JP2013501771A - Complex formation system - Google Patents

Abstract

  The present invention relates to two polypeptide helices derived from SNAP protein; one polypeptide helix derived from syntaxin; one polypeptide helix derived from synaptobrevin or a homologue thereof; and And a complex-forming system in which four polypeptide helices form a stable SNARE complex. The invention further relates to a method of creating a complex forming system and the use of the complex forming system.

Description

FIELD OF THE INVENTION The present invention relates to a complex formation system for forming a molecular scaffold based on a SNARE complex.

Background of the Invention In eukaryotic cells, vesicles fuse with membranes in a process known as vesicle fusion. For example, vesicles can be fused with cell plasma membranes or other cell compartments such as endosomes or lysosomes. The most studied form of vesicle fusion is the docking of neuronal presynaptic membranes and synaptic vesicles to release neurotransmitters to cause propagation of nerve impulses in postsynaptic neurons.

  The process of vesicle fusion is mediated by SNARE proteins (SNAREs), which are a large protein superfamily of over 60 members in yeast and mammalian cells. SNAREs are small but abundant and most are membrane-bound proteins. Although they vary greatly in structure and size, they all share a segment within their cytosolic domain called the SNARE motif that allows assembly into a dense four-helix bundle called the SNARE complex. SNARE motifs are thought to be about 60-70 amino acids in length (Jahn R and Scheller RH), but this is not well defined. SNARE complexes are divided into three proteins: syntaxin and SNAP-25 (which are resident in the cell membrane); and synaptobrevin (also called vesicle-associated membrane protein or VAMP) (which is anchored to the vesicle membrane) ).

  In neuronal exocytosis, syntaxin and synaptobrevin are anchored to their respective membranes by their C-terminal domains, whereas SNAP-25 is intact via palmitoyl chains linked by several cysteines. Tethered to the plasma membrane. The core SNARE complex is four α-helix bundles, where one α-helix is brought by syntaxin, one α-helix is brought by synaptobrevin, and two α-helices are SNAP Brought about by -25. This SNARE complex has been found to be very stable. A schematic representation of the molecular mechanism involved in vesicle fusion is shown in FIG.

The inventors have found that SNARE complexes and their formation can be used in a variety of applications. To that end, the basis of the present invention is:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein the four polypeptide helices form a stable SNARE complex.

  In various embodiments of the invention, further limitations can be added to the previous complex formation system. The present invention further relates to a method of making a stable SNARE complex using the previous complex formation system, and the use of the previous complex formation system and the resulting stable SNARE complex.

DETAILED DESCRIPTION OF THE INVENTION The following description presents many different embodiments of the present invention. However, it will be appreciated by those skilled in the art that almost all of the described limitations, preferred features and variations are suitable for other embodiments of the present invention, so long as they are all based on the complexation system described above. Will soon be understood.

In one aspect, the present invention provides a complex forming system for forming a molecular scaffold, the system comprising:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein four polypeptide helices form a stable SNARE complex; and wherein at least two of the polypeptide helices are less than 50 amino acids in length.

In other embodiments of the invention, the complex-forming system does not necessarily have only two polypeptide helices that are less than 50 amino acids in length.
The present invention allows for a controlled assembly of stable complexes that form different functional units. The advantage of having a stable complex is that it can be used in relatively severe conditions without the risk of the complex dissociating. This is because the helix (s) to which one or more cargo moieties are bound remains part of the complex, ensuring that one or more cargo moieties are dissociated from the remaining part of the complex. It means not to.

  As indicated above, the complex formation system is based on the formation of a stable SNARE complex. Therefore, the four polypeptide helices of the present invention can have some arbitrary sequence as long as they form a stable SNARE complex.

  In neurons, the SNARE complex is formed from the following proteins: SNAP-25; syntaxin; and synaptobrevin. These proteins, and other SNARE proteins, contain a SNARE motif or SNARE domain that is part of the protein involved in forming the SNARE complex. These SNARE domains or motifs are helices that are packed together to form a SNARE complex. In general, only a portion of the SNARE protein is involved in SNARE complex formation; not the entire SNARE protein. For example, syntaxin has a C-terminal transmembrane domain, also known as the head domain, a SNARE domain, and an N-terminal regulatory domain. Apparently, only the SNARE domain is involved in forming the SNARE complex.

  Terms such as “SNARE motif” and “SNARE domain” are well known to those skilled in the art. Furthermore, SNARE motifs and SNARE domains of a variety of different SNARE proteins are also well known to those skilled in the art (Jahn R and Scheller RH (2006); Sieber et al. (2006); Besteiro (2006)).

  The four polypeptide helices of the present invention are based on SNARE domains or motifs of SNARE proteins forming a SNARE complex, ie, SNAP protein; syntaxin; and synaptobrevin or homologues thereof. Thus, in one embodiment, the complexing system comprises: two polypeptide helices derived from the SNARE motif of the SNAP protein; one polypeptide helix derived from the SNARE motif of the syntaxin; and SNARE of synaptobrevin or a homologue thereof. Comprising one polypeptide helix derived from the motif; where the four polypeptide helices form a stable SNARE complex. The helix of the present invention need not be the same length as the SNARE motif or domain from the SNARE protein. When complexed with other helices of the invention, it may be shorter in length as long as it is still capable of forming a stable SNARE complex.

  The SNAP protein from which the two polypeptide helices are derived can be any SNAP protein that can form part of a SNARE complex. One skilled in the art knows various SNAP proteins that can form part of the SNARE complex. For example, the SNAP protein can be SNAP-25A, SNAP-25B, SNAP-23 (also known as syndet), or SNAP-29. Preferably, the SNAP protein is not α-SNAP. Preferably, the SNAP protein is SNAP-25, ie SNAP-25A or SNAP-25B. The SNAP protein contains two SNARE motifs. Thus, two polypeptide helices may be derived from two SNARE motifs within a particular SNAP protein. Alternatively, the two polypeptide helices may be derived from different SNAP proteins. Preferably they are derived from the same SNAP protein.

  The syntaxin protein from which the polypeptide helix is derived can be any syntaxin protein that can form part of a SNARE complex. Those skilled in the art are aware of various syntaxin proteins that can form part of the SNARE complex. For example, syntaxin proteins include syntaxin 1A, syntaxin 1B, syntaxin 2 (also known as epimorphin), syntaxin 3 and syntaxin 4, syntaxin 5, syntaxin 6, syntaxin 7, syntaxin 8, syntaxin 10, It can be selected from syntaxin 11, syntaxin 13, syntaxin 17 or syntaxin 18. Preferably, the syntaxin protein is syntaxin 1A or 3.

  The synaptobrevin protein or homologue thereof from which the polypeptide helix is derived can be any synaptobrevin protein or homologue thereof that can form part of the SNARE complex. Those skilled in the art are aware of various synaptobrevin proteins and their homologues that can form part of the SNARE complex. Synaptobrevin is a member of the vesicle-associated membrane protein (VAMP) family. Since it is known that other VAMP proteins can form SNARE complexes, it may therefore be suitable to provide a basis from which polypeptide helices can be obtained. In one embodiment, the homologue of synaptobrevin is a VAMP protein that can form part of a SNARE complex. Such VAMP proteins are well known to those skilled in the art. The synaptobrevin protein or homologue thereof can be selected from synaptobrevin 1, synaptobrevin 2, synaptobrevin 3 (also known as selbrevin) and synaptobrevin 7 (also known as TI-VAMP). Preferably, the polypeptide helix is derived from a synaptobrevin protein. Preferably, the synaptobrevin protein is synaptobrevin 1, 2 or 3.

  The organism from which the SNARE protein is derived can be any suitable organism in which the SNARE complex is utilized. For example, the protein may originate from: mammals such as humans, primates, rodents; fish; and invertebrates such as flies. Optionally, the SNARE protein may be derived from yeast (Rossi G et al. (1997)). The organism from which the SNARE protein originates may depend on the application of the complex forming system. For example, for medical applications, the SNARE protein is preferably of human origin.

  The four polypeptide helices of the present invention are derived from SNARE proteins that form a SNARE complex. The SNARE motif or domain helix of the SNARE protein forming the SNARE complex is typically about 60-70 amino acids in length. As indicated above, the four polypeptide helices of the present invention are derived from the SNARE motif or SNARE domain of the SNARE protein. The term “derived from” means that the sequence of the polypeptide helix is substantially the same as the sequence of the SNARE domain / motif or part thereof such that it can form a stable SNARE complex. To do. Preferably, the sequence of the polypeptide helix should have at least about 80% sequence identity with the sequence of the selected SNARE domain / motif or part thereof. More preferably, the sequence identity should be at least about 85%, and even more preferably at least about 90%. In one embodiment, the sequence identity can be at least about 95%, at least about 98%, or even 100%. However, in some embodiments it may be desirable for the sequence of the polypeptide helix to differ from the sequence of the selected SNARE domain / motif or part thereof. This can be beneficial for protein expression, protein purification or downstream applications. For example, and without limitation, this may include the addition of a histidine residue at either end of the sequence to allow purification, or an additional lysine for functional binding of the peptide to the surface or cargo. Or the incorporation of cysteine residues may be included.

The two SNAP derived helices may comprise a sequence selected from SEQ ID NOs: 1, 2, 5, 6, 24, 41, 48, 49, 50, 51, 64 and 65.
The syntaxin derived helix may comprise a sequence selected from SEQ ID NOs: 3, 7, 9, 25, 32, 33, 34, 35, 36, 37, 38, 46, 47, 60, 61, 62 and 66.
The helix derived from synaptobrevin or a homologue thereof has a sequence selected from SEQ ID NOs: 4, 8, 27, 28, 29, 30, 31, 42, 43, 44, 45, 52, 53, 67, 68 and 69. May be included.
Furthermore, the helix derived from the SNARE protein can consist of the previous sequence.
In addition, the helix of the present invention may comprise or consist of a sequence selected from SEQ ID NOs: 1-9, 12-15 and 18-69.

  Each polypeptide helix must be long enough so that it interacts with other helices to form a stable SNARE complex. In practice, this means that the polypeptide helix must be derived from a SNARE motif / domain part that is long enough to allow the formation of a stable SNARE complex. Testing whether a polypeptide helix is long enough to allow the formation of a stable SNARE complex is relatively simple and well within the ability of one skilled in the art. This can be done by complexing the polypeptide helix with the other three polypeptide helices and testing to see if the resulting SNARE complex dissociates under adverse conditions. Bad conditions are generally conditions that cause dissociation of protein complexes or protein-protein interactions. Such conditions will be readily apparent to those skilled in the art. For example, such an adverse condition can be exposing the complex to a strong surfactant or a disrupting detergent. In one embodiment, the SNARE complex is tested to determine whether it is stable by using SDS-PAGE performed in the presence of denatured SDS concentration (> 0.02%). Can. If the complex is dissociated by SDS in the gel so that it breaks into its components, this SNARE complex may not be stable. However, if the SNARE complex is not dissociated and remains as a single entity when it can be moved through an SDS-PAGE gel and detected, eg, using Coomassie staining, it is stable. Can be considered. Thus, in one embodiment, a SNARE complex can be considered stable if it does not dissociate using SDS. Preferably, the SNARE complex is stable even when using a gel sample buffer for SDS-PAGE containing about 2% SDS. More preferably, the SNARE complex is stable even when using a SDS-PAGE gel containing about 0.1% SDS. This can be done using a gel for SDS-PAGE containing about 0.1% SDS.

Alternatively, the stability of the SNARE complex can be confirmed by a bead pull-down method. A SNARE complex is considered stable if one component of the complexing system precipitates all interaction partners in a stoichiometric manner. The bead pull-down method is well known to those skilled in the art (Rickman C. et al. (2004)).
Surface plasmon resonance measurements of protein-protein interactions are also used and well known to those skilled in the art (Karlsson & Falt (1997)). When using the surface plasmon resonance method, the SNARE complex is considered stable if one skilled in the art does not observe the dissociation evidenced by a decrease in the surface plasmon resonance signal.

Another way to determine whether the SNARE complex is stable is to measure the dissociation constant of the complex. This is the dissociation constant for the dissociation of one helix from the complex. A stable SNARE complex should have a dissociation constant of less than 10 ⁻⁷ M. In certain embodiments, a stable SNARE complex has a dissociation constant of less than 10 ⁻⁸ , 10 ⁻⁹ , 10 ⁻¹⁰ , 10 ⁻¹¹ , 10 ⁻¹² , 10 ⁻¹³ , 10 ⁻¹⁴ , or 10 ⁻¹⁵ M. Should have. Depending on the application in which the complexing system is used, it may be beneficial to have different dissociation constants. Thus, in some embodiments, the dissociation constant can be 10 ⁻⁷ M to 10 ⁻¹¹ M or 10 ⁻⁷ M to ^{10 −10} M. Alternatively, the dissociation constant may be comparable to the antibody dissociation constant (6-10 × 10 ⁻⁸ M).
SNARE complex formation can also be assessed by binding to a complexin that binds to the fully formed SNARE complex (Hu et al. (2002)).

  In one embodiment, the polypeptide helix of the present invention is at least about 25 amino acids in length. SNARE complexes using such a helix can be formed in solution. However, they may not be SDS resistant. Preferably, the polypeptide helix of the present invention is at least about 30 or about 35 amino acids in length. More preferably, the polypeptide helix of the present invention is at least about 40 amino acids in length. SNARE complexes formed using such helices are in most cases SDS-resistant. Alternatively, the polypeptide helix can be at least about 45 amino acids in length.

  In one embodiment, at least one of the polypeptide helices is less than 50 amino acids in length. The advantage is that it is much easier to artificially produce polypeptides that are less than 50 amino acids in length. A polypeptide helix that is less than 50 amino acids can be any polypeptide helix. Preferably, at least two of the polypeptide helices are less than 50 amino acids in length. Preferably they are polypeptide helices derived from syntaxin and synaptobrevin or homologues thereof.

  In alternative embodiments, 3 or 4 of the polypeptide helices can be less than 50 amino acids in length. In such embodiments, any combination of helices can be less than 50 amino acids in length. The inventors have surprisingly found that the minimal peptide sequence of the SNARE motif of each SNARE protein is sufficient to form a high affinity stable four helix bundle SNARE complex. This is advantageous in terms of ease of peptide production. In addition, each minimal array can carry cargo so it retains functionality.

  If the two polypeptide helices are less than 50 amino acids in length, preferably it is a helix derived from syntaxin and a helix derived from synaptobrevin or a homolog thereof that is less than 50 amino acids in length. .

  The four polypeptide helices of the present invention may be approximately the same length or different lengths. For example, in one embodiment, they can all be about 45 amino acids long. Alternatively, the polypeptide helices can be of different lengths.

  In one embodiment of the invention, the polypeptide derived from syntaxin and / or the polypeptide derived from synaptobrevin or a homologue thereof is a membrane bound sequence that allows immobilization of the SNARE complex to the membrane or lipid bilayer. May further be included. Such membrane binding sequences are well known to those skilled in the art. For example, the native sequences of syntaxin and synaptobrevin contain transmembrane moieties that allow proteins to bind to vesicles and cell membranes (Kasai and Akagawa (2001) and Laage et al. (2000)). Thus, in one embodiment, the polypeptide helix derived from syntaxin may further comprise a membrane binding sequence derived from a syntaxin transmembrane domain for immobilizing the polypeptide helix to the membrane or lipid bilayer. Similarly, a polypeptide helix derived from synaptobrevin or a homologue thereof further comprises a membrane binding sequence derived from the transmembrane domain of synaptobrevin or a homologue thereof for immobilizing the polypeptide helix to the membrane or lipid bilayer. obtain.

  A stable SNARE complex is one that usually does not separate under adverse conditions that cause dissociation of protein complexes and protein-protein interactions. This can be measured using, for example, SDS-PAGE, bead pull-down method, surface plasmon resonance method of immobilized protein, dissociation constant or complexin binding. This is discussed in more detail earlier.

  Since SNARE complexes are well studied and well known to those of skill in the art, one skilled in the art can determine whether a particular protein is suitable for use in the present invention and form a stable SNARE complex of the present invention. It would be possible to establish how to manipulate these proteins and their sequences to create polypeptide helices.

