Patent Grant
11548921
This application is a 371 National Stage entry of International Application No. PCT/GB2018/051495 with an international filing date of May 31, 2018, which claims the benefit of priority to Great Britain Application No. 1708675.2 filed on May 31, 2017, the entire contents of each of which are herein incorporated by reference in their entirety for all purposes.
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named P184655_SEQUENCE_ST25.TXT, created on Nov. 27, 2019, and having a size of 29 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
The present invention relates to macromolecular complexes comprising micron-scale networks which include binding motifs thereon which allow the covalent bonding of the micron-scale networks to particles which provide nanoscale display surfaces. Further, the present invention provides for the synthesis of nano-, micro-scale functional materials, in particular synthesis of such materials without the need to use complex and restrictive linker chemistries.
One of the major goals of material science is to produce materials that are ordered on all length scales, from molecular (1-100 Å), nano (10-100 nm) to macro (1-100 micron). The development of systems with integrated catalytically active enzymes, specific antibodies or other biologics have high potential for synthesis, degradation and biosensor diagnostics of various biologically active substances.
It has been demonstrated that short peptide sequences can be incorporated into different regions of the coat proteins of virus or virus like particles (VLP—virus which does not include nucleic acid), for example at the C-termini, such that the peptides are surface displayed on the VLP after self-assembly; conferring surface functionality of interest. However, this system for surface display is limited by the size of the peptide that can be inserted.
Alternatively, functionalization may be achieved by chemical linkage approaches, whereby larger proteins and peptides may be covalently linked to addressable amino acids on the surface of a VLPs (for example carbodiimide crosslinking of carboxyl to amine groups) or by non-specific crosslinking reactions (glutaraldehyde). Additionally, these chemical linkage approaches have been used to create linkages between surface functionalized viruses/VLPs for the production of macroscale scaffolds with multienzyme utility.
However, a disadvantage of chemical linkage approaches is that they may provide too much non-specific cross-linkage which may reduce peptide/protein reactivity, or alternatively/additionally there may be the requirement for additional clean up steps. Thus, alternative strategies are required. WO2014/113203 discusses this problem and proposes asymmetrically functionalised virus particles and linker molecules made up of a long hydrophilic carbon chain or hydrophilic polymer.
WO2016/112921 discusses antigen array display scaffolds provided to spherical VLPs of a size 22 to 150 nm which appears optimal for efficient uptake by professional antigen presenting cells. In particular WO2016/112921 discusses the modification of the AP205 capsid protein to include a 116 amino acid SpyCatcher polypeptide and the production of antigens fused to a SpyTag polypeptide. The antigen can then be coupled to the AP205 capsid protein surface.
The present inventors have determined that virus and virus like particles that are able to assemble into micron structures can be modified to include a peptide tag or a binding protein of a peptide tag and binding protein pair wherein the peptide tag and binding protein pair are capable of spontaneously forming a covalent bond, for example an isopeptide bond, to provide functionalised micron-structures. Advantageously, the method provides for a ‘one-pot’ conjugation step rather than more complex chemical approaches that typically require clean up steps.
Accordingly, a first aspect of the present invention provides a macromolecular complex comprising:
The micronscale network provided by the invention may suitably be spontaneously formed. Suitably a macroscale network structure may be provided for example using a modified TMV coat protein (suitably provided by amino acid changes in the TMV to allow assembly without the presence of nucleic acid and including SpyTag or SpyCatcher). Such a micron macroscale structure provides a porous structure. Suitably there is provided a first complex comprising a self-assembled macromolecular network micron-structure derived from virus components which surface displays a motif that can spontaneously form covalent linkages with a second complex comprising a fluorescent particle wherein the fluorescent particle surface displays the motif interacting partner. Such macroscale structures comprising a fluorescent nano-display surface comprise a non-viral component.
Suitably, TMV coat protein, modified with a peptide tag or binding protein of a peptide tag (for example SpyTag or SpyCatcher) does not form spherical particles, but spontaneously forms micron-sized porous network structures which are larger than individual VLPs.
Suitably the covalent bond spontaneously formed may be an isopeptide bond between a peptide tag and a binding protein of a peptide tag and binding protein pair.
In embodiments the micron-structure can comprise a self-assembled macromolecular network structure derived from virus components. The virus components of the micron-structure can be the same or different to each other. Suitably the virus components may be selected from a virus coat protein or virus like particle. Suitably the virus coat protein or virus like particle can form networks of rods or, when expressed at high levels, aggregates of micron scale.
Various modified TMV coat protein constructs with peptide tag or a binding protein of a peptide tag (e.g. SpyTag or SpyCatcher) introduced into the expressed coat protein have been produced, expressed, and tested under multiple isolation conditions. The TMV constructs have produced micronscale networks rather than distinct 18 nm rods of different length which would be expected if SpyTag or SpyCatcher were not included. Without wishing to be bound by theory, the inventors consider that high levels of expression of coat proteins which can't fold or assemble in the appropriate manner due to an introduced tag or binding protein may be provided during expression of a construct. Partial or aborted assembly may lead to aggregates in the form of networks. It is considered that further types of VLP or virus coat protein depending on the size of the insert and insertion in the coat protein sequence would cause aggregates to form.
