Methods and materials for encapsulating proteins

Abstract

The invention provides a method for encapsulating a protein of interest, the method comprising the step of expressing a fusion protein comprising an N-terminal region of a rearrangement hot spot (RHS)-repeat-containing protein fused to the protein of interest. The invention further provides applications for the encapsulation, release and delivery of the protein of interest. The invention also encompasses the encapsulated protein of interest and compositions comprising the encapsulated protein of interest. The invention also provides uses of the encapsulated protein of interest, optionally after release from encapsulation, to control pests. The encapsulated protein of interest may for example be produced via expression in a plant to control a pest of the plant, such as an insect pest.

Claims

1. A protein of interest that is encapsulated by a shell formed by a complex consisting of: a) a TcB component of a bacterial toxin complex, and b) an N-terminal region of a TcC component of a bacterial toxin complex, wherein the N-terminal region of the TcC component extends from the N-terminus of the TcC component to a C-terminus of a RHS repeat-associated core domain, and wherein the protein of interest is heterologous to the N-terminal region of the TcC component, and the protein of interest is less than 40 kDa.

2. The encapsulated protein of interest of claim 1, wherein the RHS repeat-associated core domain conforms to the profile-HMM shown in FIG. 15.

3. A cell, composition, insecticidal composition, or pharmaceutical composition comprising the encapsulated protein of claim 1.

4. A method of controlling a pest, pest of a plant, or an insect, the method comprising contacting an encapsulated protein of claim 1 with the pest, pest of a plant, or insect wherein the protein of interest is a protein that is toxic to the pest, pest of a plant, or insect.

5. The method of claim 4 wherein the encapsulated protein is produced in the plant by expressing in the plant: a) the TcB component of a bacterial toxin complex, and b) a fusion protein comprising the N-terminal region of the TcC component of a bacterial toxin complex fused to the protein of interest, wherein the N-terminal region of the TcC component extends from the N-terminus of the TcC component to the C-terminus of the RHS repeat-associated core domain, and wherein the protein of interest is heterologous to the N-terminal region of the TcC component.

6. The encapsulated protein of claim 1 wherein the RHS repeat-associated core domain comprises the motif DXXGX, where X is any amino acid.

7. The encapsulated protein of interest of claim 1, wherein the protein of interest is less than 35 kDa.

8. The encapsulated protein of interest of claim 1, wherein the protein of interest is less than 32 kDa.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the structure of the YenB/YenC2-N complex. a, Ribbon diagram of YenB/YenC2-N. YenB is on the left in light grey and YenC is on the right in dark grey. b-c, Orthoganol views of the complex, with the central cavity shown as a translucent surface and the protein as grey ribbons, and with approximate interior and exterior diameters marked. The position of an RGD motif is shown with a circle. d, a schematic topology diagram of the structure with -helices shown as cylinders and -sheets as arrows, and the domains labelled.

(2) FIG. 2 shows structural details of the YenC2-N auto-proteolysis site. The site of auto-proteolysis in YenC. The residues immediately upstream of the cleavage point are D686, P687, D688, G689 and M690. The side chains of a selection of residues conserved in the RHS-associated core domain are shown. The distance (6.3 ) between the two conserved residues that are essential for proteolysis, D686 and D663, is shown in dark grey. The side chain of D663 is within hydrogen-bonding distance of the terminal carboxyl group of the cleaved peptide (distances (2.8 and 3.2 ) shown in light grey).

(3) FIG. 3 shows RHS repeat structure. a, A section of the shell showing the pattern of RHS repeats, viewed from the inside of the central cavity. A single RHS repeat is highlighted with a grey background box. The ordered loop made by the DxxG motif is shown at the top and the conserved pattern of hydrophobic residues on the face of the -sheet is shown in dark grey (conserved tyrosines) and light grey (other hydrophobic residues). b, The same face of the -sheet shown as a solvent accessible surface and coloured by side-chain hydrophobicity (hydrophilic as white, hydrophobic as dark grey, protein backbone as mid grey). The stripe formed by the conserved hydrophobic residues is boxed with a dashed line.

(4) FIG. 4 shows the position of the YenB/YenC2-N complex in the complete Yen-Tc particle. a-b, The YenB/YenC2-N dimer is shown as a ribbon diagram, fitted to fitted to EM class averages of the complete Yen-Tc toxin particle. c-d, Orthogonal views of the complete Yen-Tc complex. The YenB/YenC2-N dimer is shown as a ribbon diagram. The associated chitinases Chi1.sup.35 and Chi2 (PDB ID: 4DWS) are shown as pale grey ribbon diagrams. The EM map of the YenA/Chi1/Chi2 complex determined at a resolution of 17 by single particle averaging.sup.6 is shown as a light grey surface.

(5) FIG. 5 shows a topology diagram of YenB/YenC2-N. The YenB/YenC2-N structure is shown in schematic form with -helices as cylinders and -sheets as arrows. The start and end points of secondary structure elements are indicated. The domains are composed as follows: -strands 1-29, YenB SpvB domain; -strands 30-50, YenB -propeller domain; -strands 51-92 (light grey), the remainder of YenB; -strands 1-44 (dark grey), YenC2-N; -strands 45-49 (dark grey), YenC2-N RHS hyper-conserved core domain.

(6) FIG. 6 shows SAXS bead models of YenB/YenC2 and YenB/YenC2-N. a and c, a slice through the ab initio bead models produced from small-angle X-ray scattering of YenB/YenC2 and YenB/YenC2-N respectively. The model of YenB/YenC2-N has a large internal cavity shown in dark grey, absent from the model of YenB/YenC2. b and d, fit of the ab initio bead models to scattering data.