  The one or more cargo moieties can be any component that one of skill in the art may wish to attach to the polypeptide helix. One or more cargo moieties are preferably attached to the ends of the helix in the complex forming system. The cargo moiety can be selected from small molecules, small molecules, polypeptides, proteins, polymers including nucleic acids or derivatives, and particles or nanoparticles. For example, the cargo portion can be as follows:

1. Small molecules or polymers containing small molecules, such as:
An affinity tag, such as biotin;
-Therapeutic agents, such as toxins or drugs;
-Reactive groups for further / downstream crosslinking, polymerization and further derivatization, such as amino groups, carboxyl groups, sulfhydryl groups, guanidine groups, phenol groups, thioether groups, imidazole groups, indole groups, etc .;
-Spontaneously reactive groups suitable for further modified forms, for example maleimides or derivatives for crosslinking SH groups, or other chemicals suitable for crosslinking;
Molecules for binding directly to the surface, eg SH containing molecules for binding to metal surfaces;

A contrasting reagent, such as a fluorescent or absorbing component for UV, VIS, IR, Raman, NMR, MRI, PET, X-ray or other imaging methods;
-Biologically relevant ligands, such as for receptor binding / targeting;
-Biologically relevant substrates such as phosphorylation or other PTM sites;
-Biologically relevant molecules, such as lipids or carbohydrates;
A protecting group or molecule, such as PEG;
A metal chelating compound;

2. A polypeptide or protein, such as:
-Binding peptides, hormones, toxins, etc .;
A polypeptide or protein comprising a functional site, for example a protease digestion site;
-Targeting functional peptides, for example targeting different organelles, nuclear targeting (for transfection), intracellular targeting (for drug delivery), etc .;
-Peptide affinity tags, such as Flag, Myc, VSV, HA, 6xHis, 8xhis, poly-His, etc .;

A polypeptide or protein capable of forming a protein-protein interaction, eg PDZ, SH2 / 3;
An antibody, antibody fragment, antibody mimetic, RNA or peptide-based aptamer, or another affinity reagent (proteinaceous or non-proteinaceous);
-Enzymes, such as for research, diagnosis (for some applications complexation systems can be used to immobilize enzymes) and therapeutic applications, promoters, polymerases, restriction endonucleases, or others For nucleic acid synthesis or amplification including the modified enzymes of

3. Nucleic acids or derivatives such as:
DNA, RNA, or PNA for detection, immobilization, hybridization, synthetic primers, synthesis and amplification, labeling, signal detection and signal amplification, transcription and translation; and

4). Particles or nanoparticles, such as:
-Ferromagnetic particles or nanoparticles (for separation);
-Dendrimers (for labeling);
Metal particles or nanoparticles, such as gold or silver for staining or labeling;
-Semiconductor particles or nanoparticles, eg quantum dots for labeling and detection;
-Polymer micro or nano particles, such as resins, gels, etc .;
-Carbon nanotubes or nanowires.

  In one embodiment, multiple cargo moieties can be attached to the polypeptide helix terminus. The number of cargo moieties that can be attached to the polypeptide helix ends will depend on how many free ends are present in the helix. For example, if the complex-forming system contains 4 separate polypeptide helices, the helix will have 8 free ends (one at each end of each helix). Therefore, it is possible to bind a cargo moiety to each of the eight free ends. This means that 1, 2, 3, 4, 5, 6, 7 or 8 cargo moieties can be attached to the free end of the helix. If some of the helices do not have free ends, the number of free ends is small if, for example, two of the helices are one or if one helix is attached to the substrate. Become. This will reduce the number of cargo moieties that can be attached to the helix ends. In one embodiment, more than one cargo moiety can be attached to the helix ends.

  In certain embodiments, the first cargo portion is attached to the first helix end and the second cargo portion is attached to the second helix end. In certain such embodiments, the first and second cargo moieties should not be bound together to the same helix or helix-containing component. They should be bound to a separate helix or helix-containing component. For example, two cargo portions can be combined into two single independent helices.

  When a polypeptide helix derived from syntaxin is linked to a polypeptide helix derived from synaptobrevin or a homologue thereof (described in more detail later), both the first and second cargo moieties are combined in the duplex. Should not be bound to a helical component. Alternatively, the first cargo moiety can be bound to a synaptobrevin / syntaxin fusion protein and the second cargo moiety can be bound to one of the helices derived from the SNAP protein.

  When the four polypeptide helices of the complex-forming system are integrated to form two helix-containing components (discussed in detail below), the first cargo portion becomes the first helix-containing component. It should be attached and the second cargo moiety should be attached to the second helix-containing component.

  In one embodiment, the first cargo portion is an enzymatic or contrast component. In other embodiments, the second cargo moiety is a ligand for targeting the complexing system to a specific target, such as a specific cell type or a specific receptor. Preferably, in the same embodiment, the first cargo moiety is an enzymatic or imaging moiety and the second cargo moiety is complexed to a specific target, such as a specific cell type or a specific receptor. A ligand for targeting the forming system.

  The enzymatic agent or contrast agent can be any suitable agent. The contrast agent is any agent that can bind to the helix and allow the position of the helix to be imaged, such as GFP fluorescent tags, fluorescently labeled peptides, and MRI contrast agents. The enzymatic agent can be either an enzyme or a functional part thereof. In one embodiment, the enzymatic agent or contrast agent is an enzymatic agent. In a specific embodiment, the enzymatic agent comprises a botulinum toxin light chain or a functional part thereof. The function of the light chain of botulinum toxin is as a function of endopeptidase. Therefore, the functional part of the light chain of botulinum toxin is a part that retains endopeptidase activity. Preferably, the enzymatic agent comprises a botulinum toxin light chain. The light chain of the botulinum toxin can be from any botulinum toxin. There are seven different types of botulinum toxin, types A, B, C, D, E, F and G. Preferably, the light chain is from botulinum toxin type A or E, and more preferably, the light chain is from botulinum toxin type A.

  Preferably, the enzymatic agent comprises a botulinum toxin light chain or functional portion thereof and a botulinum toxin heavy chain translocation portion. The translocation of the heavy chain of botulinum toxin causes the associated light chain to be released from the vesicle into the cytosol of the cell. Those skilled in the art readily understand what the term “botulinum toxin heavy chain translocation” means, and it is also sometimes referred to as the translocation domain. The translocation of the botulinum toxin heavy chain can be from any botulinum toxin. Preferably, the translocation is from botulinum toxin A or E, and more preferably from botulinum toxin A. The light chain or its functional part and translocation can be the same or from different botulinum toxins. Preferably, the light chain or its functional part and translocation part are from the same botulinum toxin. The light chain or its functional part and translocation part may be linked by any suitable method. Preferably, they are linked via disulfide bond formation, like the naturally occurring botulinum toxin. If the light chain of botulinum toxin or a functional part thereof and the translocation part of the heavy chain of botulinum toxin are linked by a peptide bond between amino acid chains, preferably the light chain of botulinum toxin or a functional part thereof and the botulinum toxin There is a nicking site recognized by the protease to cause cleavage of the amino acid sequence between the two parts in the amino acid sequence between the translocations of the heavy chain. In one embodiment, the nicking site is a thrombin site that can be cleaved by thrombin. The light chain of botulinum toxin or a functional part thereof and the translocation part of the heavy chain of botulinum toxin can be bound to one of the polypeptide helices derived from the SNAP protein.

The botulinum toxin light chain sequence may comprise the sequence set forth in SEQ ID NO: 70.
The sequence of the botulinum toxin heavy chain translocation may comprise the sequence shown in SEQ ID NO: 71.

  When the cargo moiety is a ligand, it is any suitable ligand for targeting the complexing system to a specific target, such as a specific cell type or a specific receptor. Such ligands are well known to those skilled in the art. For example, a ligand may allow binding to a cell surface receptor. Such ligands include neuropeptides such as substance P, neuropeptide Y and VIP, growth factors such as NGF and BDNF, and hormones such as pituitary hormones such as ACTH, TSH, PRL, GH, endorphins, FSH, LH. , Oxytocin, ADH and AVP), GNRH and CGRP and the like. In certain embodiments, the ligand is a somatostatin peptide or a functional part thereof that allows the somatostatin peptide to bind to a somatostatin receptor. In other embodiments, the ligand can be a substance P peptide, or a functional part thereof that allows the substance P peptide to bind to a neurokinin receptor, or an AVP peptide, or an AVP peptide. Can be its functional part that allows it to bind to the AVP receptor. In an alternative embodiment, the ligand may be the receptor binding portion of the heavy chain of botulinum toxin. The receptor binding portion of the heavy chain of botulinum toxin is involved in neuronal ganglioside recognition and binds to the synaptic vesicle receptor SV2C and allows the toxin to be taken up into the cell. The term “receptor binding portion of the heavy chain of botulinum toxin” will be readily understood by those skilled in the art and is also referred to as the receptor binding domain. The receptor binding portion can be from any botulinum toxin. Preferably, the receptor binding portion is from botulinum toxin A or E type, and more preferably, the receptor binding portion is from botulinum toxin type A. In one embodiment, the receptor binding portion of the heavy chain of botulinum toxin is bound to a polypeptide helix derived from synaptobrevin or a homologue thereof.

The sequence of the receptor binding portion of the heavy chain of botulinum toxin can comprise the sequence shown in SEQ ID NO: 72.
The present invention further provides the above conjugates for use in therapy and / or diagnosis. The exact nature of the therapy and / or diagnosis will depend on the identity of the ligand and the enzymatic agent / contrast agent.

  In one embodiment where the first cargo portion is attached to the end of the first helix and the second cargo portion is attached to the second helix end, the first cargo portion is the light chain of the botulinum toxin. Or an enzymatic agent comprising a functional part thereof and a translocation of the heavy chain of botulinum toxin, and the second cargo moiety is a ligand that is a receptor binding part of the heavy chain of botulinum toxin. In an alternative embodiment, the first cargo moiety is an enzymatic agent comprising a botulinum toxin light chain or functional portion thereof and a translocation of the botulinum toxin heavy chain, and the second cargo moiety is A somatostatin peptide or a ligand that is a functional part thereof.

  One or more cargo moieties may be directly linked to the helix ends or may be linked via a linker. Suitable linkers are well known to those skilled in the art. For example, this can be done chemically or by genetic recombination if the cargo moiety is a protein or polypeptide. The cargo moiety can also be attached via a linker. Such linkers are well known to those skilled in the art.

  The four polypeptide helices can be four separate helices that are not integrated in any way until they form a SNARE complex. In one embodiment, two of the helices can be integrated so that the complexing system comprises three separate components (hereinafter also referred to as a ternary system). The two helices can be integrated in any suitable manner as long as the two helices can assemble into the same stable SNARE complex. The two helices can be integrated by genetic recombination means or can be chemically coupled. The two helices can be integrated via a linker. The term “linkage” refers to a linear combination of helices (as opposed to a helix combination that occurs in a parallel direction similar to “zippering”). The integration of the two helices helps to simplify the complex formation system. Any two helices can be integrated. In one embodiment, two polypeptide helices derived from a SNAP protein are integrated. The advantage is that a full-length SNAP protein comprising two polypeptides SNARE helices within the protein can be used, for example the full-length SNAP-25 protein. Alternatively, a polypeptide helix derived from syntaxin can be linked to a polypeptide helix derived from synaptobrevin or a homologue thereof.

  In other embodiments, the two helices are integrated, while the other two helices are also integrated. This creates a complex-forming system (hereinafter also referred to as a two-component system) comprising two separate components and further simplifies the system. The two sets of helices can be integrated in any suitable manner as long as they continue to form a stable SNARE complex. For example, the two sets of helices can be integrated by genetic recombination means or can be chemically coupled. One or both helices may be integrated via a linker. Any combination of helices can be integrated to form two sets of two helices. Preferably, two polypeptide helices derived from a SNAP protein are integrated, and a helix derived from syntaxin and synaptobrevin or a homolog thereof is integrated. This makes it possible to use full length SNAP proteins such as SNAP-25.

  In an alternative two-component system, three of the helices can be integrated. The three helices can be integrated in any suitable manner as long as they continue to form a stable SNARE complex with the fourth helix. For example, the three helices can be integrated by genetic recombination means or can be chemically coupled. Three helices can be integrated with the linker between two helices or all three helices. Any three helices can be integrated. Preferably, two polypeptide helices derived from a SNAP protein are integrated with a third helix, a helix derived from syntaxin or a helix derived from synaptobrevin or a homologue thereof. In certain embodiments, two polypeptide helices derived from a SNAP protein are integrated with a polypeptide helix derived from synaptobrevin or a homologue thereof. In an alternative embodiment, two polypeptide helices derived from a SNAP protein are integrated with a polypeptide helix derived from syntaxin. In one embodiment, the cargo moiety is linked to a fourth single helix, for example when two polypeptide helices derived from a SNAP protein are integrated with a polypeptide helix derived from synaptobrevin or a homologue thereof. For binding to syntaxin.

  Those skilled in the art will readily recognize that these two-component systems are two-component reagents that can be used for affinity applications similar to antibody-antigen interactions. For example, a syntaxin-derived tag is bound to a protein of interest and is composed of SNAP-25 and a synaptobrevin helix to the same extent that a FLAG epitope can be bound to the protein of interest and recognized by its cognate antibody. Can be recognized by triple helix constructs. The affinity of the interaction between the triple helix SNARE construct and a single SNARE tag can be to the extent that the protein can be immobilized or can be reversible for other applications. This is advantageous because triple helix constructs can be constructed cheaper than antibodies.

  Although the head domain of syntaxin 3 is known to protect its SNARE motif from SNARE assembly, certain surfactants and / or lipids can “open” syntaxin for SNARE assembly ( Darios and Davletov (2006); Rickman and Davletov (2005)). Thus, in one embodiment where the syntaxin-derived helix has a syntaxin head domain attached to it, the system allows the formation of a surfactant, preferably a weak surfactant, such as a stable SNARE complex. Octyl glucopyranoside and the like that further open the syntaxin molecule. This makes it possible to control the syntaxin SNARE motif and thus to regulate the formation of the SNARE complex.

  Furthermore, the system may further comprise a surfactant regardless of whether the syntaxin 3 head domain is bound to a polypeptide derived from syntaxin. The inventors have found that the presence of a surfactant helps promote the assembly of the SNARE complex of the present invention. Preferably, the surfactant is a weak surfactant. Some assembly occurs even in the absence of surfactant, but the presence of surfactant promotes more effective assembly of SNARE complexes. Preferably, the surfactant is present at a concentration above the critical micelle concentration (CMC), which is the concentration at which the surfactant begins to form micelles. Preferably, the surfactant has a carbon chain having a length of 7 to 12 carbon atoms. Preferably, the surfactant is neither Triton X-100 nor Thesit. Suitable surfactants can be selected from the group consisting of MEGA8, C-HEGA10, C-HEGA11, HEGA9, heptylglucopyranoside, octylglucopyranoside, nonylglucopyranoside, zwittergent 3-08, zwittergent 3-10, and zwittergent 3-12. Preferably, the surfactant is octyl glucopyranoside. An advantage of systems comprising surfactants, and particularly weak surfactants, is that they allow a controlled and faster assembly of SNARE complexes. Since other protein interactions are disturbed by the surfactant, the presence of the surfactant further preferentially facilitated the formation of the SNARE complex.

  In one embodiment, one of the helices can be immobilized on a substrate. Preferably, the helix is immobilized on the substrate. The helix can be immobilized by any suitable method, and such methods are well known to those skilled in the art. The helix can also be immobilized via a linker. The substrate can be any substrate suitable for immobilizing the helix. For example, the substrate can be a surface, matrix, bead, quantum dot, resin, glass, metal, polymer, microscope slide, array or nanotube, such as a carbon nanotube. In one embodiment, the cargo moiety is not bound to the helix immobilized on the substrate.

  In other embodiments, the complexing system can further comprise a single polypeptide helix derived from complexin. The polypeptide helix resulting from the polypeptide helix can be any complexin protein capable of binding to the SNARE complex. Those skilled in the art are aware of various complexin proteins that can bind to the SNARE complex. For example, the complexin protein can be selected from mammalian complexin 1 or complexin 2 isoforms. Other desirable characteristics and properties (eg, size) of the helix derived from the complexin protein are the same as for other helices. A helix derived from complexin can optionally carry one or two cargo moieties. Also, since the complexin moiety only binds to the formed SNARE complex, it can also be used to specifically purify the assembled complex.

  The helix derived from complexin can also include the sequence shown in SEQ ID NO: 63. A helix derived from complexin can also consist of this sequence.