Suitably, such virus component or virus like particles may be provided by a rod-shaped virus. Suitably the virus component or virus like particle may be provided by the rod-shaped tobacco mosaic virus (TMV), tobacco rattle virus (TRV), spherical brome mosaic virus, cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus or filamentous potato virus X (PVX). Alternatively, suitably components or virus like particles may be provided by bacteriophages such as M13, T4, T7, lambda or MS2.
Advantageously, these components or virus like particles can be obtained to high yields. Further, advantageously coat proteins (CPs) or other components (not limited to phage tail fibres, or plant virus movement proteins) can be manipulated to surface display peptides, proteins and/or enzymes which confer high catalytic/reactive activities of interest to the virion or structures surface. In a suitable advantageous embodiment, surface display of peptides/proteins/enzymes on characterised coat proteins for example, can greatly enhance the stability of the enzymes/proteins and permit provision of very dense reactive surface areas after self-assembly.
To maintain biocontainment either a virus can be rendered non-replicative, or alternatively virus-like particles (VLPs) produced from self-assembled coat proteins which lack infectious nucleic acid can be used. Given that VLPs can be readily produced from the coat proteins of many viruses and be isolated to high yield with no containment issues, they combine the features of safety and commercial viability. While some virus coat proteins can be expressed which spontaneously self-assemble into VLP structures without any requirement for the viral nucleic acid, others like tobacco mosaic virus (TMV), a major “workhorse” for nanobiotechnology (due to its genetic malleability and stability to temperatures up to 60° C. and pH ranges of 2-9) are considered to require either defined buffering conditions or genetic modification of the CP in order to assemble without nucleic acid. The latter approach is likely more commercially feasible due to less downstream processing and greater particle uniformity.
Wildtype TMV exists as 300 nm length, 18 nm diameter rods formed from 2130 identical coat protein subunits which encapsidate a plus-sense single stranded RNA genome of 6395 nucleotides. Assembly of TMV coat protein (CP) monomers into this structure requires RNA and the presence of cations in order to counteract the flanking repulsive negative charges between the CP subunits that are detrimental to oligomerization. Suitably, a VLP can be provided which includes mutations at E50Q and D77N in the CP sequence to effectively remove the repulsive groups and enable self-assembly of the CP monomers into a TMV rod like structure in the absence of nucleic acid, and when expressed in bacteria the modified CP produce RNA free TMV VLP rods of variable length. Suitably subsequent addition of a protein or “peptide tag” or further changes to the CP sequence, for example inclusion of a binding protein of a peptide tag, or bacterial induction conditions, or purification conditions may provide coat protein which can spontaneously form self assembled macromolecular micron scale network structures.
The TMV coat protein only poorly assembles without nucleic acid in the presence of cations. Mutations at E50Q and D77N overcome this limitation by replacing repulsive charges which prevent interaction between neighbouring coat proteins. This is considered to allow formation of rods which are 18 nm in diameter but can be up to several microns in length (since RNA is not present to dictate the length of 300 nm). Upon insertion of SpyCatcher, SpyTag and/or linker sequences at the C-terminal end of the E50Q D77N mutated coat protein, micronscale networks rather than distinct rods have been determined to form.
These network structures may provide the desired surface displayed functionalities being contingent upon the nature of the introduced groups; these micron scale structures may or may not be composed of obvious discrete VLPs, but may comprise aggregates of the coat protein, for example as discussed Love A., Makarov V., Sinitsyna O., Shaw J., Yaminsky I., Kalinina N., Taliansky M., A genetically modified tobacco mosaic virus that can produce gold nanoparticles from a metal salt precursor. Frontiers in Plant Science, 2015, 6, 984 doi.org/10.3389/fpls.2015.00984.
As used herein, the term “peptide tag” generally refers to a small peptide fragment, suitably 5 to 50 amino acids in length, for example 10, 20, 30, 40 or 50 amino acids in length, which may be fused to another peptide or protein, for example a VLP or a self assembled micron scale network, that may or may not be designed or derived directly from an isopeptide protein. The term “binding partner” is a protein which can form an isopeptide bond to the “peptide tag”.
In embodiments the covalently reactive peptide tag and binding protein pair can comprise SpyCatcher/SpyTag, SnoopCatcher/SnoopTag, the Streptococcus pyogenes split pilin N-terminal isopeptide tag/the Streptococcus pyogenes split pilin C-terminal isopeptide tag, or other peptide tag and protein binding pairs which provide for spontaneous formation of isopeptide bonds, for example between a between carboxyl/carboxamide and amino groups, suitably a lysine and an asparagine residue. Suitably, the covalently reactive peptide tag and binding protein pair may comprise SpyCatcher/SpyTag, SnoopCatcher/SnoopTag, the Streptococcus pyogenes split pilin N-terminal isopeptide tag/the Streptococcus pyogenes split pilin C-terminal isopeptide tag.
Suitably, the tag may be about 5 to 15 amino acids in length. Suitably, the tag or binding protein may not affect the biologically activity of the micron-structure or nanoscale display surface to which it is provided.