(7) FIG. 7 shows SAXS data for YenB/YenC2. a, purity of YenB/YenC2 sample for SAXS analysis, shown by size exclusion chromatography trace and SDS-PAGE (inset). b, SAXS data for YenB/YenC2 as a log-log plot. Inset is a Guinier plot of the low-q region. c, P(r) plot for YenB/YenC2 with D.sub.max=134 . d, scattering of YenB/YenC2 compared to the theoretical scattering of the YenB/YenC2-N crystal structure, highlighting the poor fit.

(8) FIG. 8 shows SAXS data for YenB/YenC2-N. a, purity of YenB/YenC2-N sample for SAXS analysis, shown by size exclusion chromatography trace and SDS-PAGE (inset). b, SAXS data for YenB/YenC2-N as a log-log plot. Inset is a Guinier plot of the low-q region. c, P(r) plot for YenB/YenC2-N with D.sub.max=138 . d, scattering of YenB/YenC2-N compared to the theoretical scattering of the YenB/YenC2-N crystal structure.

(9) FIG. 9 shows the effect of point mutations on YenC2 self-cleavage. When co-expressed with YenB, wild-type YenC2 (WT) self-cleaves following M690. Three point mutations (R650A, D663N, and D686N) were found to abrogate self-cleavage.

(10) FIG. 10 profile-HMM logos of RHS and YD repeats. Profile-HMM logos of an RHS repeat (a) and a YD repeat (b) show that these two repeats have the same consensus sequence.

(11) FIG. 11 shows the E. coli CNF1 catalytic domain compared with YenB/YenC2-N. The catalytic domain of E. coli CNF1 (dark grey surface representation), which is homologous to YenC1-C, is shown manually placed inside the hollow shell formed by YenB/YenC2-N (cartoon diagram). This shows that the central cavity of YenB/YenC2-N is large enough to accommodate the C-terminal toxin domain of the YenC proteins.

(12) FIG. 12. Profile-HMM that describes the RHS repeat, as defined in the Pfam database (pfam<dot>sanger<dot>ac<dot>uk; /family/PF05593).

(13) FIG. 13. Profile-HMM that describes the YD repeat, as defined in the TIGRfams database (www <dot>jcvi<dot>org/cgi-bin/tigrfams/HmmReportPage. cgi?acc=TIG R01643)

(14) FIG. 14. Composite profile-HMM that describes both the RHIS repeat and the YD repeat, constructed using the program jackhmmer (hmmer<dot>janelia<dot>org/search/jackhmmer).

(15) FIG. 15. Profile-HMM that describes the RHS repeat-associated core domain, as defined in the TIGRfams database (www<dot>jcvi<dot>org/cgi-bin/tigrfams/HmmReportPage<dot>cgi?acc=T1G R03696).

(16) FIG. 16. a, Size exclusion chromatography trace of TcB:TcC-GFP fusion at pH17.5; b, SDS-PAGE of fractions from size exclusion trace in panel a). Peak1GFP encapsulated in TcB:TcC shell, Peak 2 non bound GFP; c, left microcentrifuge tube contains protein from Peak 1 (no fluorescence) and right microcentrifuge tube contains released GFP from Peak 2 (fluorescence under UV illumination).

EXAMPLES

(17) The invention will now be exemplified with reference to the following non-limiting Examples

Example 1: Elucidation of the Structure of the Complex Formed Between the TcB and TcC Components of ABC Toxin Complexes (Tc)

(18) The ABC toxin complexes (Tc) produced by some bacteria are of interest due to their potent oral insecticidal activity.sup.1,2 and potential role in human disease.sup.3. They are composed of at least three proteins, TcA, TcB and TcC, which must assemble together in order to be fully toxic.sup.4. The carboxy-terminal section of TcC is the main cytotoxic component.sup.5, and displays remarkable heterogeneity between different Tcs. A general model of action has been proposed, in which the TcA component first binds to the cell surface, is endocytosed and subsequently forms a pH-triggered channel, allowing the translocation of TcC into the cytoplasm.sup.5, where it can cause cytoskeletal disruption in both insect and mammalian cells. Tc complexes have been visualised using single particle electron microscopy.sup.6,7, but no high-resolution structures of the components are available, and the role of TcB in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex between TcB and the conserved amino-terminal section of TcC determined to 2.3 by X-ray crystallography. These components assemble to form an unprecedented large hollow structure that encapsulates and sequesters the cytotoxic carboxy-terminal portion of TcC like the shell of an egg. The shell is decorated on one end with -propeller domain, which mediates attachment of the TcB/TcC dimer to the TcA component of the complex. Furthermore, the structure shows how TcC auto-proteolyses when folded in complex with TcB. TcC is the first known protein structure to contain RHS (rearrangement hot spot) repeats.sup.8, and illustrates the structural architecture that is likely to be conserved across this widely distributed bacterial protein family and the related eukaryotic YD-repeat-containing protein family, which includes the teneurins.sup.9. In addition to indicating the function of these protein families, the structure suggests a generic mechanism for protein encapsulation and delivery.

(19) ABC toxins were first identified, and have been best characterised, in the bacterium Photorhabdus luminescens. The entomopathogenic bacterium Yersinia entomophaga contains a related Tc locus where the TcA component is split into two ORFs (YenA1 and Yen A2), along with a single TcB gene (YenB) and two TcC genes (YenC1 and YenC2).sup.10. The TcC proteins of this and other Tcs are similar to the polymorphic toxins described by Zhang et al..sup.11 as they have a conserved RHS-repeat-containing amino-terminal region and a variable carboxy-terminal region.sup.10. The carboxy-terminal regions of the Y. entomophaga TcC proteins are predicted to have different toxic activities: YenC1-C is homologous to cytotoxic necrotising factor 1 (CNF1) from Escherichia coli.sup.12, while YenC2-C is homologous to the deaminase YwqJ from Bacillus subtilis.sup.13. When YenB and YenC1 or YenC2 proteins are co-expressed, the YenC proteins are cleaved at the boundary between the conserved amino-terminal domain and the variable carboxy-terminal domain.