An alternative embodiment of the invention is directed to a SNARE complex in which a polypeptide helix derived from syntaxin is linked to a polypeptide helix derived from synaptobrevin or a homologue thereof. In this embodiment, a complex forming system for forming a molecular scaffold is provided, which system is:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein four polypeptide helices can form a stable SNARE complex, and wherein the polypeptide helix derived from syntaxin is derived from synaptobrevin or a homologue thereof. Connected to

  This embodiment of the invention is directed to the ternary system described above, wherein a polypeptide helix derived from syntaxin is linked to a polypeptide helix derived from synaptobrevin or a homologue thereof. A polypeptide helix derived from syntaxin linked to a polypeptide helix derived from synaptobrevin or a homologue thereof, almost all of the preceding description of limitations and desirable characteristics of complex-forming systems comprising helices of length less than 50 amino acids Those skilled in the art will readily recognize that it is equally suitable for previous systems containing. For example, also two helices derived from the SNAP protein of this system can also be integrated as described above to produce a binary system. One skilled in the art will appreciate that two of the helices need not be less than 50 amino acids in length.

In another embodiment of the invention directed to the two-component system described above, a complex-forming system for forming a molecular scaffold is provided, which system is:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Where the four polypeptide helices form a stable SNARE complex, and where the polypeptide helices are integrated to form two helix-containing components.

As described above, a two-component system can be formed in two ways. First, two of the helices can be integrated, and the other two helices can be integrated to form two components each comprising two helices Can do. Alternatively, three of the helices can be integrated to obtain a component that includes three helices that can form a SNARE complex with one remaining helix.
Almost all of the above limitations for complex forming systems that include a helix that is less than 50 amino acids in length are equally appropriate for previous systems that include two components. It will be clear to those skilled in the art which parts are suitable for a two-component system. For example, a binary system need not have two helices that are less than 50 amino acids in length.

In other embodiments, the present invention provides a complex forming system for forming a binary compound comprising two cargo moieties, wherein the complex forming system is:
Two polypeptide helices derived from the SNAP protein;
A polypeptide helix derived from syntaxin; and a polypeptide helix derived from synaptobrevin or a homologue thereof,
Wherein the four polypeptide helices form a stable SNARE complex, wherein the first cargo moiety is bound to the first helix and the second cargo moiety is the second Bind to two separate helices, where the formation of the SNARE complex causes the formation of a binary compound.

  The term “binary compound” means a compound made from two separate components (cargo moieties) such that when the two parts are brought together, the compound can perform its function. The function of the compound will depend on the function or identity of the two separate parts of the compound. Preferably, the first cargo portion has a first function and the second cargo portion has a second function. As previously mentioned, the first cargo moiety can have an enzymatic or imaging function and the second cargo moiety can have a targeting function. This allows the compound to be targeted at a specific location where the enzymatic or contrast component can perform its function.

  In one embodiment, the binary compound can be a polypeptide formed from different units. The polypeptide can be a toxin formed from different units. Suitable toxins are botulinum toxin, diphtheria toxin, tetanus toxin and ricin. Those skilled in the art will readily know which peptides and toxins are suitable for use in the system of the present invention. For example, botulinum toxin is made up of three different parts: a receptor binding part; a translocation part; and an enzyme part. Therefore, when they are grouped together by a complex forming system, one part (cargo part) can be formed in the enzyme part and translocation part to form a functional botulinum toxin, and the receptor binding part The second part (cargo part) can be formed. Similarly, tetanus, ricin and diphtheria toxin can be separated into two parts so that when they are brought together, a functional toxin is formed.

  In one embodiment, the first cargo portion has a first function and the second cargo portion has a second function, and when it is combined, a fully functional peptide For example, forming toxins. The selection of peptides formed from different units and usable in the methods described above is well within the ability of one skilled in the art. For example, US2009 / 0035822 describes the formation of functional proteins from separate parts.

  Diphtheria toxin, tetanus toxin, and ricin can be used in therapy, for example in the treatment of neoplastic diseases. Therefore, complex formation systems involving these polypeptides can be used for therapy and treatment of neoplastic diseases. They can further be used in therapy comprising administering to a subject an effective amount of a composition comprising the complexing system.

The present invention further provides a method of forming a SNARE complex to form a binary compound comprising two cargo moieties, the method comprising:
Two polypeptide helices derived from SNAP protein, one polypeptide helix derived from syntaxin, and one polypeptide helix derived from synaptobrevin or a homologue thereof were combined and stabilized. Forming a SNARE complex, wherein the first cargo portion is bound to the first helix and the second cargo portion is bound to the second separate helix; and , SNARE complex formation causes the formation of a binary compound.

In another specific embodiment, the present invention is a complex forming system for forming botulinum toxin comprising the following:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof,
Wherein the four polypeptide helices form a stable SNARE complex, wherein the first cargo moiety is bound to the first helix and the second cargo moiety is the second Bound to two separate helices, wherein the first cargo moiety comprises a botulinum toxin light chain or functional portion thereof and a botulinum toxin heavy chain translocation, and a second cargo moiety Provides a complexation system comprising a receptor binding portion of the heavy chain of botulinum toxin.

This embodiment of the present invention is directed to the specific application of SNARE complex assemblies that can create botulinum toxins in separate parts, thus avoiding any risks associated with complete toxins during the manufacturing process. . The two parts are then combined using the formation of a SNARE complex to form a functional botulinum toxin.
The present invention further provides the use of the complex formation system described above. Furthermore, the present invention provides a complex formation system as described above for use in the treatment of diseases and bodily conditions that are alleviated by inhibition of neural synapses, and further for use in treatment.

  Since the use of botulinum toxin A has been widely used for medical and cosmetic therapies for many years, those skilled in the art will fully recognize diseases or physical conditions that are alleviated by inhibition of neural synapses (eg, Jankovic ( 2004) Botulinum in clinical practice. See J Neurol Neurosurg Psychiatry 75 951-957). In particular, some of the diseases or physical conditions that are alleviated by inhibition of nerve synapses are: hyperhidrosis, hypersalivation, dystonia, gastrointestinal disorders, urological disorders, facial spasms, strabismus, cerebral palsy, stuttering, chronic tension headache, tears Selected from the group consisting of hypersecretion, hyperhidrosis, spasm of the hypopharyngeal constrictor muscle, spastic bladder, pain, migraine, and cosmetic surgery such as wrinkle removal, wrinkle removal on the forehead.

A method of treatment is further provided, the method comprising administering to the subject an effective amount of the complexing system described above.
The invention further provides a method of forming a SNARE complex to form a botulinum toxin, the method comprising:
Stable SNARE complex by combining two polypeptide helices derived from SNAP protein, one polypeptide helix derived from syntaxin, and one polypeptide helix derived from synaptobrevin or its homologue. Forming a body, wherein the first cargo portion is coupled to the first helix and the second cargo portion is coupled to the second separate helix, wherein The cargo portion of the botulinum toxin comprises the light chain of botulinum toxin or a functional part thereof and the translocation of the heavy chain of botulinum toxin, and the second cargo portion comprises the receptor binding part of the heavy chain of botulinum toxin .
This method results in two parts of the botulinum toxin to be combined together, thus creating a fully functional botulinum toxin.

  Furthermore, the invention provides a component for forming a botulinum toxin, which component includes: a SNAP protein; a syntaxin; or a polypeptide helix derived from synaptobrevin or a homologue thereof, wherein the polypeptide Bound to the helix is a botulinum toxin light chain or functional portion thereof and a cargo moiety comprising a botulinum toxin heavy chain translocation. Also provided by the present invention is a component for forming a botulinum toxin, the component comprising: a SNAP protein; a syntaxin; or a polypeptide helix derived from synaptobrevin or a homologue thereof, wherein the polypeptide The helix is bound to the cargo moiety comprising the receptor binding portion of the heavy chain of botulinum toxin.

  In addition, the present invention relates to the use of the preceding components, and two polypeptide helices derived from SNAP protein: one polypeptide helix derived from syntaxin: and one polyply derived from synaptobrevin or homologues thereof. Comprising a peptide helix, wherein the four polypeptide helices form a stable SNARE complex, wherein the first cargo moiety is bound to the first helix and the second cargo moiety And wherein the first cargo moiety comprises a botulinum toxin light chain or functional portion thereof and a botulinum toxin heavy chain translocation, and a second There is provided the use of a kit wherein the cargo moiety comprises a receptor binding portion of the heavy chain of botulinum toxin.

  The previously described embodiments relating to botulinum toxins comprise further limitations, and the limitations described elsewhere with respect to other aspects and embodiments of the invention are similar to these botulinum toxin embodiments. Those skilled in the art will appreciate that it is appropriate.

In one particular embodiment related to a surfactant, the present invention provides a complex forming system for forming a molecular scaffold, which system is:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Where the four polypeptide helices form a stable SNARE complex, where the SNARE complex is formed in the presence of a surfactant.

Various features and limitations associated with the complexation system described above are equally suitable for this embodiment of the invention.
Previously, arachidonic acid was shown to allow SNARE complex formation in the presence of Munc18 (Rickman C and Davletov B (2005)). Munc18 is thought to be a negative regulator of SNARE complex formation. The inventors have made the surprising discovery that in the absence of Munc18, surfactants allow better SNARE complex formation. Preferably, in a system related to the presence of a surfactant, the system does not contain a Munc18 protein. The above system is a system that does not contain Munc18.

The present invention further provides the use of a surfactant to promote SNARE complex assembly in the absence of Munc18. Preferably, the surfactant is a weak surfactant. Preferably, the surfactant is a synthetic surfactant. In one embodiment, the surfactant has a carbon chain length of 7 to 12 carbon atoms.
All the embodiments of the invention described above relate to a complex formation system in which a single stable SNARE complex is created by one or more cargo moieties attached to the SNARE complex. This is useful for many applications, such as diagnostics. The systems further have applications in tagging applications for affinity purification of labeled proteins, protein or cell immobilization, or identification of labeled proteins (ELISA, Western blot). Immobilization of cargo on a substrate is useful in diagnostic and microarray applications. One skilled in the art will readily appreciate that the stability of the SNARE complex may be particularly useful for cargo immobilization in microfluidic or continuous flow applications. Further uses are listed below:

Use in solution:
-Construction of affinity reagents by genetic recombination including combinatorial assembly, poly-, homo-, and hetero-oligomerization;
-Targeted delivery of functional cargo, eg drug delivery (drug = small molecule, nucleic acid, protein), eg for the assembly of cargo with target and endogenous signals;

-Tagging and labeling protein molecules, molecular complexes containing protein molecules, cells, tissues, organs and organisms containing protein molecules.
-For the construction of binary compounds, eg functional proteins, enzymes, factors, cofactors (and other functional proteins), FRET labels, binary inorganic compounds and small molecules, binary organic compounds;
A more complex assembly (eg, spore) containing the self-assembled protein construct and protein for post-assembly immobilization of the protein on the surface of the self-assembled construct.

Solid surface applications:
-Arrays, surface immobilization;
-Surface modification and protein immobilization of immunoassay (eg ELISA) well plates;
-Surface modification and protein immobilization of BIA core and other SPR (surface plasmon resonance) instruments;
-Surface modification and protein immobilization of QCM (quartz crystal microbalance) instruments;

-Surface modification and protein immobilization of MALDI mass spectrometry plates;
-Surface modification of microfluidic devices and protein immobilization;
-Surface modification of capillary electrophoresis and protein immobilization;
Surface modification and protein immobilization of chromatography columns and immobilization media (eg beads);
-Surface and tip modification and protein immobilization of scanning probe microscopy;
-Surface modification and protein immobilization of micro and nano calorimetric instrument sensors;
-Surface modification of micro and nanoparticles;
-Surface modification of solid surfaces (eg gold-plated glass slides), thin films, wires (eg nanowires) and nanotubes.

Other uses:
-Nanobiotechnology, eg adhesion of self-assembled "adhesive" based biodegradable proteins and surfaces;
-Protein-based fibers and polymers;
-Tissue scaffolding and regenerative medicine.

  Furthermore, the present invention provides the use of any of the embodiments of the complex forming system described above, and in particular, in any of the applications described above. For example, the present invention uses complexation systems in diagnosis, such as arrays, assays, microfluidic devices, SPR instruments, QCM instruments, mass spectrometers, electrophoresis instruments, chromatography columns, scanning probe microscopes, or calorimetry. Provide equipment. For example, complexation systems can be used in arrays to immobilize antibodies to a substrate.

In one embodiment, the present invention provides a device having a stable SNARE complex immobilized thereon, wherein the SNARE complex is:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein said apparatus is selected from arrays, assays, microfluidic devices, SPR instruments, QCM instruments, mass spectrometers, electrophoresis instruments, chromatography columns, scanning probe microscopes, and calorimetric instruments Is done.

The present invention further provides a method of forming a SNARE complex that possesses one or more cargo moieties, the method comprising:
Stable SNARE complex by combining two polypeptide helices derived from SNAP protein, one polypeptide helix derived from syntaxin, and one polypeptide helix derived from synaptobrevin or its homologue. Forming a body, wherein one or more cargo moieties are attached to the polypeptide helix and wherein:
(I) linking a polypeptide helix derived from syntaxin to a polypeptide helix derived from synaptobrevin or a homologue thereof;
(Ii) integrate the polypeptide helices to form a component comprising two helices; or (iii) at least two of the polypeptide helices are less than 50 amino acids in length.

The previous description relating to the system and limitations of the present invention is equally appropriate for the previous method.
The invention further comprises a component comprising a helix derived from syntaxin linked to a polypeptide helix derived from synaptobrevin or a homologue thereof, wherein the two linked helices are stable. That can form part of the SNARE complex. In one embodiment, the two helices can also be integrated so that they can be assembled into the same stable SNARE complex. Alternatively, the two helices can be integrated so that they cannot assemble into the same stable SNARE complex. In this alternative embodiment, the two helices must be able to assemble into different stable SNARE complexes. The two-helix component of the present invention comprising a helix derived from syntaxin and a helix derived from synaptobrevin or a homologue thereof has the following sequences shown in SEQ ID NOs: 12, 13, 14, and 73:

Syntaxin 3 (1-260) / synaptobrevin 2 (1-84) (syntaxin including head domain and linker)-SEQ ID NO: 12
Syntaxin 3 (195-253) / synaptobrevin 2 (1-84) (syntaxin without the head domain but with a linker)-SEQ ID NO: 13
Syntaxin 3 (1-253) / Synaptobrevin 2 (29-84) (Syntaxin with head domain and no linker)-SEQ ID NO: 14
Syntaxin 3 (195-253) / Synaptobrevin 2 (29-84) (Syntaxin containing no head domain and no linker)-SEQ ID NO: 73
May be included.

The two-helix component of the present invention comprising a helix derived from syntaxin and a helix derived from synaptobrevin or a homologue thereof can also consist of one of the preceding sequences.
The two-helix component can further comprise one or more cargo moieties linked to a polypeptide helix.

  The invention further comprises a component comprising either two polypeptide helices derived from SNAP protein and a polypeptide helix derived from synaptobrevin or a polypeptide helix derived from syntaxin, wherein The helices are integrated to form a triple helix component, where the three linked helices provide a component that can form part of a stable SNARE complex. In one embodiment, the third helix is derived from synaptobrevin. In other embodiments, the three helices can be integrated so that they can assemble into the same stable SNARE complex.

  The triple helix component of the present invention comprising a SNAP-25 helix and a helix derived from synaptobrevin or a homologue thereof can also comprise SEQ ID NO: 15. Furthermore, the triple helix component can also consist of this sequence.

  Alternatively, the three helices are integrated so that the two SNAP helices can assemble into the same stable SNARE complex, but the third helix cannot assemble into the same stable SNARE complex as the two helices. Can also be done. In this alternative embodiment, the third helix should be able to assemble into a stable SNARE complex different from the two SNAP helices. The triple helix component can further comprise one or more cargo moieties attached to the polypeptide helix.

Furthermore, the present invention provides for the use of the previous component to form a stable SNARE complex, for example to form a molecular scaffold.
The present invention is a kit comprising a component comprising a polypeptide helix derived from syntaxin linked to a polypeptide helix derived from synaptobrevin or a homologue thereof, wherein Further provided are kits that allow the helix to form part of a stable SNARE complex.

  The kit may further comprise two polypeptide helices derived from a SNAP protein capable of forming a stable SNARE complex with a helix derived from syntaxin / synaptobrevin. Two SNAP helices can be integrated.