In embodiments the first member of the covalently reactive peptide tag and binding protein pair comprises a tag, for example SpyTag, SnoopTag or the Streptococcus pyogenes split pilin C-terminal isopeptide tag, ACE19—a domain of adhesion protein from E. faecalis which spontaneously forms an isopeptide bond, the collagen binding domain from S. aureus which forms an isopeptide bond, FbaB from Streptococcus pyogenes comprising a domain CnaB2, and bacteriophage HK97 subunits which spontaneously form isopeptide bonds. WO2011/098772 discusses such tags and binding protein pairs. Further examples of covalently reactive peptide tag and binding protein pair comprise homologues, for example with at least 70%, at least 80%, at least 90%, at least 95%, at least 99% sequence identity to the tag or binding protein discussed above or mutants of proteins capable of spontaneously forming one or more isopeptide bond, for example wherein the covalently reactive peptide tag and binding protein pairs discussed herein are mutated via insertion, deletion, or substitution of one more than one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues from the wild type residues (known or published sequences) of a peptide tag and binding protein pair which can spontaneously form an isopeptide bond. Suitably a mutation may be within 5 to 10 amino acids of the amino acids which form the isopeptide bond. Suitably, a mutation may be provided by substituting an amino acid(s) with an amino acid with similar properties, for example similar hydrophobicity, charge, size of side chain. Mutants which retain the ability to form an isopeptide bond can be determined using computer software or via for example phage display screening techniques.
Amino acid identity can determined by methods and computer programs as known in the art, for example using the local homology algorithm of Smith and Waterman with the default values. Software for providing such analysis is publicly available through the national Center for Biotechnology Information.
In embodiments the second member of the covalently reactive peptide tag and binding protein pair comprises a SpyCatcher, SnoopCatcher, or the Streptococcus pyogenes split pilin N-terminal isopeptide tag.
In embodiments the micron-structure can comprise a combination of first members of different covalently reactive peptide tags and binding protein pairs wherein the respective peptide tag and binding protein pair are capable of spontaneously forming a covalent bond and a nanoscale display surface can comprise a combination of second members of different covalently reactive peptide tag and binding protein pairs that are respectively capable of binding to the corresponding first member to spontaneously form a covalent bond.
In embodiments a nanoscale display surface can be a nucleic acid molecule, protein, enzyme, peptide, polysaccharide, small molecule, metal-ligand complex, nanoparticle, nanotube or polymer. In embodiments, the nanoscale display surface can be a fluorescent label, a luminescent label, or chromophore. In embodiments, a nanoscale display surface comprising a second member of a covalently reactive peptide tag and binding protein can comprise a fluorescent protein, suitably selected from GFP, CFP, RFP, YFP, mCherry, iLOV or cerulean or fragments or biologically active derivatives thereof (for example cerulean fluorescent protein and yellow fluorescent protein can be obtained from GFP via a few amino acid changes). In embodiments, multiple fluorescent proteins may be conjoined together to form nanoscale display surfaces. Suitably fluorescent proteins as would be known in the art are discussed, for example by Rebekka M Wachter, Marc-André Elsliger, Karen Kallio, George T Hanson and S James Remington; “Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein”; Structure 15 Oct. 1998, 6:1267-1277 [2] Mats Ormo, Andrew B. Cubitt, Karen Kallio, Larry A. Gross, Roger Y. Tsien, S and James Remington; “Crystal Structure of the Aequorea Victoria Green Fluorescent Protein”, Science (273) September 1996 5280, 1392-1396 and Brian G. Reid and Gregory C. Flynn; “Chromophore Formation in Green Fluorescent Protein”; Biochemistry 1997, 36, 6786-6791, and further by Wachter, King, Heim, Kallio, Tsien, Boxer and Remington; “Crystal Structure and Photodynamic behaviour of the blue emission variant Y66H/Y145F of Green.
In an embodiment, the inventors have modified the tobacco mosaic virus (exists as a 18 nm×300 nm rod) coat protein to form micron-scale networks which lack nucleic acid and which also surface display SpyTags (a peptide tag which binds covalently to a SpyCatcher protein via an amide bond (Zakeri, Bijan; Fierer, Jacob O.; Celik, Emrah; Chittock, Emily C.; Schwarz-Linek, Ulrich; Moy, Vincent T.; Howarth, Mark (2012). “Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin”. Proceedings of the National Academy of Sciences. 109 (12): E690-7)).
Further, the inventors have created nanoscale spherical Green Fluorescent Protein (GFP) particles which contain SpyCatcher motifs which can covalently bond to the SpyTags on the surface of the micron-scale networks. Such binding leads to robust and rapid decoration of these networks with spherical particles of GFP.
Each of the
are considered to provide separate aspects of the present invention.
Further, the inventors have developed a “one pot” synthesis of nano-, micro-scale functional materials, whereby, for example proteins or enzymes of any description may be attached to either the micron-structure (for example VLP or other virus derived self-assembled structure) or the nanoscale display surface (for example GFP spherical particles) via covalent interaction, suitably a spontaneously forming isopeptide bond, for example via SpyTag/SpyCatcher interaction, SnoopCatcher, or the Streptococcus pyogenes split pilin N-terminal isopeptide tag. Suitably the synthesis is relatively rapid. Suitably the synthesis does not require complex and restrictive linker chemistries. Accordingly, this provides a separate aspect of the present invention.