(20) As the function of TcB proteins in Tcs is unknown, we prepared the complex of YenB (167 kDa) with the conserved N-terminal 76 kDa portion of YenC2 (YenC2-N) by co-expression and incubation at low pH (Supplementary methods), and solved its structure using X-ray crystallography (Table S1). The structure reveals a remarkable, intimately associated heterodimer formed by YenB and YenC2-N that cooperatively fold into a large, hollow shell (FIG. 1a). An immediately striking feature is the single long -sheet, comprised of 76 -strands derived from both proteins, that constitutes the majority of the shell structure. The shell is completed by a second -sheet formed by 14 strands contributed by YenC2-N, bringing to 90 the total number of -strands that wrap around what is a substantial central cavity (FIG. 1 b & c; FIG. 5). The carboxy-terminus of YenB is in close proximity to the amino-terminus of YenC2, suggesting that the two proteins could be produced as a single polypeptide. Evidence in support of this can be found in the bacterium Burkholderia rhizoxinica where a single ORF (tcdB2) encodes an apparent TcB-TcC fusion protein.

(21) The central cavity is a solvent-accessible space approximately 42 wide and 87 long, with a total enclosed volume of approximately 59,000 .sup.3. The shell is closed at both endsthe YenB end by a -propeller domain inserted into the loop between strands 29 and 51, and the YenC2 end by another short strip of -sheet (strands 145-149) that spirals inwards, forming a plug. The overall shape is reminiscent of a hollow egg.

(22) The carboxy-terminal end of YenC2-N (i.e. the cleavage site between the two portions of YenC2) lies inside this shell. We therefore propose that the complete YenB/YenC complex encapsulates YenC2-C within the shell of -sheets created by YenB and YenC2-N. This proposal is supported by small-angle X-ray scattering data, which are consistent with a hollow spheroid for the YenB/YenC2-N complex, but with a solid spheroid for the complete YenB/YenC2 complex (Supplementary FIGS. 2-4 and Tables 8-11). This explains how YenC2-C remains tightly associated with the complex after auto-proteolysis, in the absence of any covalent linkage between the proteins. In broader terms, this also explains how generally cytotoxic proteins encoded by the C-terminal regions of TcC proteins, such as deaminases, can be safely produced without intoxication of the producing cell. According to the current model, the toxic payload, in this case YenC2-C, remains sequestered until exposure to a change in pH triggers its release.sup.5.

(23) The amino acids immediately before the YenC2 cleavage site are clearly visible in the electron density, allowing us to suggest a mechanism of auto-proteolysis. We propose that two aspartate residues (D663 and D686) positioned either side of the last residue prior to cleavage (M690), form the catalytic site for proteolysis (FIG. 2). In our structure, they are too far apart to form the canonical aspartic protease arrangement (6.2 between carboxyl oxygens), but as we have visualised the post-cleavage state, it is possible that prior to cleavage these residues may adopt a slightly different conformation. To establish their role, we made point mutations that replaced the conserved aspartates with asparagines (D663N & D686N), and showed that mutation of either residue completely abolished auto-proteolytic activity (FIG. 9). These residues are part of the strongly conserved RHS repeat-associated core domain.sup.14 (TIGRfam TIGR03696, Interpro IPR022385), which is widely distributed across the archaea, bacteria and eukaryota. Our structure therefore shows this domain to be a cryptic aspartic protease.

(24) RHS repeats themselves are present in many polymorphic toxin complexes that are found across a diverse range of bacterial species. Until now, RHS-repeat containing proteins have been structurally intractable, making this structure of YenC-N the first of any protein containing RHS repeats. Individual RHS repeat proteins can vary in size, and the overall sequence conservation across the family is low, but a consensus sequence for the repeat has been previously defined: GxxxRYxYDxxGRL(I/T).sup.15. When this is mapped onto the structure of YenC-N (FIG. 3), it is clear that each RHS repeat corresponds to a single strand-turn-strand motif, multiple copies of which make up the extended -sheet of the shell. Although the initial glycine is not especially well conserved, it marks the hairpin facing the YenC2-end of the shell. The central DxxG creates the hairpin facing the YenB-end, with the aspartic acid hydrogen-bonding to the backbone amides of the glycine and adjacent arginine. This glycine is largely conserved, but the aspartic acid can be replaced by a glutamic acid, threonine or serine and typically the interactions formed remain the same. The YxY motif places the two tyrosine sidechains inside the shell (coloured magenta in FIG. 3a) where they sit parallel to each other, and also stack with the post-hairpin arginine from an adjacent strand. The conserved hydrophobic amino acids at the C-terminal end of the repeat (coloured yellow in FIG. 3a) also lie inside the shell, forming a continuous hydrophobic stripe along the face of the -sheet composed of tyrosines and leucines/isoleucines on alternating strands (FIG. 3b).