  In an alternative embodiment, the invention comprises a kit comprising two polypeptide helices derived from a SNAP protein and a polypeptide helix derived from syntaxin or a polypeptide helix derived from synaptobrevin. Where the three helices are integrated to form a triple helix component, and wherein the linked three helices can form part of a stable SNARE complex. provide.

  The kit may further comprise a single polypeptide helix derived from a fourth SNARE protein. Therefore, if the triple helix component comprises two SNAP-derived helices and a synaptobrevin or homologue-derived helix, the kit will generate a syntaxin that can form a stable SNARE complex with the linked SNAP / synaptobrevin-derived helix. It may further comprise a single polypeptide helix derived from. Alternatively, if the triple helix component comprises two SNAP-derived helices and a syntaxin-derived helix, the kit may be synaptobrevin or a homologue thereof capable of forming a stable SNARE complex with the linked SNAP / staxin-derived helix. It may further comprise a single polypeptide helix derived from.

The kit of the present invention may further comprise reagents suitable for use with the kit.
Additional features of the kit helix and SNARE complex are as described above.

  The complex formation system can also be used to form multimers of SNARE complexes. This is an integrated multiple SNARE complex. In one embodiment, two of the helices are integrated in such a way that a stable SNARE complex cannot be formed as the same complex. However, the two helices should be integrated in such a way that they can each form a stable SNARE complex, although they are different SNARE complexes. This can be done by directly integrating the helices without using any type of linker so that the two helices cannot assume the correct conformation in the same SNARE complex. Alternatively, each helix of such a two-helix ligation construct can be complexed with three other polypeptide helices to form two stable SNARE complexes. The two helices can be combined by any suitable method, such as genetic recombination means or chemical coupling. For example, a two-component system in which two helices derived from a SNAP protein are integrated and linked in a restricted manner such that two helices derived from syntaxin and synaptobrevin cannot assemble into the same SNARE complex. Adopt. Starting from a syntaxin-synaptobrevin fusion protein, the SNAP fusion protein will form a complex with the syntaxin helix. The synaptobrevin helix from another syntaxin-synaptobrevin fusion protein will then bind to the SNAP-syntaxin complex to form a 4-helical SNARE complex. In this complex, there will be a syntaxin helix and a synaptobrevin helix protruding from it, one from each syntaxin-synaptobrevin fusion protein. These single helix overhangs can be the basis for the formation of additional SNARE complexes. Therefore, a multimer of SNARE complex is formed. In the previous example, the SNAP helix need not be integrated. In addition, every two helices could be integrated for the system to function as long as both of them could not be assembled into the same complex to form a stable SNARE complex.

The invention further provides multimers produced by the previous system.
Alternatively, multimers can be formed by controlling the reaction conditions of the system. Two of the system's helices must be integrated. This can be done in any suitable way. The two helices need not be limited in any way as in the previous example, but can be limited if desired. The system further comprises a single helix derived from the same SNARE protein as one of the two linked helices. For example, in a system comprising a syntaxin-synaptobrevin fusion protein, the system further comprises a single syntaxin helix. In order to create multimers, a single syntaxin helix is introduced into the solution. This single helix may be free in solution or immobilized on a substrate as described above. Two helices (which may or may not be linked) from the SNAP protein are added to the solution. These bind to the syntaxin helix to form a syntaxin-SNAP triple helix complex. After this, syntaxin-synaptobrevin fusion protein is added into the solution. The synaptobrevin helix from this fusion protein will bind to the triple helix complex to form a stable SNARE complex, while the syntaxin helix will remain unbound and protrude from the SNARE complex. . By repeating the same steps as before, this syntaxin overhang is used to form additional SNARE complexes, thus creating multimers. Since previous systems require the helices to be added in a particular order, it may be necessary to ensure that there are no unwanted molecules after a particular step. This can be done, for example, by immobilizing a single syntaxin helix and performing a wash after each binding step. The invention further provides multimers produced by the previous system.

Furthermore, the present invention provides a multimer comprising a plurality of integrated stable SNARE complexes, wherein each SNARE complex is:
Two polypeptide helices derived from the SNAP protein;
A polypeptide helix derived from syntaxin; and a polypeptide helix derived from synaptobrevin or a homologue thereof,
Where a helix from one SNARE complex is linked to a helix from another SNARE complex to integrate the SNARE complexes, and wherein one or more cargo moieties are Bind to polypeptide helix.

  The multimer of the present invention can be linear. If it is linear, each SNARE complex is bound to the other two SNARE complexes on both sides to form a long linear chain resembling a series of beads. Thus, the two helices within the SNARE complex are bound to the helices within the two different SNARE complexes. Apparently, the SNARE complex at the end of the chain is bound to only one other SNARE complex by one polypeptide helix.

  Alternatively, the multimer may be branched. This can be done, for example, by linking two syntaxin-derived helices in any order to a synaptobrevin-derived helix. The synaptobrevin-derived helix from this triple-linked construct binds to the syntaxin / SNAP-derived triple helix intermediate to form a SNARE complex with the two syntaxin-derived helices that remained unbound. When a SNAP-derived helix, such as SNAP-25, is added, two linked syntaxin / SNAP-25 intermediates are formed. Addition of a “steady state” synaptobrevin-syntaxin (double helix) derived construct forms two linked SNARE complexes with two syntaxin derived helices protruding. This introduces two branches for further extension. Thus, joining three helices allows for branching (as opposed to two joined helices used for linear polymerization). Thus, if a multimer is branched, one SNARE complex in the multimer is bound to three other SNARE complexes rather than bound to two other SNARE complexes like a linear chain, A branch point is formed. Three of the helices in the SNARE complex are attached to the helices in three different SNARE complexes at the bifurcation point.

  Each multimer can have a cargo moiety attached to the end of the helix. Multimers can have multiple cargo moieties attached to the multimer at the ends of the helix. In one embodiment, the multimer may have a cargo moiety on each SNARE complex. A multimer can have multiple cargo moieties attached to each SNARE complex. If the multimer has a plurality of cargo moieties, these may be the same or different. By providing a SNARE domain that carries a different cargo after each wash step, the addition and ordering of multiple cargos can be controlled.

The present invention further provides a method for producing a multimer, which method comprises the following steps:
1) providing a first polypeptide helix derived from a first SNARE helix;
2) The second and third polypeptide helices are joined to the first polypeptide helix to form a triple helix intermediate complex, wherein the second and third polypeptide helices are second and second From 3 SNARE helices;
3) Bind the fourth polypeptide helix to the triple helix bundle to form a stable SNARE complex, where the fourth polypeptide helix is derived from the fourth SNARE helix, and A fourth polypeptide helix is linked to a fifth polypeptide helix derived from the first SNARE helix; and 4) repeating steps 2) and 3) to form a multimer, wherein 1 or Linking multiple cargo moieties to a polypeptide helix;
Comprising.

The previous description relating to complex formation systems, multimers, and various limitations is also appropriate for the previous methods.
Steps 2) and 3) can be repeated as many times as necessary to create a multimer of the required length.

  The previous method refers to the first, second, third, and fourth SNARE helices. As described above, the SNARE complex is formed from four helices. Two of these helices are provided by SNAP proteins, one is provided by syntaxin, and one is provided by synaptobrevin or a homologue thereof. Therefore, in the previous method, these four helices have no particular order and are the first, second, third and fourth SNARE helices. This is because the identity of a particular helix is not important as long as four helices are used in the SNARE complex. Optionally, the SNARE complex can also be bound to complexin as described above.

  In one embodiment, the first helix is a syntaxin derived helix; the second helix is a SNAP derived helix; the third helix is a SNAP derived helix; the fourth helix is a synaptobrevin derived helix; and The fifth helix is a syntaxin derived helix.

  Due to the fact that four helices are used in the SNARE complex in no particular order, the fifth polypeptide helix need not be derived from the same SNARE helix as the first polypeptide helix. Similarly, when the formation of the second, third, fourth, fifth, sixth SNARE complex, etc. in the multimeric state is repeated as a step of the method, the second, third and fourth There is no requirement that the polypeptide helix is derived from the same SNARE helix as the second, third and fourth polypeptide helices in the first SNARE complex. The only requirement is that all four SNARE helices correspond to each SNARE complex to form a stable SNARE complex.

  Since the previous method involves a multi-step process, as long as they form a stable SNARE complex, this recognizes the possibility of introducing different polypeptide helices in subsequent iteration steps compared to the first step. . For example, in the first cycle of the step, one of the polypeptide helices derived from the SNAP protein can be a full-length SNAP-25 helix, whereas in the next cycle of the step, the polypeptide helix is 45 amino acids. Of length SNAP-25 helix. An important aspect is that both polypeptide helices are derived from the same SNARE helix, eg a particular SNAP helix, so that all four SNARE helices correspond to the SNARE complex.

  One or more cargo moieties are attached to the polypeptide helix so that the resulting multimer has a cargo moiety attached thereto. This can be done by attaching the cargo moiety to the polypeptide helix prior to use in the method. Preferably, the cargo moiety is attached to the polypeptide helix terminus. Multiple cargo parts can be introduced into the body. This can be done by attaching the cargo moiety to a number of polypeptide helices that are subsequently incorporated into the multimer using the previous method. The cargo portions may be the same or different.

  In order to make the method easier, it is desirable that the uniqueness of the first, second, third and fourth SNARE helices is maintained in the iteration step. Thus, for example, in the first cycle of the step where the first SNARE complex occurs, if the first SNARE helix is syntaxin, the second and third SNARE helices are derived from the SNAP protein and the fourth SNARE helix The helix is synaptobrevin or a homologue thereof, and in subsequent cycles of the step resulting in further SNARE complexes, the first SNARE helix is always syntaxin and the second and third SNARE helices are always derived from the SNAP protein And the fourth SNARE helix is always synaptobrevin or a homologue thereof.

  Furthermore, it is desirable that the uniqueness of the second, third, fourth and fifth polypeptide helices be maintained in the iteration step. Thus, for example, in the first cycle of the step resulting in the first SNARE complex, the second and third SNARE helices are derived from the SNAP protein, the fourth SNARE helix is derived from the synaptobrevin SNARE helix, and the fifth The polypeptide helix of the first polypeptide helix is derived from syntaxin, and in subsequent cycles of the step yielding additional SNARE complexes, the fifth polypeptide helix (which forms the first helix with the next SNARE complex) is always syntaxin. Yes, the second and third SNARE helices are always derived from SNAP-25, and the fourth SNARE helix is always a synaptobrevin SNARE helix.

In one embodiment, the second and third polypeptide helices are integrated so that they can assemble together in the same SNARE complex. Preferably, the second and third polypeptide helices are full length SNAP proteins, such as SNAP-25.
When the uniqueness of the polypeptide helix is the same in all subsequent steps, and when the second and third polypeptide helices are integrated, this is a rapid quantity using only two building blocks. Allows body formation.

In other embodiments, the first polypeptide helix is immobilized on a substrate. Suitable substrates are well known to those skilled in the art and are discussed above.
The method may further include a washing step after each binding step to remove any unbound helix. This ensures that these unbound helices cannot interfere with multimer formation in subsequent steps. Preferably, the multimer formed by said method is immobilized before the washing step is used.

  The method can be modified to introduce branching into the multimer. To do this, a sixth polypeptide helix from the first SNARE helix is linked to one of the second, third, fourth or fifth polypeptide helix, thereby allowing another Provide a helix in which a stable SNARE complex is formed. In one embodiment, the sixth polypeptide helix is linked to the second or third polypeptide helix. In an alternative embodiment, the sixth polypeptide helix is linked to the fourth or fifth polypeptide helix. This allows two additional SNARE complexes to be derived from a particular SNARE complex, thereby introducing branching into the multimer.

  This can be done, for example, by linking a synaptobrevin-derived helix to two syntaxin-derived helices in any order, as described above. Alternatively, the syntaxin helix is linked to SNAP-25 (which is the second and third helix) to initiate the formation of the second SNARE complex from the originating SNARE complex, so that the originating SNARE It means that the complex is bound to all three other SNARE complexes to form a branch point.

  Furthermore, multiple branches can be introduced by introducing additional helices into a particular SNARE complex in addition to a sixth helix that is linked to other helices of the SNARE complex. For example, synaptobrevin-derived helices can be linked to three or more syntaxin-derived helices in any order. Alternatively, two or more syntaxin helices are linked to SNAP-25 (which is the second and third helix) to form a third, fourth SNARE complex, etc. from the originating SNARE complex. Since it can be initiated, it means that the originating SNARE complex is bound to all four or more other SNARE complexes to form multiple branch points.

The required number of branches can be introduced into the multimer at one particular point and over the entire length of the multimer.
The invention further provides a multimer produced by the previous method.
The multimers described above are useful in many applications, such as diagnostics and the applications described below:

Use in solution:
-Construction of affinity reagents by genetic recombination including combinatorial assembly, poly-, homo-, and hetero-oligomerization;
-Targeted delivery of functional cargo, eg drug delivery (drug = small molecule, nucleic acid, protein), eg for the assembly of cargo with target and endogenous signals;
-Tagging and labeling protein molecules, molecular complexes containing protein molecules, cells, tissues, organs and organisms containing protein molecules.
-For the construction of multi-component compounds, eg functional proteins, enzymes, factors, cofactors (and other functional proteins), FRET labels, multi-component inorganic compounds and small molecules, multi-component organic compounds;
A more complex assembly (eg, spore) containing the self-assembled protein construct and protein for post-assembly immobilization of the protein on the surface of the self-assembled construct.

Solid surface applications:
-Arrays, surface immobilization;
-Surface modification and protein immobilization of immunoassay (eg ELISA) well plates;
-Surface modification and protein immobilization of BIA core and other SPR (surface plasmon resonance) instruments;
-Surface modification and protein immobilization of QCM (quartz crystal microbalance) instruments;

-Surface modification and protein immobilization of MALDI mass spectrometry plates;
-Surface modification of microfluidic devices and protein immobilization;
-Surface modification of capillary electrophoresis and protein immobilization;
Surface modification and protein immobilization of chromatography columns and immobilization media (eg beads);
-Surface and tip modification and protein immobilization of scanning probe microscopy;
-Surface modification and protein immobilization of micro and nano calorimetric instrument sensors;
-Surface modification of micro and nanoparticles;
-Surface modification of solid surfaces (eg gold-plated glass slides), thin films, wires (eg nanowires) and nanotubes.