The micron-structure of (i) or a nanoscale display surface (ii) of the invention may be provided by chemical synthesis or recombinant cells.
Suitably, there is provided a vector comprising a nucleic acid sequence encoding a micron-structure of (i) or a nanoscale display surface (ii) the invention. The vector may include a control sequence operably linked to the tag and or binding protein. Suitably the control sequence can comprise a promoter, enhancer, transcription initiation site, termination site, or a combination of thereof. The vector may further comprise a selective marker gene or means to aid the incorporation of the nucleic acid into a cell, or the transcription or expression of the nucleic acid into the cell.
Suitably, the present invention provides a kit comprising at least one of (i) a micron-structure comprising a first member of a covalently reactive peptide tag and binding protein pair wherein the peptide tag and binding protein pair are capable of spontaneously forming a covalent bond, and (ii) a nanoscale display surface comprising a second member of a covalently reactive peptide tag and binding protein pair that is capable of binding the first member of (i) to spontaneously form a covalent bond. Suitably the kit may comprise a vector(s) able to express at least one of
(i) a micron-structure comprising a first member of a covalently reactive peptide tag and binding protein pair wherein the peptide tag and binding protein pair are capable of spontaneously forming a covalent bond, and (ii) a nanoscale display surface comprising a second member of a covalently reactive peptide tag and binding protein pair that is capable of binding the first member of (i) to spontaneously form a covalent bond. Suitably the kit may further comprise cells into which the vector may be introduced. Suitably the kit may comprise cells transformed with a vector or vectors of the invention.
In embodiments a nucleic acid sequence encoding the peptide tag or binding partner may be operable linked to respective control sequences in a vector of interest, for example to express a VLP (or a self assembling virus derived component which may form a VLP and/or micron scale network) and tag, or to express a fluorescent protein and binding protein.
It is considered the development of biomimetic 2d and 3d nanodevices and nanomaterials from virus and other protein nanoparticles have potential applications in medicine, veterinary medicine, agriculture and biocatalysis.
The SpyTag/SpyCatcher system has been developed from the collagen adhesion domain of Streptococcus pyogenes, in particular the SpyTag (13 amino acids) peptide can form a covalent isopeptide bond with its interacting partner, namely the SpyCatcher (116 amino acids) peptide. This bond formation is typically spontaneously rapid and specific. This bond formation reaction can occur in diverse conditions and is relatively unaffected by temperature and pH variation. Moreover, it is believed that bond formation can occur with SpyCatcher irrespective of whether the SpyTag is located in the N-, C-termini or central regions of the host protein.
In an embodiment of the present invention, SpyTag can be introduced into the C-terminal end of an E50Q and D77N modified TMV CP. Suitably, the modified TMV CP may be expressed in bacteria. It was expected that this would form discrete 18 nm rods of varying length; however surprisingly it was determined that large macromolecular networks were obtained.
To demonstrate the SpyTag was surface displayed on this macroscale structure, the 116 amino acid SpyCatcher sequence was fused to the N-terminal end of GFP in order to test the interaction between the SpyCatcher-GFP with the TMV CP-SpyTag network.
When expressed alone, the SpyCatcher-GFP unexpectedly formed fluorescent 50-100 nm spherical particles. These particles did not include a long C-terminal extension which has previously been indicated as being required for formation of fluorescent spherical particles.
When both the SpyTag displayed macroscale structure was incubated with the SpyCatcher-GFP particles, they formed rapid covalent bonds mediated via the interaction of the SpyCatcher with its SpyTag partner. This confirmed that both respective Spy components were surface displayed and capable of forming a covalent interaction. It is considered that virus derived macromolecular complexes provide a macroscale platform on which nanoscale display particles, for example SpyCatcher-GFP particles can be provided for the rapid surface presentation of proteins (or potential enzymes) of interest.
Suitably, a macroscale network may be derived from TMV coat proteins or TMV VLPs assembled from coat proteins. In embodiments the macroscale network can be formed from a microstructure comprising viral coat proteins or VLPs, suitably, wherein the viral coat proteins may comprise motifs which allow the viral coat proteins or VLPs to coalesce to form networks. The networks formed may not appear to comprise obvious virus like particles and may appear to be aggregated strings of coat proteins. These networks may be formed by at least one of insertion of amino acid sequences or motifs into the coat protein, induction of bacterial expression and protein isolation. Suitably such structures may be modified by introducing a SpyTag into the C-terminal end of the TMV CP. Advantageously, introduction of a SpyTag into the C-terminal end of the TMV-CP may provide large TMV network structures (devoid of nucleic acid), when expressed in bacteria. SpyTag at the C-terminal end of TMV CP can suitably form covalent linkages with its SpyCatcher interacting partner which may be suitably surface displayed on GFP spherical particles. Suitably networks, including SpyTag, may be decorated with surface displayed SpyCatcher, for example GFP spherical particles to provide different functional proteins (or enzymes), effectively producing bioactive 2d/3d macroscale nanomaterials.