(25) The RHS structural motif is present in YenB as well as YenC2-N, albeit with less sequence conservation. The YenB sequence contains more insertions and extensions within the RHS repeats than the YenC sequence, which makes identifying the RHS pattern difficult by sequence conservation alone. However, inspection of the structure reveals many examples of DxxG turns and tyrosine or phenylalanine sidechains arranged in an equivalent fashion. Using this structural conservation as a guide, we were able to produce a refined consensus sequence for the RHS repeat (FIG. 10) and show that the pattern of conservation is identical to that seen in YD repeats (TIGRfam TIGR01643; FIGS. 10, 12, 13, 14) that are found in many bacterial and eukaryotic proteins, notably in the extracellular domains of teneurins, which are developmental signalling proteins conserved from flies to mammals and required for synaptic partner matching.sup.16,17. We propose that RHS and YD repeats represent a conserved structural motif that will always give rise to an extended -sheet, forming a shell structure similar to that seen here. Support for this proposal can be found in previous low-resolution EM images of the extracellular domains of mouse teneurin, which revealed globular domain of similar dimensions to the YenB/YenTc-C complex.sup.18. We predict that the YD-repeat containing domains of eukaryotic teneurins will encapsulate their C-terminal regions, the teneurin C-terminal associated peptides (TCAPs), which are known to be active extracellular signalling components in mice.sup.19,20.

(26) Previous visualisations of complete ABC Tcs from Y. entomaphaga.sup.6 and P. luminescens.sup.7 have shown that the TcB/TcC complex sits in the vestibule of the channel-forming domain of TcA, positioned at the end of the Tc complex furthest from the membrane. Fitting the YenB/YenC-N structure into the 25 EM map of the P. luminescens Tc unambiguously places the five-bladed -propeller domain of the TcB/TcC as a point of interaction with the TcA pentamer (FIG. 4). In Tcs such as Yen-Tc, where the TcA component is encoded by two separate ORFs, this represents an interaction with YenA2. We are now able to model the complete Yen-Tc complex for the first time (FIG. 4) by docking both the YenB/YenC-N complex and both associated chitinase enzymes, Chi1 and Chi2, onto the previously determined 16 EM structure of the Y. entomaphaga YenA pentamer.sup.6.

(27) A general model for the injection mechanism of Tcs has been proposed, based on the EM structure of a membrane-bound form of the P. luminescens Tc, in which the C-terminal domain of TcC is translocated in an unfolded state through a transmembrane pore, 15 in diameter, formed by TcA. It remains unclear whether the toxic C-terminal region of YenC is encapsulated in a folded or unfolded state within the YenB/YenC-N shell, but the central cavity is large enough to contain the C-terminal region of YenC2 in a folded state, assuming it adopts the same fold as other deaminases (FIG. 11). As the pore of the TcA channel has not yet been visualised in an active conformation, it remains a possibility that the translocation state of the toxin contains an open pore wide enough to allow the passage of a folded protein. On the other hand, the overall architecture of the YenB/YenC-N shell, with its conserved RHS repeats producing an interior hydrophobic pattern of tyrosine, leucine and isoleucine residues, is reminiscent of the protein chaperone GroEL.sup.21, perhaps implying that the function of TcB/TcC proteins, and of RHS and YD repeats more generally, is to encapsulate unfolded proteins. There is support for this idea in the observation that many polymorphic toxins have predicted proteases as their toxic components, which would need to be contained in an inactive state to prevent proteolysis of the shell itself.

(28) Release of the encapsulated TcC-C from the TcB/TcC-N shell will require a conformational change, as there are no gaps in the structure large enough for a polypeptide to pass though. Two possibilities exist the -propeller blades could separate, allowing extrusion of an unfolded polypeptide through the middle of the propeller, or the propeller domain could swing aside like a bottle-top, hinged on the 29/30 and 50/51 loops, which form the only covalent connections between the -propeller and the main body of the shell. Either mechanism is likely to be dependent on both a pH-driven tigger and mechanical interactions with the TcA component of the toxin.

(29) The structure of the YenB/YenC-N complex presented here reveals how the cytotoxic TcC components of ABC-type Tc complexes are processed and contained, demonstrates the function of the TcB component within the Tc and provides a framework for further experiments to build a complete mechanistic model of action for this class of toxins. More broadly, it also illuminates the function of the widely distributed RHS and YD repeat families of proteins, which had until now been unknown.

(30) Methods Summary

(31) The YenB/YenC2 protein complex was produced by co-expression in E. coli and purified using Ni-affinity and size exclusion chromatography. The YenB/YenC2-N protein complex was obtained by dialysing YenB/YenC2 against acetate buffer at pH 4.5, filtration and size exclusion chromatography. Crystallisation was carried out by hanging-drop vapor diffusion with microseeding in drops containing 18% (w/v) PEG 3350, 0.15 M KH.sub.2PO.sub.4 pH 4.8. X-ray diffraction data was collected to a resolution of 2.26 at beamline MX2 at the Australian Synchrotron.sup.22, integrated using XDS.sup.23 and scaled and merged using Aimless.sup.24 (Fables 6 and 7). Phasing was accomplished by a combination of MAD and SAD using Ta.sub.6Br.sub.12 soaked and selenomethionine-substituted crystals.sup.25,26. Structure refinement and analysis was performed using Phenix.sup.27 and diagrams were produced using PyMol.sup.28 and Chimera.sup.29

(32) Supplementary Methods.

(33) YenC-C Dissociates at Low pH

(34) When YenB and either YenC1 or YenC2 were co-expressed in E. coli, YenC1 and YenC2 auto-proteolysed into two fragments as described previously (ref). In both cases, although all three protein fragments co-eluted as a single complex when purified by size-exclusion chromatography, we were unable to crystallise the purified complexes. As the current model for Tc cell entry involves exposure to low pH in the acidified endosome.sup.5, we tested the behaviour of YenB+YenC1/YenC2 complexes at a range of pH conditions from 4.5 to 9.5. At low pH (4.5-5.0), the complexes began to precipitate, with the C-terminal domains of the YenC proteins showing differential precipitation, allowing purification of complexes containing just YenB and the N-terminal portions of YenC.

