method according to any one of the preceding claims, wherein prior to a clarification step, a cell lysate is incubated in the presence of a nuclease such as DNase (e.g. for about 30 min at 37° C or 30 min at room temperature such as 18-21°C).

44. The method according to any one of the preceding claims, further comprising analysing the collected fractions (e.g. by transmission electron microscopy) to confirm that the fractions are enriched for the pre-contraction state PVC Nanosyringes.

45. The method according to any one of the preceding claims, wherein said binding moieties (step b) are quaternary amine binding moieties.

46. An isolated sample / fraction of (e.g. recombinant) pre-contraction state PVC Nanosyringes obtainable by a method according to any preceding claim, e.g. wherein the isolated sample / fraction is enriched for pre-contraction state PVC Nanosyringes; preferably wherein the isolated sample / fraction is substantially free of endotoxin.

47. An isolated sample / fraction of (e.g. recombinant) pre-contraction state PVC Nanosyringes, wherein the isolated sample / fraction is substantially free of postcontraction state PVC Nanosyringes; preferably wherein the isolated sample / fraction is substantially free of endotoxin.

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