Other uses:
-Nanobiotechnology, eg adhesion of self-assembled "adhesive" based biodegradable proteins and surfaces;
-Protein-based fibers and polymers;
-Tissue scaffolding and regenerative medicine.
The present invention will now be described in detail by way of example only and with reference to the drawings:

1 is a schematic representation of the molecular mechanism that drives vesicle fusion in neurotransmitter release. The core SNARE complex is formed by four α-helices provided by synaptobrevin, syntaxin, and SNAP-25. Synaptotagmin functions as a calcium sensor and closely regulates SNARE zipping during vesicle fusion. Figure 2 is a schematic representation of the binding of multifunctional units in an irreversible and site-selective manner as bundles or linear multimers. Arrows represent a binding scaffold; functional units are represented by geometric figures. A four-stranded SNARE bundle made up of four polypeptides having a length of at least 80 amino acids is shown (Button et al., 1998). The SNARE motif used to design the 40 and 45 amino acid peptides is shown. The shaded area is a hydrophobic layer and there is a central layer highlighted in dark gray. It is an SDS-PAGE gel showing that a 45 amino acid polypeptide can form an irreversible SNARE complex (Panel A), while a 40 amino acid peptide cannot (Panel B). For brevity, the inventors refer to this aggregate as TetriCS (four-helix combinatorial scaffold). FIG. 6 is a graph showing that streptavidin was able to bind to 45aa Tetrics peptide bound to either glutathione beads or nickel beads in a very specific manner. 1 is a schematic representation of a three-component SNARE bundle comprising a full-length SNAP-25 molecule (amino acids 1-206) with two SNARE helices and a separate syntaxin and synaptobrevin SNARE helix. Since both 40 (panel A) and 45 aa (panel B) peptides can assemble with SNAP25B, a 40 aa long peptide is sufficient for irreversible binding to full length SNAP-25 in a ternary system. An SDS-PAGE gel demonstrating this is shown. The control reaction with GST-SNAP25 alone on the beads shows very little binding, whereas the addition of 40aa syntaxin and synaptobrevin peptide with Myc tag and S tag, respectively, resulted in anti-glutathione beads FIG. 5 is a graph showing that the formation of a ternary complex is evidenced by tight binding of both Myc antibody and S protein. FIG. 5 is a sensogram showing binding of 40 aa syntaxin and synaptobrevin peptide to immobilized SNAP25B. Anti-myc antibody (Myc) binds to the syntaxin-myc epitope but can be eluted with 0.1% SDS. Bound syntaxin / synaptobrevin peptides cannot be separated by SDS. FIG. 2 is a schematic representation of a binary SNARE bundle comprising a SNAP-25 molecule with two SNARE helices and a syntaxin / synaptobrevin fusion protein. FIG. 6 is a schematic diagram showing the binding of rat syntaxin 1A SNARE motif (1955-254 amino acids) indicated by gray arrows to rat synaptobrevin 2 SNARE motif (25-84aa). FIG. 2 is an SDS-PAGE gel showing a rapid assembly of syntaxin 3-synaptobrevin 2 fusion protein and rat SNAP-25B into an irreversible complex. 1 is a schematic diagram showing the head domain and SNARE motif (1-260 amino acids) of rat syntaxin 3 fused to rat synaptobrevin 2 sequence 1-84 via a short stretch of amino acids. It is an SDS-PAGE gel showing that octyl glucopyranoside can “open” syntaxin 3 molecules, allowing the formation of binary assemblies. SDS-PAGE gel showing octyl glucopyranoside surfactant promotes dense SNARE complex assembly at concentrations above CMC (critical micelle concentration-CMC is 0.6% for this surfactant) It is. Some assembly takes place in the absence of surfactant, but effective assembly includes detergents to “open” syntaxin 3 molecules to allow the formation of binary assemblies, Requires octyl glucopyranoside. FIG. 5 is a surface plasmon resonance sensogram showing unique resistance for syntaxin-synaptobrevin fusion protein ( ^* ) binding and assembly between syntaxin / synaptobrevin fusion protein and SNAP-25 immobilized on a CM5 chip. Washing with 2 M NaCl (1), glycine pH 2.5 (2), 1% SDS (3), 0.1 M NaOH (4), 0.1 M phosphoric acid (5) is a two-component capture The reagent cannot be broken. Note, step 3 was repeated twice. Figure 2 is a schematic representation of a binary SNARE bundle comprising a triple helix molecule (black) and a fourth helix (grey). Showing that both 40 and 45aa syntaxin 1A peptides can assemble with a triple helix fusion protein composed of SNAP-25B and synaptobrevin 2 (SNAP-25B (22-206) / Syb2 (1-84)) In a binary system, an SDS-PAGE gel demonstrating that a 40 aa long peptide is sufficient for the formation of an irreversible SNARE complex. Note, the SDS-resistant complex moves faster than the triple helix protein in the SDS gel, probably due to its compact structure. SDS-PAGE gel showing that GST-SNAP25 binds to Sepharose beads with syntaxin 3 / synaptobrevin 2 fusion protein in a very specific manner. Note, syntaxin 3 / synaptobrevin 2 fusion protein is covalently bound to BrCN-Sepharose beads and therefore cannot be eluted and visualized on SDS-PAGE gels. Lane 1-Bacterial extract made using zwittergent 3-08. Lane 2-GST-SNAP-25 purified by beads with syntaxin 3 / synaptobrevin 2 fusion protein. 1 is a schematic illustration of a supramolecular device in the form of an infinitely strong, linear multimer formed by SNARE protein. 1 is a schematic representation of the process of forming long linear multimers using SNARE protein. (A) Coomassie stained gel showing a gradual increase in the amount of both syntaxin 3-synaptobrevin 2 fusion and SNAP-25 bound to the beads. Note, the amount of GST-syntaxin 3 used for addition to the beads remains constant, while there is a gradual increase in the amount of SNAP-25 and syntaxin 3-synaptobrevin 2 fusion proteins bound. To indicate the amount of binding material, the sample was boiled to destroy the SDS-resistant nature of the assembly. (B) Similar to panel A, but the sample was not boiled before SDS-PAGE. Note, increase in molecular weight of SDS resistant polymer consistent with additional polymerization steps. 1 is a schematic representation of a branched multimer formed from SNARE proteins. Figure 2 is a schematic representation of a fusion construct formed from syntaxin residue (1-253) fused directly to synaptobrevin 2 residue (29-84) (no linker). It is an SDS-PAGE gel showing that SNARE bundles can multimerize in solution by simple mixing. It has been shown that SNARE tagging allows delivery of Hc-mediated quantum dots to nerve endings. a. Illustration showing SNARE binding of the Hc and LcHN parts of BoNT / A. Individual subunits are shown in the same way as the structural model (source Lacy et al, 1998). b. Schematic showing SNARE tagging scheme for binding of SV2C binding site of botulinum neurotoxin (Hc) and streptavidin-coated quantum dots. Biotin (star)-syntaxin peptide (blue) allows quantum dot SNARE tagging, while Hc is fused to the synaptobrevin SNARE motif (purple). SNAP25 (green and red) allows Q-dot binding to Hc. c. Coomassie-stained SDS gel showing irreversible assembly of Hc-synaptobrevin, SNAP25 and biotinylated syntaxin 3 peptide into SDS-resistant complex, Hc-SNARE-biotin. d. Hc-SNARE-Q-dots show synaptic binding as evidenced by immunostaining of synaptophysin, a synaptic vesicle marker, during axonal elongation of cultured hippocampal neurons. Omission of SNAP25 during assembly prevents targeting of Q-dots to synaptic ends. We show that SNARE tagging allows for the gradual assembly of individual parts of BoNT / A into a single molecular entity. a. Schematic showing the positions of LcHN and BoNT / A Hc component disulfide bonds and SNARE tagging. b. LcHN tagged with SNAP25 can be purified and degraded to Lc and HN-SNAP25 after treatment with 50 mM dithiothreitol (DTT). Coomassie stained SDS gel. c. SNAP25 tagged LcHN that can bind to synaptobrevin tagged Hc by addition of syntaxin 3 peptide as evidenced by Coomassie stained and fluorescently imaged SDS gels. SNARE-binding botulinum neurotoxin has been shown to exhibit synaptic localization and cleave its intrasynaptic target. a. Fluorescein-labeled LcHN-SNARE-Hc binds to axonal extensions of hippocampal neurons. Immunostaining with anti-synaptophysin antibody reveals presynaptic terminals of cultured hippocampal neurons. b. Immunoblot showing cleavage of intrasynaptic SNAP25 by assembled neurotoxins in a manner similar to native BoNT / A. It has been shown that SNARE-conjugated botulinum neurotoxin inhibits neurotransmitter release. a. Fluorometric measurements for glutamate release from isolated rat cerebral nerve endings (synaptosomes) show comparable inhibition between LcHN-SNARE-Hc and BoNT / A. Following 1 hour incubation of synaptosomes with toxin, real-time glutamate release graph (upper panel) and dose dependency graph (lower panel, evaluated after 15 min stimulation with 35 mM KCl and 2 mM CaCl ₂ . ) b. As assessed after 15 minutes of stimulation, individual SNARE-tagged neurotoxin parts do not interfere with glutamate release after 1 hour incubation with synaptosomes. c. Graph showing dose-dependent inhibition of isometric contraction of mouse diaphragm by LcHN-SNARE-Hc. Error bars represent SEM; n = 3. LcHn was tagged with syntaxin (195-253), indicating that Hc was tagged with synaptobrevin (25-84). Two botulinum parts were mixed in the presence of SNAP-25 and toxin formation was visualized on a Coomassie stained SDS-gel. FIG. 5 shows that after 1 hour incubation with LcHnSix3-SNAP25-synaptobrevin Hc toxin from panel A, the blockade of glutamate release from rat brain synaptosomes was evaluated. FIG. 5 shows that cleavage of catalytic part Lc neuronal targets after application of the toxin LcHnSix3-SNAP25-synaptobrevin HcA from panel A to hippocampal neurons was assessed by immunoblotting using anti-SNAP-25 antibody. . The reassembled toxin has an activity similar to natural botulinum neurotoxin (BoNT / a) in cleaving SNAP-25. Note, LcHnSyx3-SNAP25-synaptobrevin HcA is more effective than LcHnSyx3-SNAP25-synaptobrevin HcD in this neuronal assay. It shows that SNARE tagging of the synaptobrevin 40 amino acid motif with the somatostatin peptide Ac-RLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDADAL-Ahx-Ahx-AGCKNFFFWTFTSC-OH enables the creation of somatostatin-quantum dots. Streptavidin-coated quantum dots were incubated with biotinylated syntaxin peptide and then mixed with somatostatin-synaptobrevin and SNAP-25. Assembled somatostatin-Q-dots are applied to cultured hippocampal neurons and their invasion into neurons using Q-dot fluorescence and anti-SNAP-25 antibodies that label many intracellular proteins Visualized by counterstaining. It has been shown that SNARE tagging of the synaptobrevin 40 amino acid motif with a somatostatin peptide allows the creation of a functional somatostatin-botulinum construct after mixing with LcHn syntaxin 3 and SNAP-25. The activity of LcHn syntaxin 3-SNAP25-synaptobrevin somatostatin (SS-LcHN) was assessed by cleavage of SNAP-25 in cultured hippocampal neurons after 20 hours of application of the assembled toxin. Figure 6 is an SDS gel showing the formation of a stable SDS resistant complex using a SNARE motif with an N-terminal truncation. 6 is an SDS gel showing the formation of a stable SDS resistant complex using a SNARE motif with C-terminal truncation. 6 is an SDS gel showing the formation of a stable SDS resistant complex using syntaxin SNARE peptide in which the internal methionine is replaced by non-oxidizable norleucine. Many SDS gels showing the formation of stable SDS-resistant complexes using a variety of different SNARE peptides. 1 is an SDS gel showing the formation of a stable SDS resistant complex using a truncated SNARE peptide. 1 is an SDS gel showing the formation of a stable SDS resistant complex using a truncated SNARE peptide. 1 is an SDS gel showing the formation of a stable SDS resistant complex using a truncated SNARE peptide. 2 is an SDS gel showing the formation of a stable SDS resistant complex when a neuropeptide is complexed with one of the SNARE peptides. 6 is an SDS gel showing the formation of a stable SDS-resistant complex when arginine / vasopressin peptide (AVP) is complexed with the N-terminus or C-terminus of the syntaxin peptide. 6 is an SDS gel showing the formation of a stable complex using a SNARE peptide containing less than 40 amino acids. FIG. 2 is an SDS gel showing maleimide-based cross-linking of proteins to SNARE peptides. The absorbance at 650 nm for the TMB (3,3 ', 5,5'-tetramethylbenzidine) substrate is shown. It is a part of a film that emits light. No original description. No original description.

Introduction A bundle or linear scaffold made of SNARE-derived polypeptides for controlled, irreversible, non-chemical binding of functional units.

  The inventors have developed a method for combining functional or structural units by simple mixing. The creation of nanometer-sized, well-defined, functional supramolecular structures by self-assembly provides a means for performing programmed work in life-biology, pharmacy and nanotechnology. Specifically, the present invention provides an unmet need for controlled binding of multiple functional units in an irreversible and site-selective manner of bundles or linear multimers shown in FIG. About. At the heart of this new technology is the exploitation of the unique properties of SNARE proteins, and indeed any envisioned chemical, for the binding of protein domains. Originally, these proteins are small to the plasma membrane by forming a ternary complex composed of syntaxin, SNAP-25, and synaptobrevin (also known as VAMP-vesicle-binding membrane protein). Drives cell fusion. This SNARE complex comprises two helices from SNAP-25, one helix from syntaxin and one helix from synaptobrevin; each helix is ˜60-70 amino acids in length (Jahn and Scheller (2006)) A coiled-coil bundle of four helices (Fig. 3). This four-helix bundle is abnormally stable even in SDS-a direct indicator of the irreversible nature of SNARE assembly (Hu, K et al., 2002).

  Various strategies have been used so far for dimerization, oligomerization and multimerization of ligands and other functional groups. Among these, chemical cross-linking of small (<5 kDa) peptides (Pillai et al., 2006; Tweedle, 2006), transglutaminase-catalyzed heterodimerization (Tanaka et al., 2004), and tetramer strepto of biotinylated ligands Avidin binding (Leisner et al., 2008) provides some examples of binding functional units. In addition, coiled coils have attracted considerable interest as design templates for oligomerization in various applications including protein engineering, biotechnology, biomaterials, basic research and pharmacy (Engel and Kammerer, 2000; O ' Shea et al., 1993; Scherr et al., 2007). An example of a useful oligomerization domain is the leucine zipper of GCN4 comprising a 33-residue and a 46-residue homopentamer coiled-coil COMPcc that form parallel coiled-coil homodimers (Engel and Kammerer, 2000 ). The selection of coiled coils for various applications can have several characteristics: length of coiled coil polypeptides, their solubility, their ability to allow homo- or hetero-oligomerization; and the concentration of coiled coils, ie Depends on ability to withstand dissociation under steady and adverse conditions.

  The unique properties of SNARE coiled coil bundles, such as heterotetramerization and the irreversible nature of SNARE assembly, have not yet been considered for use. The core concept of the use of SNARE bundles relates to a means of generating diagnostic / therapeutic / biotechnological proteins that must carry a combination of different cargo (fluorescence, radioactivity, immunity, chemical, affinity, etc.). The inventors have used syntaxin, SNAP-25 and synaptobrevin protein-based for the production of well-defined, organized heterotetrameric supramolecular structures capable of self-assembly from different individual components. We proposed the use of genetically engineered polypeptides. We first define a minimum core of four and three component bundles; second, we describe a two component capture system for irreversible binding; and third, The inventors have shown the usefulness of SNARE bundles in producing linear multimers.

Example 1: Four component bundles The inventors have shown that a shortened SNARE helix can be used in a set of functional units as a four component bundle. The use of a truncated SNARE helix is essential for the addition of chemicals to the SNARE peptide via a synthetic pathway. At present, peptide synthesis is sufficiently reliable for ˜50 amino acids and can be realized financially. Therefore, if the SNARE helix is shortened, it would be advantageous to allow the addition of additional peptide sequences or other chemicals. It is known that shortening a single SNARE motif leads to irreversible SNARE aggregation (Hao et al., 1997). The inventors have (1) cleavage of all four helices into 45 amino acid peptides still leaves a stable four-helix complex, and (2) various functional groups in these peptides It was found that it can be added to either end of This simplifies multivalent complexes in bundles for a variety of applications, including affinity reagents and kits or multivalent therapeutic agents (wherein an array of ligands or receptor multimerization is desired) Making it possible. The free ends of the four individual helices can be used for the addition of up to 8 separate ones in a desired spatial combination by synthetic or genetic engineering means.
For brevity, we refer to the irreversible hetero-oligomeric protein complex as the four-helix combinatorial scaffold (TetriCS).

The inventors synthesized a TetriCS peptide containing either 40 or 45 amino acids.
40 amino acids (aa):
Rat SNAP25A helix 1 (amino acids 28-67) containing biotin:
STRMLQLVEEESDADGIRTLVLMLDEQGEQLDRVEEGMNHIGGSGGG-biotin (SEQ ID NO: 1)
(40 amino acid SNARE sequence is bold);
Rat SNAP25A helix 2 (amino acids 149-188) containing 6-histidine label:
NLEQVSGIIGNLRHMALDMGNIDTQNRRQIDRIMEKADSNGSGGGGHHHHHH (SEQ ID NO: 2)
(40 amino acid SNARE sequence is bold);

Rat syntaxin 1A (amino acids 201-240) 6-histidine labeled and cysteine-containing:
EIIKLENSIRELHDMFDMDMALVESQGEMIDRIEYNVEHAGSGGGHHHHHHC (SEQ ID NO: 3)
(40 amino acid syntaxin sequence is bold);
Contains S-tag epitope for rat synaptobrevin-2 (amino acids 31-70) monoclonal antibody recognition:
RLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDDRADALGSKKETAAKFERQHMDS (SEQ ID NO: 4)
(40 amino acid SNARE sequence is bold);

In parallel, we tested a 45 amino acid long TetriCS peptide:
Rat SNAP25A helix 1 (amino acids 28-72) containing biotin:
Biotin-STRRMLQLVEESKDAGIRTLVMLDEQGEQLDRVEEGMNNHINQDMKC (SEQ ID NO: 5)
(45 amino acid SNARE sequence is bold);
Rat SNAP25A helix 2 (amino acids 149-193) containing cysteine and 6-histidine labeling:
CNEMDENLEQVSGIIGNRLRHMALDMGNIDTQNRQIDRIMEKADSNKTRIGGHHHHHHH (SEQ ID NO: 6)
(45 amino acid SNARE sequence is bold);

Rat syntaxin. 1A (amino acids 201-245) N-terminal antibody epitope and cysteine content:
Ac-AEDAEIIKLENSIRELDMFDMDMAMLVESQGEMIDRIEYNVEHAVDYVEC (SEQ ID NO: 7)
(45 amino acid SNARE sequence is bold);
Contains N-terminal epitope for recognition of rat synaptobrevin 2 (31st-75th amino acids) antibody:
MSATAATPPPAAPAGEGGPPPPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDDRALQAGAS (SEQ ID NO: 8)
(The 45 amino acid SNARE sequence is bold).