(35) SAXS Data Collection and Processing

(36) Small-angle X-ray scattering data were collected at the SAXS/WAXS beamline at the Australian Synchrotron (FIGS. 6, 7 and 8; Tables 8, 9, 10 and 11). Samples were purified to homogeneity by IMAC, and SEC and exhaustively dialysed against sample buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl. The dialysate was used as the solvent blank. Data collection was carried out at 291 K with 1 or 2 second exposures. Sample was flowed across the beam at 4 l/s to avoid radiation damage. Multiple concentrations were tested for each protein, and images for each concentration were compared, averaged and buffer-subtracted using the ScatterBrain software provided by the Australian Synchrotron. Scattering data were placed on the absolute scale by measuring the scattering of a water sample.sup.30. Ab initio bead models were created by running dammif.sup.31 20 times, superimposing and averaging the resulting models with damaver.sup.32, and using this as input for a final refinement run of dammin.sup.33. SAXS data were compared with the theoretical scattering of the YenB/YenC2-N crystal structure using crysol.sup.34.

(37) TABLE-US-00006 TABLE 6 Data collection and refinement statistics for native YenB/ YenC2-N dataset. Wavelength () 0.9537 Resolution range () 49.55-2.26 (2.40-2.26) Space group P2.sub.12.sub.12.sub.1 Unit cell () 133.7 147.6 274.4 90 90 90 Total reflections 3,380,388 (245,257) Unique reflections 245,036 (33,576) Completeness (%) 97.3 (83.2) Multiplicity 13.8 (7.3) Mean I/(I) 9.81 (0.81) CC.sub.1/2 (%) 99.1 (15.7) Wilson B-factor 37.69 R-measure 0.3129 (2.4219) R-factor/R-free 0.2075/0.2574 Number of atoms 35,548 macromolecules 33,027 ligands 54 water 2,467 Protein residues 4,240 RMS (bonds) () 0.005 RMS (angles) () 0.99 Ramachandran favored (%) 96.00 Ramachandran outliers (%) 0.14 Clashscore 12.16 Average B-factor 48.20 macromolecules 48.30 solvent 46.80 Statistics for the highest-resolution shell are shown in parentheses.

(38) TABLE-US-00007 TABLE 7 Data collection and refinement statistics for selenomethionine protein crystals. Combined Ta.sub.6Br.sub.12 Dataset SeMet 1 SeMet 2 SeMet 3 SeMet 4 SeMet soak Wavelength 0.979100 0.979100 1.258000 () Resolution 94.11-2.78 96.36-2.92 96.32-2.91 96.53-2.77 Dmid 44.79-3.17 range () (2.95-2.78) (3.09-2.92) (3.08-2.91) (2.94-2.77) 21.45 (3.20-3.17) (2.79) Space group P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 Unit cell () 134.6, 134.3, 134.3, 134.9, 135.1, 149.7, 150.5, 150.4, 150.3, 152.9, 276.0, 90, 276.7, 90, 276.6, 90, 276.3, 90, 276.3, 90, 90, 90 90, 90 90, 90 90, 90 90, 90 Total 375,898 907,911 917,907 2,030,940 4,226,680 2,584,590 reflections (43,908) (136,715) (140,953) (227,949) (41,245) (112,511) Unique 238,027 234,490 237,191 271,549 141,963 89,256 reflections (33,377) (36,658) (37,874) (40,699) (5,905) (4,841) Completeness 87.5 99.4 99.7 98.6 99.2 (84.4) 90.6 (%) (75.8) (96.1) (98.4) (91.3) (94.8) Multiplicity 1.6 (1.3) 3.9 (3.7) 3.9 (3.7) 7.5 (5.6) 29.8 (7) 29.0 (23.2) Mean I/(I) 9.12 5.91 6.35 10.67 14.6 (1.1) 13.9 (1.0) (1.64) (0.71) (0.75) (1.18) CC.sub.1/2 (%) 99.5 97.4 97.9 98.8 99.5 (47.2) 99.7 (74.8) (20.5) (23.2) (60.1) (38.8) Anomalous 22 (4) 12 (2) 13 (3) 17 (0) 13.8 (0) 52.5 (9.5) correlation (%) Anomalous 11.1 6.7 6.6 5.5 5.0 5.68 resolution.sup.1 () Statistics for the highest-resolution shell are shown in parentheses. .sup.1Anomalous resolution defined as the point at which CC.sub.anom drops below 0.3.

(39) TABLE-US-00008 TABLE 8 Data collection and scattering derived parameters for YenB/YenC2 SAXS. Data-collection parameters Instrument Australian Synchrotron SAXS/WAXS beamline Beam geometry point Wavelength () 1.12713 q range (.sup.1) 0.009-0.614 Exposure time 2 s Concentration range (mg/ml) 5-0.15 Temperature (K) 291 Structural parameters.sup.1 I(0) (cm.sup.1) [from P(r)] 0.190 R.sub.g () [from P(r)] 42.0 I(0) (cm.sup.1) [from Guinier] 0.189 R.sub.g () [from Guinier] 41.8 D.sub.max () 134 Porod volume estimate (.sup.3) 395,300 Molecular mass determination.sup.1 Partial specific volume (cm.sup.3/g) 0.7425 Contrast ( 10.sup.10/cm.sup.2) 2.1 Molecular mass [from I(0)] (kDa) 261.5 Molecular mass [from SAXS-MoW] (kDa) 253.8 Calculated monomeric M.sub.r from sequence (kDa) 276.3 Software employed Primary data reduction ScatterBrain Data processing GNOM Ab initio analysis DAMMIF & DAMMIN Validation and averaging DAMAVER Three-dimensional graphics representations PyMOL .sup.1data reported for 5 mg/ml concentration.