In embodiments larger micronscale networks can be created by combining two populations of SpyTag TMV derived networks and utilizing a SpyCatcher linker to join the two networks together. Alternatively SpyTag networks could be mixed with SpyCatcher networks, to form larger network structures. As will be appreciated, a combination of peptide tags and binding proteins to respective peptide tags may be used to allow multiple microscale networks to be combined to form larger structures/networks. Details of further peptide tags and binding proteins which may be used for example in combination with each other or with SpyTag and SpyCatcher are provided below.
The SnoopTag/SnoopCatcher system was developed from the RrgA adhesin from Streptococcus pneumoniae, where the SnoopTag constitutes residues 734-745, and the SnoopCatcher constitutes residues 749-860. The system was further optimized by incorporating mutations at G842T and D848G (Veggiani et al. 2016).
The Isopeptag system was developed from the Streptococcus pyogenes major pilin protein by splitting and separating the covalently interacting domains, whereby the N-terminal pilin domain reacts with the split pilin N-terminal isopeptide tag and the split C-terminal domain interacts with the C-terminal isopeptide tag (Veggiani et al. 2016).
In embodiments the covalent linkages/functionalities of the micron-structures or nanoscale display particles may be provided by the insertion of derivatives, components, and sequence elements of these covalent linkage systems into the protein sequences which provide the micron-structures or nanoscale display.
In embodiments, the micron-structure can be provided with more than one covalent linkage systems, for example a virus coat protein, e.g. modified TMV coat protein which can generate micron or macroscale network complexes can be provided with at least two of SpyTag, SnoopTag and a member of the Isopeptag system with isopeptide bonding activity. This can allow selective attachment to particular tags.
In embodiments, the tag or binding protein may be provided to the micro-structure or nanoscale surface display via a linker or spacer. For example a glycine/serine rich spacer may be provided. In embodiments, the spacer may include a site for specific proteolysis to allow release or the binding pair member from the micron-structure.
Suitably, the isopeptide bond may be a chemical bond or a covalent bond which forms between the tag and the binding partner as described herein which can be stable after heating to 70° C., yet more suitably 80° C., yet more suitably 90° C., yet more suitably 95° C. Suitably the bond can form from pH 2 to 10 and in a temperature range from −20° C. to 100° C., suitably 4° C. to 40° C. at a pH in the range from 5 to 8.
In an embodiment a SpyTag/SpyCatcher can be formed from an amino acid sequence as described by NCBI Entrez Code AAK33238 (encoded by nucleic acid described by NCBI Entrez Accession code AE004092) wherein the isopeptide bond is formed between the lysine at position 179 and asparagine 303. Thus the peptide tag preferably comprises the reactive asparagine of position 303 and the binding partner (SpyCatcher) suitably comprises a fragment with the lysine at position 179.
In an embodiment as described herein ACE19, comprising an amino acid sequence as described by NCBI Entrez Accession code NP_814829, and encoded by the nucleic acid sequence described by NCBI Entrez Accession Code NC_004668 can be used wherein the isopeptide bondy occurs between a lysine residue at position 181 and an asparagine residue at position 294. Thus the tag preferably includes the reactive asparagine at position 294 and the binding partner can comprise a fragment comprising the lysine at position 181.
Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.
As used herein, the articles “a” and “an” refer to one or to more than one (for example to at least one) of the grammatical object of the article.
“About” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.
Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
Embodiments of the invention will now be described by way of example only, with reference to the accompanying figures.
Bacteria which expressed either TMVc (TMV CP with the modified E50Q and D77N groups), TMVc* (TMVc into which a SpyTag was fused to the C-terminal end for surface display after assembly) or GFP+ (SpyCatcher fused to the N-terminus of GFP) were lysed and the lysates were mixed and then purified via a 20% sucrose cushion, prior to western blot analysis with GFP and TMV specific antibodies. Western blots probed with the TMV antibody (
In summary, TMVc and TMVc* are present in their respective lysates, during incubation and also after the sucrose cushion purification.
Prior to incubation of the lysates it was found that the GFP+ reacted strongly with the GFP antibody (
No reactivity was observed in the lysates derived from bacteria which expressed TMVc* or TMVc only, indicating specificity of the antibody. After incubation of the TMVc lysate with the GFP+, the GFP+ band size and pattern did not change relative to the GFP+ only control (
Expressed SpyTag-TMV coat proteins exist as networks and SpyCatcher-GFP forms spherical nanoparticles, which can interact to produce macroscale structures
Samples of the TMVc*, TMVc and GFP+ after lysis, mixing and incubating and centrifugation were taken and assessed using transmission electron microscopy (TEM). Analysis of the TMVc bacterial lysates revealed rods which were 18 nm in diameter with a variable length which ranged from 40 nm to several microns (
In contrast, the TMVc* lysates contained network-like structures (
The GFP+ lysates formed “spherical” particles which ranged in size from 50-100 nm in diameter (
TMVc/GFP+ and TMVc*/GFP+ lysates were incubated and then purified using ultracentrifugation on sucrose cushions as described previously. The obtained structures were obtained using TEM with immunogold labelling with GFP antibodies. It was determined the GFP+ only particles were predominantly lost after ultracentrifugation on sucrose cushions (TEM image not shown) which is confirmatory to the data in
Structural associations were confirmed using immunotrapping TEM analysis. GFP+, TMVc or TMVc* were incubated separately with GFP antibody coated TEM grids. It was found that the GFP+ particles became immunotrapped (
The TMV derived networks and GFP containing SpyCatcher or SpyTag can form covalent links with proteins containing the interacting partner, which may promote protein stability without abolishing protein activity.