  40 or 45 amino acid peptides were incubated for 60 minutes at 20 ° C. and their assembly was analyzed by SDS-PAGE. FIG. 4 shows that the 45 amino acid Tetrics peptide was able to form an irreversible SNARE complex (panel A) while the 40 amino acid peptide was not (panel B).

  The functionality of the assembly was tested in a pull-down experiment. The inventors have included GST-tagged synaptobrevin (45 amino acid SNARE sequence, produced by genetic recombination in bacteria), biotin chemically bound to SNAP-25 helix 1 and helix 2 of SNAP-25 as functional units. A conjugated 6-histidine tag was used. We have bound the TetriCS assembly to glutathione beads (for GST binding) or nickel beads (for 6-histidine tag binding) followed by binding of fluorescent streptavidin (for binding to biotin). Was tested. FIG. 5 shows that streptavidin was able to bind to 45aa TetriCS bound in a very specific manner to either glutathione or nickel beads.

  These results indicate that 45aa TetriCS peptides can be used to attach various functional groups without compromising their functional properties. Since the 4 Tetrics peptides have 8 free ends, it is possible to attach 8 different groups. Thus, TetriCS develops functional supramolecular devices that are defined as structurally organized and functionally integrated systems built from appropriately designed molecular components that perform a given action. Make it possible (Lehn, 2007).

Example 2: Ternary Bundle In cases where less than 8 groups are to be attached, it is useful to have a simplified TetriCS assembly. In fact, it is possible to utilize a full-length SNAP-25 molecule (amino acids 1-206) that already has two SNARE helices (see FIG. 6).
The inventors therefore tested whether the previously described 40, 45aa rat syntaxin 1A and synaptobrevin 2 peptides could form irreversible aggregates with full-length rat SNAP-25B. FIG. 7 shows that both 40 (panel A) and 45aa (panel B) peptides can assemble with SNAP25B, and in a ternary system, a 40aa long peptide is sufficient for irreversible binding to full length SNAP-25. It is proved that.

The inventors tested the functionality of a 40aa Tetrics assembly containing:
(I) Full length rat SNAP25B (amino acids 1-206) including substitution of cysteine 84, 85, 90, 92 with alanine. This is known to aid in the expression and purification of SNAP-25 (Fasshauer et al., 1999).
(Ii) Synthetic syntaxin-myc peptide (40 amino acid syntax sequence is bold)
EIIKLENSIRELDFMDMDMAMLVESQGEMIDRIEYNVEHA-GSGEQKLISEEDLC (SEQ ID NO: 9).
(Iii) Synthetic synaptobrevin-S tag peptide (40 amino acid synaptobrevin sequence is bold)
RLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDDRADAL-GSKETAAAKFERQHMDS (SEQ ID NO: 4).

  Following binding of GST-fused SNAP-25 to glutathione beads, the functionality of the assembly was tested by the ability of assembled ternary TetriCS to bind to fluorescent anti-Myc antibody and fluorescent S-protein (FIG. 8). The control reaction with a single GST-SNAP-25 on the beads showed little binding, while the addition of 40aa syntaxin and synaptobrevin peptide resulted in glutathione for both anti-Myc antibody and S-protein. • resulted in the formation of a ternary TetriCS, as evidenced by tight binding to the beads.

  The inventors have concluded that the 40aa synthetic peptide allows for the assembly of various tags within a single irreversible complex. The ternary system refers to a self-assembled set of two short polypeptides and a SNAP-25 molecule, and allows simple six-functional tagging of proteins.

  The inventors then used the surface plasmon resonance method to test the ability of 40aa ternary TetriCS to withstand harsh processing. The inventors chemically coupled GST-tagged SNAP-25B to a Biacore CM5 chip, followed by binding of 40aa syntaxin and synaptobrevin peptide, followed by antibody binding. FIG. 9 shows that the peptide binding is stable and allows the binding of anti-myc antibody to the surface of the chip. Addition of 0.1% SDS removed the antibody but did not remove the peptide from the immobilized SNAP-25.

Example 3: Binary bundles We further simplified the irreversible SNARE assembly into a binary system. Two-component affinity-based tools are the basis for all basic research and are invaluable in the development of drugs and diagnostics (Uhlen, 2008). Applications include affinity chromatography, microarray technology, microplate-based screens, and many biotechnology processes. The main factor for success in these applications relies on the tight and irreversible immobilization of the protein in a defined orientation on either a solid surface or a three-dimensional matrix. Several recent reviews have revealed many disadvantages of existing immobilization techniques (Kohn, 2009; Tomizaki et al., 2005). For example, in the case of chemical protein coupling, one skilled in the art can achieve irreversible surface immobilization, but the product may be in a non-functional state due to orientation problems or chemical modifications. . In contrast, proteins are bound to the corresponding surface (glutathione, metal, antibody-covered) by site-selective methods using various tags (GST, His tag, anti-myc and other antibody recognition epitopes) But in all of these cases, immobilization is not permanent (all existing peptide affinity tags dissociate) and / or very expensive (antibody-based affinity surface). Obviously, an ideal immobilization technique should allow for both irreversible coupling and site-selective orientation of the target protein, in addition to much more than current antibody-based approaches. It must be cheap. Separately, irreversible binding of two proteins in a functional orientation is possible if they are expressed with peptides that bind irreversibly.

  For the two-component system, we used a newly designed syntaxin / synaptobrevin fusion protein with double helix SNAP-25 (see FIG. 10).

  In the first embodiment, the inventors combined the rat syntaxin 1A SNARE motif (1955-254 amino acids) with the rat synaptobrevin 2 SNARE motif (25-84aa) as illustrated in FIG. . A short series of amino acids between the syntaxin and synaptobrevin sequences (GILDSMGRRELKL (SEQ ID NO: 10), small arrow) is due to the multiple cloning site of the hybrid plasmid.

  The inventors have purified the fusion protein but found that it tends to aggregate via a syntaxin 1 motif that prevents the formation of SNARE complexes with SNAP-25. The inventors then generated a fusion of synaptobrevin 2 and rat syntaxin 3. Within this chimera, the inventors fused the rat syntaxin 3 SNARE motif (amino acids 195-253) to the rat synaptobrevin 2 sequence 1-84 via a short series of amino acids. The short series of amino acids between the syntaxin and the synaptobrevin sequence (GILDSMGRLELKL) is due to the multiple cloning site of the hybrid plasmid that allows the insertion of a functional unit between syntaxin 3 and synaptobrevin 2. When mixed with rat SNAP-25B, this syntaxin 3-synaptobrevin 2 fusion protein quickly assembled into an irreversible complex as illustrated in FIG.

  In the next chimera, we use both the rat syntaxin 3 head domain and SNARE motif (amino acids 1-260) fused to rat synaptobrevin 2 sequence 1-84 via a short series of amino acids. (See FIG. 13). The short series of amino acids between the syntaxin and synaptobrevin sequences (GILDSMGRRELKL (SEQ ID NO: 10)) is due to the multiple cloning site of the hybrid plasmid that allows the insertion of a functional unit between syntaxin 3 and synaptobrevin 2.

  While the head domain of syntaxin 3 protects the SNARE motif from SNARE assembly, certain lipids are known to be able to “open” syntaxin for SNARE assembly (Darios and Davletov, 2006; Rickman and Davletov, 2005). Unpublished studies by the inventors have shown that a weak surfactant called octyl glucopyranoside can also “open” syntaxin 3 molecules. Thus, the inventors can control the syntaxin SNARE motif and form SNARE complexes in a controlled manner. FIG. 14A shows an example of a controlled binary set as tested in an SDS gel. The inventors further have other surfactants or similar lipid compounds such as MEGA8, C-HEGA10, C-HEGA11, HEGA9, heptylglucopyranoside, octylglucopyranoside, nonylglucopyranoside, zwittergent 3-08, zwittergent 3-10 and zwittergent It turned out that 3-12 etc. have the same effect.

Surface plasmon resonance experiments demonstrated the excellent resistance of this binary set to various disrupting agents (Figure 15).
As an alternative two-component system, the inventors have created a two-component affinity reagent containing one small tag, in which a short syntaxin helix (<5 kDa) is newly shown schematically in FIG. 16A. Can be irreversibly bound to the triple helix SNARE fusion protein (~ 27-31 kDa) designed in the above. In this chimera, the inventors fused two SNAP-25 SNARE helices (amino acids 22-206) to the SNARE motif 1-84 of the synaptobrevin 2 sequence. The linker GSGSEQKLISEEDLG (SEQ ID NO: 11) between SNAP-25 and the synaptobrevin sequence has a myc tag epitope. When mixed with the 40 or 45 amino acid peptides of syntaxin described above, the triple helix fusion protein quickly assembled into an irreversible complex, as illustrated in FIG. 16B.

  These two-component systems are useful alternatives to current affinity tags (Terpe, 2003). Both tags of the two-component capture system are expressed in bacteria and are easily added to any protein for recombinant production in a site-selective manner—this can be biotin / streptavidin or similar Different from high affinity systems (biotin cannot be expressed as part of the protein). Rapid capture from highly diluted solutions is currently possible due to the irreversible nature of the binary affinity reagents-no other such system currently exists. If desired, any of the tags in the two-component system can be chemically coupled to the surface of the bead, chip, microarray plate and modified by chemical or genetic introduction of functional groups.

  As an example of using a two-component system for the purification of proteins from bacterial extracts, we first described a rat fused to rat synaptobrevin 2 sequence 1-84 (above) via a short series of amino acids. -A double helix fusion protein containing the SNARE motif (amino acids 195-253) of syntaxin 3 was immobilized on BrCN-Sepharose beads. The inventors further fused an enzyme called glutathione-S-transferase (GST) to SNAP-25B and expressed the latter fusion protein in E. coli. After disruption of the bacterial cell membrane using 2% detergent (called zwittergent 3-08), the bacterial extract was loaded onto Sepharose beads containing syntaxin 3 / synaptobrevin 2 fusion protein. After washing, the beads were analyzed by SDS-PAGE and Coomassie staining. FIG. 17 shows that GST-SNAP25 binds in a very specific manner to Sepharose beads with syntaxin 3 / synaptobrevin 2 fusion protein.

Example 4: Controlled Linear Polymerization Reaction In addition to binding multiple groups in bundle form, SNARE protein is also highly advanced in the form of an infinitely long rigid linear multimer as shown in FIG. It also offers the possibility of creating supramolecular devices.
In that direction, the inventors' collection means a unique approach in which biomaterials assemble in molecular units to produce a novel linear supramolecular structure (for a review of potential applications (Hinman et al, 2000; Lehn, 2007; Ryadnov and Woolfson, 2003; Zhang, 2003)).

  In this case, the inventors used the syntaxin 3-synaptobrevin 2 fusion protein described above for the binary system (rat syntaxin fused to rat synaptobrevin 2 sequence 1-84 via a short series of amino acids). 3 (amino acids 1-260)). However, instead of mixing SNAP-25 and syntaxin 3-synaptobrevin 2 fusion in solution, we have used a single syntaxin 3 polypeptide (a GST fused to GST) that contains a head domain to initiate the polymerization reaction. A solid support with 1-260 amino acids) was used. The inventors first immobilized a GST-syntaxin trimolecular simple substance on glutathione beads via a GST tag. The inventors then added a SNAP25B molecule (amino acids 1-206) that allows the formation of a syntaxin 3 / SNAP-25 binary complex in the presence of 0.8% octylglucopyranoside. After washing the beads to remove unbound SNAP-25, we added syntaxin 3-synaptobrevin 2 fusion protein. This process of adding two building blocks was repeated as many times as necessary. The process is shown in FIG.

  FIG. 20A shows that the amounts of both syntaxin 3-synaptobrevin 2 fusion and SNAP-25 increase stepwise over the course of the previous step. While the amount of GST-syntaxin 3 used for binding to the beads remains constant, the amount of bound SNAP-25 and syntaxin 3-synaptobrevin 2 fusion protein increases gradually. To show the amount of bound material, the sample was boiled to disturb the SDS-resistant nature of the aggregate. FIG. 20B shows the increase in molecular weight of the SDS-resistant polymer along an additional polymerization step when the sample was not boiled before SDS-PAGE.

  Since it is controlled step by step in the previous process, the necessary cargo at the desired location can be added to the individual SNAP-25 or syntaxin 3-synaptobrevin fusion protein in any polymerization step. SNAP-25 and syntaxin 3-synaptobrevin are building blocks for the controlled fabrication of various molecular structures. Applications include the fabrication of functionalized nanofibers, multiple ligand microarrays, supermolecular enzyme assemblies, new electronic devices and biomaterials for use in biotechnology and pharmacy (for a review of potential applications) (Zhang, 2003)).

As previously described, syntaxin-synaptobrevin-syntaxin or SNAP25-syntaxin fusion proteins can also be used to form branches within a linear scaffold at specific points as shown in FIG.
Finally, the inventors explored the possibility of making a linear polymer by simply mixing the two components in solution rather than on the surface. The inventors utilized a modified form of syntaxin 3-synaptobrevin 2 fusion protein in which the linker region between the two SNARE proteins was removed. This fusion construct has a syntaxin 3 residue (1-253) fused directly to a synaptobrevin 2 residue (29-84). See FIG.

We mixed rat SNAP-25B (1-206aa) with the fusion protein and analyzed the 60 minute reaction by SDS gel electrophoresis. FIG. 23 shows that SNARE bundles can be multimerized by simple mixing in solution.
Thus, the two-component system described above can be multimerized in solution, which provides a way to bind 1-4 protein functional domains in a linear shape. In addition, protein-based fabrication of fully SDS-resistant linear polymers can be used to construct biodegradable fibers with properties superior to multi-component assemblies of joro spiders or current coiled-coil nanofibers. For reviews, see Hinman et al., 2000; Ryadnov and Woolfson, 2003.

Polypeptide sequences used in Examples 1-4 (with corresponding drawing indications):
Syx3 (1-260) / Syb2 (1-84) (FIGS. 13, 14 and 20) (syntaxin including head domain and linker) —SEQ ID NO: 12
Syx3 (195-253) / Syb2 (1-84) (FIGS. 12 and 17) (syntaxin without a head domain but containing a linker) —SEQ ID NO: 13
Syx3 (1-253) / Syb2 (29-84) (FIG. 22) (syntaxin containing head domain but no linker) —SEQ ID NO: 14
SNAP-25B (20-206) / Syb2 (1-84) (FIG. 16B) —SEQ ID NO: 15

Example 5: SNARE tagging allows stepwise assembly of botulinum neurotoxins Overview The creation of nanometer-sized, defined functional supramolecular structures by controlled attachment of suitable building blocks is And can provide a new perspective on applied technology. Current binding strategies often rely on limited chemical methods and have not been able to obtain all the benefits of recombinant technology. Here, we use three SNAREs to form a stable four-helix complex to drive intracellular membrane fusion as a universal tag for irreversible binding of genetically engineered and synthetic functional units. Protein was used. The inventors have shown that SNARE tagging allows for the stepwise production of a botulinum neurotoxin type A, commonly known as BOTOX, a functional supramolecular medical toxin. The fusion of the receptor binding region and the synaptobrevin SNARE motif allowed delivery of the working part of a botulinum neurotoxin tagged with SNAP25 into neurons. The data shows that SNARE-tagged toxin was able to cleave its neuronal molecular target and inhibit neurotransmitter release. These results demonstrate that SNARE four-helix coiled coils allow controlled coupling of various building blocks to functional nanomachines.