(40) TABLE-US-00009 TABLE 9 Concentration dependence of SAXS data for YenB/YenC2. Concentration Guinier R.sub.g standard (mg/ml) range R.sub.g () deviation (%) I.sub.0/concentration 5 5-18 41.8 0 0.189 2.5 3-17 42.7 0 0.193 1.25 7-17 42.7 0 0.176 0.31 7-17 42.9 1 0.161 0.15 16-38 41.8 1 0.147

(41) TABLE-US-00010 TABLE 10 Data collection and scattering derived parameters for YenB/YenC2-N SAXS. Data-collection parameters Instrument Australian Synchrotron SAXS/WAXS beam Beam geometry point Wavelength () 1.12713 q range (.sup.1) 0.004-0.255 Exposure time 1 s Concentration range (mg/ml) 1-0.016 Temperature (K) 291 Structural parameters.sup.1 I(0) (cm.sup.1) [from P(r)] 0.20 R.sub.g () [from P(r)] 44.1 I(0) (cm.sup.1) [from Guinier] 0.20 R.sub.g () [from Guinier] 44.2 D.sub.max () 138 Porod volume estimate (.sup.3) 365,000 Molecular mass determination.sup.1 Partial specific volume (cm.sup.3/g) 0.7425 Contrast ( 10.sup.10/cm.sup.2) 2.1 Molecular mass [from I(0)] (kDa) 277.0 Molecular mass [from SAXS-MoW] (kDa) 257.6 Calculated monomeric M.sub.r from sequence 243.3 (kDa) Software employed Primary data reduction ScatterBrain Data processing GNOM Ab initio analysis DAMMIF & DAMMIN Validation and averaging DAMAVER Computation of model intensities CRYSOL Three-dimensional graphics PyMOL representations .sup.1Data reported for 1 mg/ml concentration.

(42) TABLE-US-00011 TABLE 11 Concentration dependence of SAXS data for YenB/YenC2-N. Concentration R.sub.g standard (mg/ml) Guinier range R.sub.g deviation I.sub.0/concentration 1 20-40 44.20 3% 0.200 0.5 24-45 43.87 11% 0.216 0.25 21-45 44.06 10% 0.216 0.125 8-30 46.95 10% 0.208 0.063 18-43 43.95 7% 0.190 0.031 5-41 45.82 9% 0.161 0.016 15-43 44.71 56% 0.125 1. Bowen, D. et al. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129-2132 (1998). 2. ffrench-Constant, R. H. & Waterfield, N. R. Ground control for insect pests. Nat. Biotechnol. 24, 660-661 (2006). 3. Hares, M. C. et al. The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells. Microbiology 154, 3503-3517 (2008). 4. Waterfield, N., Hares, M., Yang, G., Dowling, A. & ffrench-Constant, R. Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria. Cell. Microbiol. 7, 373-382 (2005). 5. Lang, A. E. et al. Photorhabdus luminescens Toxins ADP-Ribosylate Actin and RhoA to Force Actin Clustering. Science 327, 1139-1142 (2010). 6. Landsberg, M. J. et al. 3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity. Proc. Nat. Acad. Sci. USA 108, 20544-20549 (2011). 7. Gatsogiannis, C., Lang, A. E., Meusch, D. & Pfaumann. A syringe-like injection mechanism in Photorhabdus luminescens toxins. Nature 495, 520-523 (2013). 8. Hill, C. W., Sandt, C. H. & Vlazny, D. A. Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein. Mol. Microbiol. 12, 865-871 (1994). 9. Minet, A. D., Rubin, B. P., Tucker, R. P., Baumgartner, S. & Chiquet-Ehrismann, R. Teneurin-1, a vertebrate homologue of the Drosophila pair-rule gene ten-m, is a neuronal protein with a novel type of heparin-binding domain. J. Cell Sci. 112, 2019-2032 (1999). 10. Hurst, M. R. H., Jones, S. A., Binglin, T., Harper, L. A. & Glare, T. R. The main virulence determinant of Yersinia entomophaga MH96 is a broad host range insect active, Toxin Complex. J. Bacteriol. (2011). 11. Zhang, D., de Souza, R. F., Anantharaman, V., Iyer, L. M. & Aravind, L. Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics. Biol. Direct 7, 18 (2012). 12. Buetow, L., Flatau, G., Chiu, K., Boquet, P. & Ghosh, P. Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1. Nat. Struct. Biol. 8, 584-588 (2001). 13. Iyer, L M., Zhang, D., Rogozin, I. B. & Aravind, L. Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems. Nucleic Acids Res. 39, 9473-9497 (2011). 14. Jackson, A. P., Thomas, G. H., Parkhill, J. & Thomson, N. R. Evolutionary diversification of an ancient gene family (rhs) through C-terminal displacement. BMC Genomics 10, 584 (2009). 15. Wang, Y. D., Zhao, S. & Hill, C. W. Rhs elements comprise three subfamilies which diverged prior to acquisition by Escherichia coli. J. Bacteriol. 180, 4102-4110 (1998). 16. Mosca, T. J., Hong, W., Dani, V. S., Favaloro, V. & Luo, L. Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice. Nature 484, 237-241 (2012). 17. Hong, W., Mosca, T. J. & Luo, L. Teneurins instruct synaptic partner matching in an olfactory map. Nature 484, 201-207 (2012). 18. Feng, K. et al. All four members of the Ten-m/Odz family of transmembrane proteins form dimers. J. Biol. Chem. 277, 26128-26135 (2002). 19. Chand, D. et al. C-terminal processing of the teneurin proteins: Independent actions of a teneurin C-terminal associated peptide in hippocampal cells. Mol. Cell. Neurosci. 52, 38-50 (2013). 20. Tucker, R. P. & Chiquet-Ehrismann, R. Teneurins: a conserved family of transmembrane proteins involved in intercellular signaling during development. Dev. Biol. 290, 237-245 (2006). 21. Xu, Z., Horwich, A. L. & Sigler, P. B. The crystal structure of the asymmetric GroEL-GroES-(ADP)_7 chaperonin complex. Nature 388, 741-750 (1997). 22. McPhillips, T. M. et al. Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines. J. Synchrotron Radiat. 9, 401-406 (2002). 23. Kabsch, W. XDS. Acta Cystallogr. D 66, 125-132 (2010). 24. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760-763 (1994). 25. Panjikar, S., Parthasarathy, V., Lamzin, V. S., Weiss, M. S. & Tucker, P. A. Auto-rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment. Acta Crystallogr. D 61, 449-457 (2005). 26. Panjikar, S., Parthasarathy, V., Lamzin, V. S., Weiss, M. S. & Tucker, P. A. On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination. Acta Crystallogr. D 65, 1089-1097 (2009). 27. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Cystallogr. D 66, 213-221 (2010). 28. Schrodinger, L. L. C. The PyMOL Molecular Graphics System, Version 1.3r1. (2010). 29. Pettersen, E. F. et al. UCSF Chimeraa visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605-1612 (2004). 30. Orthaber, D., Bergmann, A. & Glatter, O. SAXS experiments on absolute scale with Kratky systems using water as a secondary standard. J. Appl. Crystallogr. 33, 218-225 (2000). 31. Franke, D. & Svergun, D. I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Cystallogr. 42, 342-346 (2009). 32. Volkov, V. V. & Svergun, D. I. 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Example 2: Demonstrating Activity by Expressing Toxin Proteins in E. coli and Feeding to Insects