To demonstrate the potential utility of the platforms, their capacity to interact with proteins of interest which were fused to either SpyTag or SpyCatcher were tested. In one such embodiment the interaction of GFP+ with a SpyTag fused to the N-terminal of Uridine 5′-diphosphogalactose 4-epimerase (GalEst), an economically important enzyme which can convert UDP-galactose into UDP-glucose, was tested. GalEst was expressed and purified from bacteria as discussed. In one embodiment nickel column isolated GFP+ was incubated with equimolar amounts of nickel column purified GalEst for different periods of time (5 minutes-16 hours). The formation of covalent bonds between the GalEst and GFP+ was assessed using denaturing coomassie stained SDS-PAGES. The isolated and unmixed GalEst and GFP+ samples gave distinct bands of the appropriate size (see
In order to test that the GalEst-GFP+ complexes were still enzymatically active, these structures were tested in their capacity to convert UDP-galactose into UDP-glucose by using a coupled assay with which the level of conversion is stoichiometrically linked to changes in NADH, which can be detected by measuring absorbance at 340 nm (
In another embodiment we produced a variant of the TMVc* in which a linker sequence (3× repeat of GGGGS (residues 160 to 174 of SEQ ID NO: 11 or SEQ ID NO: 12)) is positioned between the C-terminal of the TMV CP and the SpyCatcher-6× his sequence (TMV CP-GGGGS-SpyCatcher His (SEQ ID NO: 11)) or a SpyTag-6× his sequence (TMV CP-GGGGS-SpyTag His (SEQ ID NO: 12)). These constructs were designed to give the additional utility of potential nickel column isolation of the complexes and also to investigate whether inclusion of additional sequences can influence the expected formation of the network structures. The bacterially expressed constructs gave the correct size on western blots probed with a 6× His antibody (
In summary, fusion of the SpyTag to the C-terminal of the E50Q and D77N modified TMV CP leads to formation of network like structures when expressed in bacteria. Moreover insertion of linker sequences (3× repeat of GGGGS (residues 160 to 174 of SEQ ID NO: 11 or SEQ ID NO: 12)), a 6× his tag and/or replacement of SpyTag with SpyCatcher in the TMV derived constructs does not compromise this network formation; indicating plasticity of the networks to support significant sequence alteration. In addition these can covalently bind GFP or an 11-20 sequence derived from a P. ovis antigen sequence fused to SpyCatcher, the interacting partner of the SpyTag. It is considered this can provide simplified production of large networks functionalized with proteins and/or enzymes, which do not involve crosslinker chemistries or multiple complex clean up steps. This work also indicates that GFP-Spycatcher spherical particles could have utility as a surface presentation platform.
Using methods as disclosed herein GFP particles of at least 500 nm in size can be provided. Fusing the SpyCatcher sequence to the N-terminal end of GFP without other modifications at the C-terminus led to production of smaller particles around 50-150 nm. Without wishing to be bound by theory, it is considered this size difference is likely a consequence of the shorter period of induction of expression in bacteria (typically less than 24 hours); however, the size difference may be influenced by the nature of the SpyCatcher protein fused to the GFP. The GFP particles still reacted with the GFP antibody, indicating that the presence of the SpyCatcher does not completely mask the GFP surface. The SpyCatcher-GFP also interacted with the SpyTag on the surface of the TMV coat protein derived networks to form covalent interactions. It is considered the GFP-SpyCatcher particles produced could act as a fluorescent novel surface display system which may be decorated with any protein or enzyme which is fused to a SpyTag at its C-terminus; as was exemplified by the capacity of the GFP-SpyCatcher particles to bind and stabilize a uridine 5′-diphosphogalactose 4-epimerase, which retained its activity. Such interactions may also occur with proteins that have a SpyTag inserted into the central region or fused to the N-terminus. Although the GFP particles can act as a nanoscale surface display platform, it also has the advantage that it can covalently integrate into larger 3d structures (substrates/scaffolds). Suitably the integration of the GFP particles onto a larger 3d substrate/scaffold can be monitored by exploiting the fluorescence of the particles. Suitably the GFP particles may have multiple available SpyCatcher motifs which may not be fully utilized/occupied upon binding 3d macroscale scaffolds. These “free” Spycatcher sites can permit the further attachment of proteins of interest; effectively producing complexes with multifunctional decoration which may be controlled by GFP particles.
It is considered SpyTag can react with its interacting partner even under a wide range of temperatures (at least 4-37° C.), pH (at least 5-8), cation concentrations and non-ionic detergent conditions.