Introduction The advent of molecular biology and genetically modified protein production has transformed science. The use of recombinant polypeptides, functional fragments of proteins and whole enzymes is currently widespread in the pharmaceutical, diagnostic, nanotechnology, and consumer bio industries. Despite the apparent success of recombination techniques, protein size remains an obstacle to creating higher performance proteins as a single functional unit. We believe we can overcome this bottleneck by combining multiple functions in a supramolecular unit rather than in individual proteins. Clearly, achieving these objectives for the construction of nanofactories or nanomachines is highly dependent on our ability to combine various functional units on demand and with high precision. Current efforts in this promising area still rely on conjugation techniques invented decades ago: biotin-streptavidin pairing, antibody-epitope recognition, chemical coupling with amino and sulfohydryl groups, It's amazing. These approaches are often limited by the need for chemical modification of recombinant proteins or the complexity of antibody-based techniques. Recently developed “click” chemistry addresses some of these problems, but remains dependent on inorganic compounds, and binding of recombinant proteins to a given supramolecular assembly has been achieved so far Was not. Alternative approaches based on DNA self-assembly or polypeptide oligomerization are further subject to these limitations in designing multifunctional genetically engineered assemblies. Here, the inventors have assembled SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein assembly discovered nearly 20 years ago to achieve irreversible binding of recombinant polypeptides to functional units. Explored the possibility of use.

  The SNARE protein drives cell membrane fusion in any eukaryotic cell by forming a heteromeric four-helix coiled coil. The brain-derived SNARE complex consists of three proteins: synaptobrevin, syntaxin and SNAP25. Syntaxin and synaptobrevin each provide a single helix, whereas SNAP25 provides two helices to form a tetrameric coiled coil. The four SNARE motifs are 55 amino acids long and have eight characteristic heptade repeats. The brain SNARE complex is extraordinary in stability and is resistant to chaotropic reagents, strong surfactants, proteases, and high temperatures. The inventors decided to investigate whether fusing SNARE to a recombinant protein would allow controlled construction of supramolecular entities.

  As an example of a multifunctional molecule, the inventors focused on a botulinum neurotoxin described as “a nanomachine that combines recognition, trafficking, unfolding, translocation, refolding and catalysis”. Botulinum neurotoxin type A, (BoNT / A) has proven to be of great medical importance due to its ability to cause neuromuscular paralysis for a very long time by local injection of trace amounts (1 pM concentration) (Montecucco, C. et al. (2009)). BoNT / A is a 150 kDa protein consisting of three main modules: a 50 kDa catalytic part (light chain, Lc) linked to the so-called heavy chain via a disulfide bridge, and the heavy chain results in It consists of an N-terminal 50 kDa translocation part (HN) and a C-terminal 50 kDa part (Hc), the latter involved in the recognition of neuronal ganglioside and synaptic vesicle receptor SV2C (Mahrhold, S. et al. al. (2006) and Dong, M. et al. (2006)). The three main modules are recognized as separate structural units in the X-ray model (source, Lacy et al. (1998), FIG. 24a). When the catalytic part is in the synaptic cytosol, it proteolyzes its neuronal target SNAP25 with excellent specificity, leading to long-term block of neurotransmission (Schiavo, G. et al. (1993 ) And Blasi, J. et al. (1993)). BoNT / A-mediated removal of 9 amino acids from the C-terminal end of SNAP25 does not reduce the stability of SNARE assembly using syntaxin and synaptobrevin (Hayashi, T. et al. (1994)).

Results The inventors first fused the SV2 binding part (Hc) to synaptobrevin (FIG. 24b) and determined whether this fusion can carry quantum dots (Q-dots) into the nerve endings. Tested. The Hc-synaptobrevin fusion was able to form a SNARE complex with SNAP25 and a 52 amino acid syntaxin 3 peptide labeled with biotin for binding to streptavidin-coated Q-dots. We chose to use the syntaxin 3 SNARE motif rather than syntaxin 1. This is because syntaxin 1 tends to homo-oligomerize. FIG. 24c shows that the Hc-synaptobrevin fusion was able to form an irreversible (SDS resistant) SNARE complex in the presence of SNAP25 even though there was a modified syntaxin 3 motif. . Q-dots pre-conjugated with biotinylated syntaxin peptide are incubated with Hc-synaptobrevin in the presence of SNAP25 and delivery of Q-dots into nerve endings is evaluated in cultures of hippocampal neurons from mice did. Q-dot-carrying Hc-SNARE accumulated at synaptic contact points as confirmed by staining with vesicular protein synaptophysin (FIG. 24d). This indicates that the targeting part of BoNT / A can still recognize its synaptic receptor and can retain large cargo after genetically engineered fusion with the SNARE tag.

  Next, we prepared a fusion of enzyme part, translocation part and SNAP25 (FIG. 25a). We introduced thrombin cleavage (instead of the natural trypsin sensitive site) between the enzyme part (Lc) and the translocation part (HN) and remained still bound by disulfide bonds during the isolation procedure. To facilitate cleavage between the two parts. The LcHN-SNAP25 fusion was obtained from E. coli. It was successfully expressed in E. coli and could be purified to homogeneity. When treated with dithiothreitol, this fusion was divided into two parts showing significant disulfide bond functionality (FIG. 25b). We then tested whether the SNARE tag allows for the assembly of LcHN and Hc parts into a single body. FIG. 25c shows that in the presence of syntaxin peptide, the combination of the two recombinant fusions led to the appearance of the new molecular entity LcHN-SNARE-Hc within 60 minutes as evidenced by SDS gel. • Indicates that this was not the case in the absence of peptide. To help visualize the targeting of reassembled BoNT, we used a fluorescein-labeled version of the syntaxin peptide, and indeed LcHN-SNARE-Hc could be visualized as a fluorescent protein (FIG. 25c).

  When LcHN-SNARE-Hc was applied to cultured hippocampal neurons, fluorescent molecules colocalized to a significant extent with the vesicle marker synaptophysin, indicating binding of BoNT / A to the natural target (FIG. 26a). . Importantly, immunoblotting of neurons treated with anti-SNAP25 antibody demonstrated that SNAP25 was cleaved in the same manner as when neurons were treated with native BoNT / A molecules (FIG. 26b). This indicates that the enzyme part was successfully released into the neuronal cytosol upon entry of LcHN-SNARE-Hc into synaptic vesicles. To test the effect of LcHN-SNARE-Hc on neurotransmitter release, we used a 96-well glutamate release test that allows simultaneous comparison of multiple factors (Darios, F. et al. (2009)). . FIG. 27a shows that LcHN-SNARE-Hc was able to inhibit calcium and KCl dependent release of glutamate from isolated brain nerve endings with a dose dependency similar to native BoNT / A. Show. The degree of inhibition of glutamate release from central nerve endings is in good agreement with previously obtained values (McMahon, HT et al., (1992)) and all central synapses carry the SV2C receptor for BoNT / A This suggests that this is not the case (Dong, M. et al., (2006)). Importantly, mixing SNARE-tagged LcHN and Hc in the absence of bound syntaxin peptide yields an inactive molecule, and complete SNARE assembly is achieved upon binding of the recombinant part to the functional entity. It is confirmed that this is an important factor (FIG. 27b). Treatment of LcHN-SNARE-Hc with dithiothreitol inactivated the assembled toxin, suggesting the functionality of the disulfide bond between Lc and HN (FIG. 27b). Finally, we tested the ability of LcHN-SNARE-Hc to paralyze muscles. We applied several concentrations of collective neurotoxin on the isolated mouse diaphragm and tested the paralytic response of the phrenic nerve. The time required for the amplitude to decrease to 50% of the initial value (paralysis half-life) was measured. FIG. 27c shows that LcHN-SNARE-Hc paralyzes the diaphragm muscle within 72 minutes at sub-nanomolar concentrations (190 pM). No paralysis was observed in the absence of bound syntaxin peptide (data not shown).

  Here, the inventors have demonstrated that SNARE tagging allows for the stepwise assembly of a medical toxin, commonly known as BOTOX, BoNT / A (Davletov, B. et al. (2005)). Although LcHN-SNARE-Hc was less effective than native BoNT / A in blocking neuromuscular junctions (Mahrhold, S. et al. (2006)), this is a distant active region within a long neuromuscular junction. Can be explained either by the low ability of the dilating toxins to reach or by the large presynaptic terminals in the diaphragm muscle leading to reduced efficacy. However, in the context of synaptic contact between neurons, we have observed similar efficacy between LcHN-SNARE-Hc and natural neurotoxins. Such preferential effects on the inhibition of interneuronal synapses, but not neuromuscular junctions, may be advantageous in developing pain-inhibiting treatments that avoid the side effects of paralyzing muscles. There are many meanings of our observations, but here are a few. First, it is now possible to express the active forms of bacterial multi-module medical toxins in a safe manner; in fact, the use of our “locking” peptides is additional Enable safe functions. It is also possible to use SNARE tagged Hc parts by tagging them with SNARE counterparts to provide contrast agents and future therapeutics (Binz, T et al. (2009 )). Furthermore, when producing anti-botulinum serum, it is also possible to oligomerize the Hc part to elicit a stronger immune response (Webb, R.P. et al. (2007)). SNARE tagging of the LcHN part will further allow easy retargeting of the active component of BoNT / A to specific neuroendocrine cells (Dolly, J. O. et al. (2009)). Here, one skilled in the art can target neuropeptides or growth factor receptors by creating the corresponding SNARE-tagged ligand. Such SNARE tagging allows for convenient combinatorial mixing of various functional units with the goal of finding the most beneficial combination (s) to silence a particular subset of neurons (Foster, KA ( 2009)).

  The inventors used BoNT / A as an example of a refined “nanomachine”, but that SNARE tagging can be used in building many additional supramolecular assemblies in a highly controlled manner. it is obvious. The creation of nanometer-sized, well-defined, functional supramolecular structures through the controlled binding of suitable building blocks appears to provide a new perspective in many areas (Lehn, J. M. (2007)). As evidenced by biotin and fluorescein incorporation during assembly of botulinum neurotoxins, the relatively short SNARE motif allows for the combination of both polypeptides and inorganic molecules created by genetic recombination. The greatest advantage of the SNARE coiled coil is its heterotetrameric nature that allows the binding of up to 8 different functions. This capability will be used in future medicine and applied technologies.

Methods Plasmid and protein reactions. All proteins were expressed as E. coli as glutathione-S-transferase (GST) fusions. It was expressed in the BL21 strain of E. coli. A plasmid for expression of LcHN-SNAP25 was generated as follows: BoNT / A Lc (amino acids 1-449) cDNA was amplified by PCR and within the SmaI and EcoRI restriction sites with the pGEX-KG vector. Inserted (Guan, KL et al. (1991)). The codon-optimized cDNA of the translocation domain HN of BoNT / A (450th to 872 amino acids, ATG Biosynthetics, Germany) was inserted at the 3 end of the light chain. A thrombin cleavage site (amino acid LVPRGS (SEQ ID NO: 16)) was inserted between the light chain and the translocation domain of BoNT / A. Finally, rat SNAP25B (1-206aa) cDNA was inserted into the 3 ′ end of HN. A plasmid allowing the expression of Hc-Syb was generated as follows: cDNA of rat synaptobrevin (amino acids 25-84) was amplified by PCR and inserted between the BamHI and EcoRI sites of the pGEX-KG vector. did. BoNT / A heavy chain (876-1296 amino acids) cDNA was amplified by PCR and inserted into the 3 ′ end of synaptobrevin. A peptide of syntaxin SNARE motif (amino acids 200-250) was chemically synthesized with either biotin or fluorescein (Peptide Synthetics, UK). The protein fused to GST was purified by glutathione sepharose beads (GE Healhcare, USA) and eluted from the beads in 20 mM Hepes, pH 7.3, 100 mM NaCl using thrombin. Supramolecular complexes were assembled by mixing SNARE-tagged protein with syntaxin peptide for 1 hour at 22 ° C.

Neuroimaging and immunoblotting. Mouse anti-synaptophysin antibody (clone 7.2) is from Synaptic Systems, and mouse anti-SNAP25 antibody (clone SMI81) is from Sternberger Monoclonals. Streptavidin conjugated Q-dots 525 are manufactured by Invitrogen. Primary cultures of hippocampal neurons were prepared as described (Darios, F. et al. (2009)) and used in vitro after 7-10 days. Neurons were exposed to SNARE tagged or native toxin for 2 hours, fixed with 4% PFA, and then immunostained with anti-synaptophysin antibody. Fluorescence emission was observed with a Radiance Confocal system (Zeiss / Bio-Rad; Hemel Hempstead, Herts., UK) linked to a Nikon Eclipse fluorescence microscope. Alternatively, neurons are incubated with aggregate toxin or native BoNT / A for 20 hours and lysed in 60 mM Tris, pH 6.8, ₂ mM MgCl ₂ , 2% SDS, Benzonase (Novagen, 250 U / ml) SNAP25 cleavage was then analyzed by immunoblotting using anti-SNAP25 antibody.

Blocking neurotransmitter release. Rat brain synaptosomes were freshly isolated as described (Darios, F. et al. (2009)). Synaptosomes (0.5 mg / ml protein) were buffered with the indicated concentrations of LcHN-SNARE-Hc (132 NaCl, 5 KCl, 20 HEPES, 1.2 NaH ₂ PO ₄ in mM, (1) MgCl ₂ , 0.15 Na ₂ EGTA, 1 MgSO ₄ , 5 NaHCO ₃ , 10 D-glucose) at 37 ° C. for 1 hour.

An equal volume of buffer A containing glutamate dehydrogenase (15 units / ml, Sigma) and 3 mM NADP (Sigma) was added over 10 minutes. Glutamate release was triggered by the addition of KCl (35 mM) in the presence of ₂ mM CaCl ₂ and followed by fluorescence emission (Exc. 340 nm, Em. 460 nm) (Darios, F. et al. (2009) and McMahon, HT et al. (1992)). The Phrenic Nerve hemidiaphragm assay was performed as previously described (Mahrhold, S. et al. (2006)). Mouse phrenic nerves were obtained from Naval Medical Research Institute (NMRI) mice. The phrenic nerve was continuously stimulated at 5-25 mA with a frequency of 1 Hz and a pulse width of 0.1 ms. Isometric contractions were converted using a force transducer and recorded with VitroDat Online software (FMI GmbH). The time required for the amplitude to decrease to 50% of the initial value (paralysis half-life) was measured.

Additional information Botulinum neurotoxin is the most powerful toxin produced by nature. These toxins are produced by Clostridium bacteria to cause persistent paralysis and death. Over the last 30 years, some members of the Botulinum family have successfully used botulinum neurotoxin type A (BoNT / A), also known as BOTOX, for medical and cosmetic purposes. These toxins can silence neuromuscular junctions and block neurotransmitter release from many types of neurons. In fact, any part of the human body other than the brain can be treated with BOTOX. Since neuromuscular junction paralysis is reversible, continuous relaxation of the muscle requires repeated injections every 3-4 months. BoNT / A blocks innervation not only for striated muscle but also for smooth muscle. In addition, cholinergic junctions of the autonomic nervous system that control sweating, salivary secretion, and other types of secretion are as sensitive to BOTOX as the neuromuscular junction. As a result, BOTOX-based treatments have recently expanded to include nearly 100 medical conditions ranging from dystonia to gastrointestinal and urinary disorders, which are staggering.

  The effectiveness of BoNT / A in clinical medicine has led to increased attention to other members of the Botulinum family. A comparative study has demonstrated that BoNT / A has the longest paralytic effect among the seven immunologically distinct serotypes of BoNTs (AG), and therefore, especially in the treatment of neurological disorders. The usefulness of / A was demonstrated. All BoNTs are synthesized by bacteria as a single polypeptide chain with a molecular weight of 150 kDa. Following bacterial death and lysis, the toxin is “scored” by bacterial proteases to produce a 50 kDa light chain and a 100 kDa heavy chain that are tethered by disulfide bonds. The two chains that are still linked by disulfide bonds cross the small intestinal epithelial cells by transcytosis, enter the bloodstream, and eventually bind to the peripheral cholinergic nerve endings.