(43) Cloning and Expression

(44) The encapsulated proteins of the invention can be expressed using commercially available non-conjugative vectors such as pET in E. coli (GATEWAY technology, Invitrogen).

(45) Once the gene expression has been established, by methods well-known to those skilled in the art, the transformed E. coli (as a bacterial cell in broth culture) can be used in a standard bioassay against, for example, diamondback moth (DBM) and other insects, to demonstrate insecticidal activity.

(46) The kit uses Transform One Shot Chemically Competent E. coli. The pET vectors carry a bacteriophage T7 promoter, transcription and translation signals. The source of T7 RNA polymerase is provided by the host cells.

(47) Bioassay Methods

(48) Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae), Cabbage White Butterfly, Pieris rapae (Lepidoptera: Pieridae) and Cabbage Looper Moth, Trichoplusia ni (Lepidoptera: Noctuidae)

(49) Diamondback moth larvae can be reared on brassica (cabbage plants), or the strains resistant to Cry1A and Cry1C and a susceptible (G88) strain can be obtained and tested at the New York State Agricultural Experiment Station, College of Agriculture and Life Sciences at Cornell University, located in Geneva, N.Y., USA. Cabbage white butterfly larvae can be field-collected.

(50) Ten 2.sup.rd-3.sup.rd instar larvae can be used and placed on 3 cm disc of cabbage leaf treated with either 20 l of transformed E. coli solution, or dipped in the solution. A wetting agent, Siliwet L-77 (Momentive Performance Materials, New York, USA) or Triton X-100 (Rohm and Hass Co, Philadelphia, USA) is used at <0.05%. Each treatment can be replicated 3-5 times (3-50 larvae per treatment). Treated larvae remain on the cabbage leaf at 23 C. 16L:8HD (Lincoln) or at 27 C. 16hL:8hD (USA) and are checked daily for dead larvae.

Example 3: Demonstrating Activity in Transgenic Plants and in Plant Bioassay

(51) Cloning

(52) Genetic Constructs Used in the Transformation Protocol

(53) Sequences for expressing the encapsulated proteins of the invention can be cloned into suitable constructs and vectors for transformation of plants as is well known by those skilled in the art, and disclosed herein.

(54) For example, the plant transformation vector, pHZBar is derived from pART27 (Gleave 1992, Plant Mol Biol 20: 1203-1207). The pnos-nptII-nos3 selection cassette has been replaced by the CaMV35S-BAR-OCS3 selection cassette with the bar gene (which confers resistance to the herbicide ammonium glufosinate) expressed from the CaMV 35S promoter. Cloning of expression cassettes into this binary vector is facilitated by a unique NotI restriction site and selection of recombinants by blue/white screening for -galactosidase.

(55) The polynucleotide sequences encoding the encapsulated proteins of the invention can be cloned by standard techniques into pART7 downstream of the 35S promoter. A unique NotI fragment can then be shuttled into pART27 (Gleave, 1992, Plant Mol Biol 20: 1203-1207) for transformation of various plant species. This binary vector contains the nptII selection gene for kanamycin resistance under the control of the CaMV 35S promoter. Genetic constructs in pART27 can be transferred into Agrobacterium tumefaciens strain GV3101 or EHA105 as plasmid DNA using freeze-thaw transformation method (Ditta et al 1980, Proc. Natl. Acad. Sci. USA 77: 7347-7351). The structure of the constructs maintained in Agrobacterium can be confirmed by restriction digest of plasmid DNA's prepared from bacterial culture. Agrobacterium cultures can be prepared in glycerol and transferred to 80 C. for long term storage. Genetic constructs maintained in Agrobacterium strain GV3101 can be inoculated into 25 mL of MGL broth containing spectinomycin at a concentration of 100 mg/L. Cultures can be grown overnight (16 hours) on a rotary shaker (200 rpm) at 28 C. Bacterial cultures can be harvested by centrifugation (3000g, 10 minutes). The supernatant is removed and the cells resuspended in a 5 mL solution of 10 mM MgSO.sub.4.

(56) Plant Transformation

(57) Plants can be transformed to express the encapsulated proteins of the invention by numerous methods well known to those skilled in the art and disclosed herein.