For the SpyTag-TMV (TMVc*), the TMV E50Q and D77N group modified coat protein sequence (TMVc; Brown et al., 2013) was fused at its C-terminal end to the SpyTag sequence (Long et al., 2013). For the SpyCatcher-GFP sequence (GFP+), SpyCatcher (Long et al., 2013) was fused to the N-terminal end of the eGFP sequence (GenBank: AAG27429.1). For the GalEst, this consisted of a UDP-glucose 4-epimerase sequence (GenBank: EIE36183.1) to which a 6 His and SpyTag sequence was attached at its N-terminal end. For the TMV CP-GGGGS SpyCatcher His (SEQ ID NO: 11), the TMVc* sequence had a 3×GGGGS linker-6 His-SpyCatcher fused to its C-terminal end. For the TMV CP-GGGGS-SpyTag His (SEQ ID NO: 12), a 3×GGGGS linker-SpyTag-6 His, was fused to the C-terminal end of the TMVc*. For the 11-20 SpyCatcher (SEQ ID NO: 13), a small Psoroptes ovis peptidic sequence was fused to the C-terminal of the SpyCatcher. These were codon optimized for bacterial expression and cloned into a pET-21a(+) vector by Genscript (Piscataway, USA). The TMV-VLP (TMVc) control (with no tags) was produced in a similar manner. Plasmids were transformed into BL21(DES)pLysS (ThermoFisher Scientific, Paisley, UK) or Lemo 21 (New England Biolabs, Hitchin, UK) competent cells by following the manufacturers recommendations.
Bacteria containing the plasmids were grown at 37° C. with shaking at 200 rpm in LB media supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Once cultures reached an OD 600 of 0.5, IPTG was added to a final concentration of 0.1 mM, and induction proceeded overnight at 20° C. with shaking at 200 rpm. Bacteria were pelleted by centrifugation at 4000 g for 15 minutes, the supernatant was removed and the cells were lysed by freeze-thawing 3 times using liquid nitrogen and a 37° C. water bath, before proceeding with protein extraction using the B-PER Complete reagent (ThermoFisher Scientific, Paisley, UK). Lysates obtained with the B-PER Complete reagent were centrifuged at 10,000 g for 20 minutes to remove debris. The lysates were combined (TMVc* or TMVc were mixed with GFP+) in equal volume and incubated at room temperature for 1 hour prior to centrifugation on a 20% sucrose cushion, after which the pellet was resuspended in water for western blot analysis. Alternatively, the lysates were kept separate and more rigorously purified for future electron microscope analysis.
For isolation of TMV CP-GGGGS-SpyCatcher His (SEQ ID NO: 11), TMV CP-GGGGS-SpyTag His (SEQ ID NO: 12), and 11-20 SpyCatcher (SEQ ID NO: 13), B-PER complete cell lysates were obtained and further purification was carried out using standard nickel columns according to manufacturers protocols (Qiagen, Manchester, UK). The TMV CP-GGGGS-SpyCatcher His (SEQ ID NO: 11) and TMV CP-GGGGS-SpyTag His (SEQ ID NO: 12) were subsequently analyzed using TEM as described below, or were mixed with 11-20 SpyCatcher (SEQ ID NO: 13) and observed on His antibody probed western blots.
Material for the enzymatic assays (GalEst and GFP+) was isolated from bacterial lysates using generic nickel column and anion exchange purification procedures.
To the centrifuged TMV structure lysates, PEG 8000 and NaCl were added to a final concentration of 2% and 1% respectively, and the solutions were incubated at 4° C. overnight to ensure precipitation. These were centrifuged at 10000 g at 4° C. for 20 minutes to pellet the virus structures. The pellets were resuspended in 25 mM tris-HCl (pH 7.8), centrifuged at 10000 g to clarify and the supernatants were collected. Another two rounds of resuspension and centrifugation was carried out to obtain maximal virus structure yields, while minimizing debris. The clarified supernatants were then loaded onto 2 ml 20% sucrose 25 mM Tris-HCl (pH 7.8) cushions, which was then centrifuged in swing out rotors (SW41 Beckman Coulter; Beckman Coulter, California, USA) at 32000 rpm for 2 hours at 4° C. Pellets were resuspended in 0.01 M Tris-HCl (pH 7.8), ultracentrifuged at 32000 rpm for 2 hours at 4° C. in a SW41 rotor, with the pellet resuspended in water. Virus structures were retained for future electron microscopic analysis.
TMVc*, TMVc, GFP+, TMVc/GFP or TMVc*/GFP, or TMV CP-GGGGS-SpyCatcher His (SEQ ID NO: 11) and TMV CP-GGGGS-SpyTag His (SEQ ID NO: 12), were diluted in sterile filtered distilled water, and 30 μl was placed onto carbon-coated copper grids for 1 minute, wicked off and stained with 2% uranyl acetate, washed twice with 20 μl water, dried and examined in a JEOL 1400 transmission electron microscope (TEM) at 80 kV, or were used for GFP antibody work where appropriate. For detection of GFP+ in the prepared GFP+, TMVc/GFP+ or TMVc*/GFP+ grids, 50 μl of blocking buffer (1% BSA, 0.05% TWEEN, 1×PBS) was applied for 0.5 hours prior to a 1 hour room temperature incubation with a rabbit GFP antibody (Abcam, Cambridge, UK; ab6556) diluted 1/50 in blocking buffer. This was followed by three 50 μl washes in washing buffer (0.25% BSA, 0.05% TWEEN, 1×PBS) before incubation for 1 hour with 50 μl of a goat anti-rabbit 5 nm gold conjugated antibody (ab27235; abcam, Cambridge, UK) diluted 1/50 in blocking buffer. Grids were then washed by rinsing with 50 μl washing buffer 3 times, followed by a single 50 μl wash in water prior to drying and then observation under TEM.