(58) Tobacco Transformation

(59) Tobacco can be transformed via the leaf disk transformation-regeneration method (Horsch et al. 1985). Leaf disks from sterile wild type W38 tobacco plants are inoculated with an Agrobacterium tumefaciens strain containing the appropriate binary vector, and cultured for 3 days. The leaf disks are then transferred to MS selective medium containing 100 mg/L of kanamycin (or 5 mg/L of glufosinate) and 300 mg/L of cefotaxime. Shoot regeneration occurs over a month, and the leaf explants are placed on hormone free medium containing kanamycin or glufosinate for root formation.

(60) Sorghum Transformation

(61) The constructs described above can also be used to transform Sorghum. A suitable protocol for transforming Sorghum is found in Howe et al., 2006, Plant Cell Reports, Volume 25, No 8, 784-791.

(62) Cotton Transformation

(63) The constructs described above can also be used to transform cotton. A suitable protocol for transforming cotton is found in U.S. Pat. No. 5,846,797.

(64) Wheat Transformation

(65) The constructs described above can also be used to transform wheat. A suitable protocol for transforming wheat is found in Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877

(66) Maize Transformation

(67) The constructs described above can also be used to transform maize. A suitable protocol for transforming maize is found in U.S. Pat. No. 8,247,369.

(68) Transformation of Other Plants

(69) Transformation protocols for other plants are well-known to those skilled in the art, and are disclosed herein.

(70) ELISA Assay

(71) ELISA analysis according to the method disclosed in U.S. Pat. No. 5,625,136 can be used for the quantitative determination of the level of the encapsulated proteins in transgenic plants, or parts thereof.

(72) Bioassay

(73) Various parts of the transformed plants, or whole plants can be used in standard insect bioassay procedures to test the activity of the encapsulated proteins of the invention, and the resistance of the transformed plants to various insects.

(74) Numerous suitable methods are known to those skilled in the art and are described for example in the following US patents: U.S. Pat. Nos. 8,247,369; 8,216,806; 8,173,872; 8,034,997; 7,858,849; and 7,803,993; 7,655,838 and 7,919,609; all of which are incorporated herein by reference.

Example 4: Demonstrating Encapsulation of a Foreign Protein

(75) The applicants have created versions of the TcB/TcC (BC) complex in which the C-terminal region of YenC2 (TcC) has been replaced with green fluorescent protein (GFP). Two versions of this construct have been created, the native-GFP version, in which GFP has a net negative charge, and a version containing a modified GFP with a net positive charge (GFP+6), mimicking the charge of the normal TcC.

(76) Both constructs have been expressed in E. coli and purified by standard procedures (immobilized metal ion affinity chromatography [IMAC], and size exclusion chromatography [SEC]). The GFP protein is produced as a fusion of the N-terminal region of YenC2 (YenC2NTR) and GFP. This fusion protein was expected to self-cleave at the boundary between these two proteins, analogous to the cleavage that occurs in the native complex. This cleavage occurs with both the native GFP and GFP+6 variants, and the protein complex consisting of YenB, the N-terminal region of YenC2 (YenC2NTR), and GFP co-purify and form a single peak on size exclusion. This indicates that GFP is being encapsulated within the BC shell, in a similar manner to the native TcC. This protein complex does not fluoresce, suggesting that GFP is encapsulated in an unfolded state. After storage for several days, fluorescence was observed with the native-GFP-containing complex. When this was again subjected to SEC, a major peak consisting of all three complex proteins (YenB, YenC2NTR, GFP) was observed (FIG. 16 A-B), which did not fluoresce (FIG. 16 C). A smaller peak was also observed consisting of GFP alone, which did fluoresce (FIG. 16 B-C). This indicates that there has been some slow leakage of GFP from the complex, at which point GFP folds and is able to fluoresce. This leakage occurred at a reduced rate in the positively-charged GFP+6 variant.

(77) Materials and Methods

(78) Yersinia entomophaga YenB and YenC2 were cloned into the pETDuet-1 co-expression vector using standard cloning techniques. Expression was performed in E. coli Rosetta2(DE3) cells using ZYM-5052 auto-induction medium (Studier, 2005). Freshly transformed cells were grown in 5-ml LB cultures overnight and used to inoculate 500-ml ZYM-5052 cultures in 2 litre baffled flasks. These were incubated at 37 C. for 4 hours, followed by 18 C. for 24 hours. Cultures were harvested by centrifugation at 4,680 RCF for 30 minutes and cell pellets were either frozen at 20 C. or used immediately. Cell pellets were resuspended in his-0 buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM 2-mercaptoethanol) with the addition of Roche Complete mini EDTA-free protease inhibitor tablets, according to the manufacturer's directions. Cells were lysed by passage through a continuous-flow cell disrupter (Microfluidics microfluidizer M-110P) at a pressure of 18 MPa. Cell lysate was clarified by centrifugation at 27,000 RCF for 30 minutes followed by filtration. The protein complex was purified by IMAC using a 5-ml Talon HiTrap column. The protein complex was washed with his-0 buffer and eluted with his-150 buffer (identical to his-0 with the addition of 150 mM imidazole). This eluted fraction was concentrated and dialysed against his-0 buffer overnight at 4 C. with the addition of TEV protease (Blommel & Fox, 2007) to remove the his-tag. This protein was subsequently applied to the same Talon HiTrap column and the flow-through collected. This was then concentrated and applied to a HiLoad 16/60 Superdex 200 size exclusion column (GE) attached to an kta prime FPLC system. His-0 buffer was pumped over the column at a rate of 1 ml/minute, and fractions were collected and analysed by SDS-PAGE. Analytical size exclusion was performed using a Superdex 200 10/300 analytical column (GE). GFP fluorescence was determined by illumination with blue light and observation under a yellow filter.

REFERENCES

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