For immunotrapping experiments, mouse GFP antibodies (ab1218; abcam, Cambridge, UK) were diluted 1/1000 in 1×PBS and 30 μl was applied to carbon coated grids and incubated for 1.5 hours at 37° C. These grids were subsequently washed twice in 1×PBS by incubating them each time for 10 minutes on a rotating table. Grids were then floated on GFP+ (in 1×PBS) or purified TMVc or TMVc* for 2 hours at room temperature prior to washing in 1×PBS, staining in 2% uranyl acetate, washed twice in water and viewing under EM.
Grids onto which GFP+ had been immunotrapped (prepared as above but not uranyl acetate treated), were floated on dilutions of TMVc or TMVc* for 1.5 hours in 1×PBS at room temperature. After washing in washing buffer (on a rotating table as before), the grids were floated on TMV rabbit antisera which was diluted 1/200 in blocking buffer and incubated for 1 hour at room temperature. This was followed by washing in washing buffer, and then a 1 hour incubation with a goat anti-rabbit 5 nm gold conjugated antibody diluted 1/50 in blocking buffer. Grids were then washed by rinsing with 50 μl washing buffer 3 times, followed by a single 50 μl wash in water prior to drying and then observation under TEM.
The following reagents were combined per 1 ml reaction: 0.125 M Bicinate pH 8.5, 1.25 mM NAD+, 2 μg UDP-glucose dehydrogenase (UGDH-729H: Creative Biomart, USA), 1-10 μg GalEst or GalEst-GFP+. These were equilibrated for 5 minutes at 27° C. prior to addition of 0.8 mM UDP-galactose substrate. The reaction kinetics were determined over a 10 minute period by measuring the A prior.
Protein samples were mixed 1:4 parts with 4× Laemmli loading buffer and then boiled for 10 minutes to denature the proteins. Twenty microliters of each of these samples were loaded onto 15% SDS-PAGE gels, along with 15 μl of PageRuler Plus prestained ladder (ThermoFisher Scientific, Paisley, UK). Gels were either coomassie stained using established methods or were electroblotted onto Immobilon-P membrane (Millipore, Watford, UK) by following the manufacturer's recommendations. The Immobilon-P membrane was blocked by incubating in 1×PBS, 1% BSA and 0.05% TWEEN, for 1 hour. Anti-TMV or anti-GFP (abcam, Cambridge, UK; ab6556) antibodies raised in rabbits, or anti-his antibodies raised in mouse (H1029—0.2 ml; Sigma Dorset) were added to the membranes in blocking solution at either 1/10000 or 1/5000 dilutions and incubated with shaking at room temperature for 1 h. After washing, an anti-rabbit IgG alkaline phosphatase conjugate (A8025—1 ML; Sigma, Dorset, UK) or anti-mouse IgG alkaline phosphatase conjugate (A9316—0.25 ml) was added to the blots at a concentration of 1/1000 in blocking buffer. After incubation at room temperature under shaking conditions the blots were washed and covered with BCIP/NBT (B1911; Sigma, Dorset, UK). The blots were left to develop for 10 min, after which banding was visible. The developed blots were scanned and saved as jpeg images.
Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.
MHHHHHHAHIVMVDAYKPTK
MRVLVTGGSGYIGSHTCVQLLQNGHDVIILDNLCNSKRSVLPVIERLGGKH
PTFVEGDIRNEALMTEILHDHAIDTVIHFAGLKAVGESVQKPLEYYDNNVN
GTLRLISAMRAANVKNFIFSSSATVYGDQPKIPYVESFPTGTPQSPYGKSK
LMVEQILTDLQKAQPDWSIALLRYFNPVGAHPSGDMGEDPQGIPNNLMPYI
AQVAVGRRDSLAIFGNDYPTEDGTGVRDYIHVMDLADGHVVAMEKLANKPG
VHIYNLGAGVGNSVLDVVNAFSKACGKPVNYHFAPRREGDLPAYWADASKA
DRELNWRVTRTLDEMAQDTWHWQSRHPQGYPD
PILPTTKAKIIAKIIG
| Number | Date | Country | Kind |
|---|---|---|---|
| 1708675 | May 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/051495 | 5/31/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/220386 | 12/6/2018 | WO | A |
| Number | Date | Country |
|---|---|---|
| WO 2016112921 | Jul 2016 | WO |
| WO 2017070742 | May 2017 | WO |
| Entry |
|---|
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| Brune et al., “Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization,” Scientific Reports, 6:19234, (2016). |
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| WIPO Application No. PCT/GB2018/051495, PCT International Search Report and Written Opinion of the International Searching Authority dated Sep. 28, 2018. |
| Number | Date | Country | |
|---|---|---|---|
| 20200190148 A1 | Jun 2020 | US |