  The intense toxicity of BoNTs indicates that peripheral nerve endings carry molecules that can function as high affinity receptors for BoNTs. In fact, several synaptic vesicle proteins have been shown to function as receptors for BoNTs. Although the heavy chain is involved in the binding of BoNTs to the nerve endings, the light chain is a powerful endopeptidase that attacks the vesicle fusion mechanism and therefore must enter inside the nerve endings. BoNTs accomplish this role by hijacking the vesicular endocytic pathway. BoNTs undergo major conformational changes as the pH inside the recycling vesicles decreases. This allows the translocation part of the heavy chain (known as HN) to form a putative channel across the vesicle membrane by sliding the partially unfolded light chain into the cytosol. To do. Upon entry into the cytosol, reduction of the disulfide bond releases the light chain from the heavy chain.

  The BoNT light chain is a potent endopeptidase that attacks many isoforms of the three SNARE proteins that mediate vesicle fusion and thereby transductor release. It is now known that BoNT / A and BoNT / E proteolyze SNAP-25, and BoNTs B, D, F and G cleave VAMP on synaptic vesicles. SNAP-25 shortened to 9 amino acids by BoNT / A maintains the ability to interact with plasma membrane syntaxin and vesicular synaptobrevin, but cannot mediate the normal vesicle fusion process . Details regarding botulinum neurotoxins (BoNTs) can be found at: Davletov, B., Bajohrs, M. and Binz, T., Trends Neurosci 28, 446-452 (2005); Johnson, EA (1999) Annu Rev Microbiol 53, 551-575; Jankovic, J. (2004) J Neurol Neurosurg Psychiatry 75 (7), 951-957; Aoki, K, R. and Guyer, B. (2001) Eur J Neurol 8 Suppl 5, 21 -29; Simpson, LL (2004) Annu Rev Pharmacol Toxicol 44,167-193; Dolly, O. (2003) Headache 43 Suppl 1, SI6-24; and Montecucco, C. and Schiavo, G. (1993) Trends Biochem Sci 18 (9), 324-327. The complete sequence information of BoNT / A (BOTOX) was published in Binz, T. et al. (1990) JBiol Chem 265 (16), 9153-9158.

Retargeting strategy:
So far, the benefits of BoNTs have been limited to the treatment of neuromuscular diseases and autonomic nervous system disorders. However, BoNTs can also block neurotransmitter release of central neurons, making them available for use in experimental neuroscience and future neurology dealing with higher brain functions. Studies on brain slices, cultured neurons and synaptosomes demonstrated that BoNTs can stop not only acetylcholine but also the release of neurotransmitters such as glutamate, glycine, noradrenaline, dopamine, serotonin, ATP and various neuropeptides (Ashton) et al. (1988), Capogna, M. et al. (1997), Sanchez-Prieto, J. et al. (1987), Verderio, C. et al. (2004), Luvisetto, S. et al. 2004), and Costatin, L. et al. (2005)). BoNTs light chains are naturally delivered by their partner heavy chains, although alternative delivery means such as liposomes and genetically engineered fusion constructs are also effective (de Paiva, A. et al. (1990)). Chadock, JA et al. (2004), and Duggan, MJ et al. (2002)). Genetically engineered chimeras of lectins with BoNT / A light chains have recently enabled delivery of BoNT / A light chains into nociceptive afferents or dorsal root ganglia (Chaddock, JA et al. (2004)). ). Importantly, delivery effectiveness can be easily tracked if the light chain is fused to a GFP fluorescent label, allowing for the marking of silenced cells. The ability of BoNTs heavy chains to target synapses and transport those light chains to nerve endings provides another tool available in neurobiology (Goodnough, MC et al. (2002) and Bade, S et al. (2004)). Delivery of various molecules, particularly enzymes, using BoNT heavy chains may be feasible for manipulation of synaptic physiology. Indeed, it has recently been demonstrated that BoNT / D can deliver genetically engineered enzymes to nerve endings (Bade, S. et al. (2004)).

The inventors have the following two parts:
A light chain (LC) comprising a translocation domain (HN), wherein the translocation domain carries a SNARE tag (syntaxin, SNAP-25 or synaptobrevin) at its C-terminus; and a SNARE tag (syntaxin, at its N-terminus) The receptor binding part of the heavy chain (HC component) carrying SNAP25 or synaptobrevin),
It was shown that functional botulinum neurotoxin can be obtained by recombining the toxin from

SNARE tags allow for irreversible binding of functional units.
These two parts (LCHN and HC) can be produced separately in a protein-producing strain without fear of compromising health. Each part can be purified by safe methods. When the two parts are mixed, they produce an active neurotoxin within 1 hour. The neurotoxin can also cleave its molecular target SNAP-25 in exposed neurons and block neurotransmitter release from nerve endings.

  Since the LCHN part with SNARE tag is functional (ie, blocking of SNAP-25 nerve cleavage and neurotransmitter release is observed), it is accompanied by the SNARE tag due to irreversible assembly of LCHN / ligand moieties. It is possible to direct this part of action to specific neurons or endocrine cells by adding additional ligands. An example is a somatostatin peptide conjugated to a SNARE tag. The intended binding of the ligand to the cell is due to the transfer of LCHN into the cell with subsequent light chain release and thereby damage to SNAP-25 with subsequent cessation of neurotransmitter or hormone release. Can bring.

  Since the HC component with the SNARE tag is functional (ie, the observation of blockage of neurotransmitter release after binding of the LCHN / SNARE tag donor), SNARE is used for delivery of other enzymes or imaging moieties into the neuron. It is possible to use a tagged HC part. It is also possible to use other receptor binding compounds (eg, somatostatin neuropeptides) with SNARE tags to deliver drugs, imaging reagents, etc. into specific cells carrying the required receptors.

The sequence of LCHN tagged with the SNARE motif and the sequence of HC tagged with the SNARE motif are shown below:
BoNT / A light chain (No. 1-449aa), translocation domain (No. 449-872aa) and cDNA encoding SNARE protein were fused by molecular biology techniques. A thrombin site LVPR-GS (SEQ ID NO: 17) was inserted between the light chain (LC) and translocation domain (HN) of BoNT / A to mimic the natural nicking of the toxin by trypsin (within the previous sequence). "-"). The protein fused to GST was purified as described. The protein was eluted from the beads in 20 mM Hepes, pH 7.3, 100 mM NaCl using thrombin. In the case of synaptobrevin 2 (25-84) BoNT / A Hc (876-1296), 0.8% octyl glucoside was present in the elution buffer. After elution, we obtained a protein with the following sequence:

LcHN-SNAP25: BoNT / ALc (1-449) Thrombin HN (449-872) -SNAP25B (C to A)-SEQ ID NO: 18
LcHN-Syx: BoNT / ALc (1-449) Thrombin HN (449-872) -Syx3 (195-253) -SEQ ID NO: 19
LcHN-Syb: BoNT / ALc (1-449) Thrombin HN (449-872) -Syb2 (1-96; WWK-AAA) -SEQ ID NO: 20

Syb-HcA: Syb2 (25-84) BoNT / A Hc (876-1296) -SEQ ID NO: 21
Syb HcD: Syb2 (25-84) BoNT / D Hc (863-1276) —SEQ ID NO: 22
Nanolocking HcA: Syx3 (195-253) Syb2 (1-84) BoNT / A Hc (876-1296)-SEQ ID NO: 23
SNAP25B (C to A)-SEQ ID NO: 24
Syntaxin 3 (195-253)-SEQ ID NO: 25

In addition to the recombinant protein obtained by bacterial production (all above), the following synthetic peptides were also used:
Syx3 peptide (45aa, 52aa-FITC)
Somatostatin-Synaptobrevin peptide Somatostatin-Syntaxin peptide Somatostatin peptide-SEQ ID NO: 26
Ac-RLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDDRAL-Ahx-Ahx-AGCKNFFFWKTSC-OH.

  In order to bind the catalytic part to the receptor binding part, we include a working part (LcHN-Syx, LcHN-SNAP25 or LcHN-Syb) fused to one SNARE protein and a second SNARE protein. Mixed with receptor binding part for 1 hour. The assembly was locked by the addition of a third SNARE partner. Examples of combinations are shown in the following table:

Example 6
The following example demonstrates the possibility of obtaining a stable SDS-resistant complex by cleaving the N-terminal end of the SNARE motif:
Synaptobrevin 2 complete SNARE motif (N-terminal to C-terminal):
RLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDDRADALQAGASQFETSAAKLA (SEQ ID NO: 27)
The following synthetic synaptobrevin peptides, which are N-terminal truncations of the SNARE motif of synaptobrevin 2, were tested:
1) FITC-Ahx-AQVDEVVDIMRVNVDKVLERDQKLSELDDDRADALQAGASQFETSAAKLA (SEQ ID NO: 28) (49 amino acids);

2) FITC-Ahx-DIMRVNVDKVLERDQKLSELDDDRADALQAGASQFETSAAKLA (SEQ ID NO: 29) (42 amino acids);
3) FITC-Ahx-DKVLERDQKLSELDDRDRADALQAGASQFETSAAKLA (SEQ ID NO: 30) (35 amino acids); and 4) FITC-Ahx-KLSELDRADALQAGASQFETSAAKLA (SEQ ID NO: 31) (27 amino acids).

  FIG. 33 shows that the synaptobrevin SNARE motif can still be reduced to 42 amino acids, so it still forms an SDS resistant complex with syntaxin 1 (stx1) and SNAP25 (S25). The 35 amino acid synaptobrevin SNARE motif also forms a complex that "melts" during gel electrophoresis.

Example 7
As illustrated by the shortening of the syntax SNARE motif, the SNARE motif can also be reduced from both the N- and C-termini. In addition, substitution of internal residues is tolerant to SNARE assemblies for tailor-made applications.

The complete SNARE motif for syntaxin 1:
EIIKLENSIRELHDMFDMDMALVESQGEMIDRIEYNVEHAVDYVERAVSDTKKA (SEQ ID NO: 32).

The following synthetic syntaxin peptides were tested:
1) FITC-Ahx-EIIRLENSIRELHDMFMDMAMLVESQGEMIDRIEYNVEHAVDYVERA- Ahx-KK-NH 2 ( SEQ ID NO: 33) (47 amino acids);
2) Ac-HHHHHH-Ahx-EIIRLENSIRELHDMFDMADMALVESQGEMIDRIEYNVEHAVDYVEC (SEQ ID NO: 34) (45 amino acids);
3) EIIKLENSIRELHDMFDMDMALVESQGEMIDRIEYNVEHAGSGGGHHHHHHC (SEQ ID NO: 35) (40 amino acids);

4) Flu-GGEIIRLENSIRELHDMFDMDMAMLVESQGEMID (SEQ ID NO: 36) (31 amino acids);
5) EIIRLENSIRELHDMFMDMAMLVESTGEMIDRIEYNVEHA-NH ₂ (SEQ ID NO: 37) (40 amino acids); and 6) Bio-NSIRELHDMFMDMAMLVESQGEMIDRIEYNVEHAVDYVEC (SEQ ID NO: 38) (39 amino acids).

  The results are shown in FIG. Syntaxin 1 with 40 amino acids is sufficient for complex formation. A 39 amino acid peptide truncated from both ends and carrying an additional cysteine useful for further modification at the N-terminus forms a stable complex as shown by SDS gel. Note, it is possible to replace the internal lysine of syntaxin (K204) with arginine and still maintain strong complex formation with SNAP25 and synaptobrevin. Such substitution is advantageous if lysine is to be added on the N- or C-terminus for peptide modification or immobilization on the surface.

It is also possible to replace all internal methionines with non-oxidizable norleucine to obtain a stable version of the syntaxin SNARE peptide:
Wild-type (Met) -FITC-Ahx-EIIKLENSIRELHDMFDMADMALVESQGEMIDRIEYNVEHAVDYVE-NH ₂ (SEQ ID NO: 39)
Norleucine peptide-FITC-Ahx-EIIKLENSIRELDnFnDnAnLVESQGEnIDRIEYNVEHAVDYVE-NH ₂ (SEQ ID NO: 40).

  The SDS gel of FIG. 35 shows that comparable assemblies can be made from SNAP-25, synaptobrevin 45aa peptide, and the two syntaxin peptides described above.

Example 8
Many combinations of SNARE motifs are available for complex formation systems. As visualized in the SDS gel shown in FIG. 36, examples of stable SDS-resistant complexes and their melting temperatures (Tm) were calculated using recombinant syntaxins 1 and 3, SNAP23 and SNAP25, and VAMPs 2, 4, Provided for SNARE complexes made of 5, 7 and 8.
The strongest complex (higher melting temperature, Tm) is obtained using syntaxin 1, VAMP2 and either SNAP25 or SNAP23.

The amino acid sequences of additional SNARE isoforms produced by bacteria and used here are as follows:
SNAP-23-SEQ ID NO: 41
VAMP4-SEQ ID NO: 42
VAMP5-SEQ ID NO: 43
VAMP7-SEQ ID NO: 44
VAMP8—SEQ ID NO: 45.

Example 9A
The complexing system binds three distinct truncated polypeptide SNARE motifs.
Recombinant synaptobrevin (VAMP2) fused to glutathione-S-transferase can bind to 40 and 45 amino acid syntaxin 1 and SNAP25 peptides. The SNAP25 peptide is designated as helix 1 (S25H1) or helix 2 (S25H2).
Syxl 45aa: SEQ ID NO: 46;
Syxl 40aa: SEQ ID NO: 47;
S25H1 45aa: SEQ ID NO: 48;
S25H1 40aa: SEQ ID NO: 49;
S25H2 45aa: SEQ ID NO: 50; and S25H2 40aa: SEQ ID NO: 51.
The results are shown in FIG. 37, in which the lane marked + contains samples boiled in SDS and the lane marked-contains samples boiled in SDS.

Example 9B
The recombinant S25H2 protein can bind to 40 and 45 amino acid syntaxin 1, VAMP2, and S25H1 peptide.
S25H1 45aa: SEQ ID NO: 48;
S25H1 40aa: SEQ ID NO: 49;
Syxl 45aa: SEQ ID NO: 46;
Syxl 40aa: SEQ ID NO: 47;
Syb2 45aa: SEQ ID NO: 52; and Syb2 40aa: SEQ ID NO: 53.
The results are shown in FIG. 38, in which the lane marked + contains samples boiled in SDS and the lane marked-contains samples boiled in SDS.

Example 9C
The recombinant S25H1 protein can bind to 40 and 45 amino acid syntaxin 1, VAMP2, and S25H2 peptides.
S25H2 45aa: SEQ ID NO: 50;
S25H2 40aa: SEQ ID NO: 51;
Syxl 45aa: SEQ ID NO: 46;
Syxl 40aa: SEQ ID NO: 47;
Syb2 45aa: SEQ ID NO: 52; and Syb2 40aa: SEQ ID NO: 53.
The results are shown in FIG. 39, in which the lane marked + contains the sample boiled in SDS and the lane marked-contains the sample not boiled in SDS.

Example 10
Neuropeptide complex formation can be achieved in various combinations.
The following peptides are used to provide examples:
Syntax tag-somatostatin: (SEQ ID NO: 54)
Ac-EIIKLENSIRELDFMDMDMAMLVESQGEMIDRIEYNVEHA-Ahx-Ahx-AGCKNFFFWKTSC-OH
Brevin Tag-Somatostatin: (SEQ ID NO: 55)
Ac-RLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDDRAL-Ahx-Ahx-AGCKNFFFWKTSC-OH

Syntaxin tag-Substance P: (SEQ ID NO: 56)
Ac-EIIKLENSIRELHDMFDMDMAMLVESQGEMIDRIEYNVEHAVDYVE-Ahx-Ahx-RPKPQQFFGLM-NH ₂
Brevin Tag-Substance P: (SEQ ID NO: 57)
Ac-RLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGAS-Ahx-Ahx- RPKPQQFFGLM-NH 2.

  Combining these peptides provides an alternative stable combination as illustrated in FIG. In FIG. 40, lanes 1 and 2: Brevin tag-substance P was mixed with syntaxin tag-somatostatin in the presence of SNAP25. The stable complex contains both substance P and somatostatin (heterodimeric peptide). Lanes 3 and 4: Brevin tag-somatostatin was mixed with syntaxin tag-somatostatin in the presence of SNAP25. The stable complex contains two somatostatins (homodimeric peptides). Note, lanes 1 and 3 showed stable complexes using tagged neuropeptides.

Example 11
Neuropeptides can bind to other functional groups, such as botulinum neurotoxin parts, in different orientations that can affect binding to cell surface receptors and translocation of the peptide. In this example, the following sequence was used:
45aa syntaxin motif-arginine / vasopressin peptide (AVP) (SEQ ID NO: 58)
Ac-AEIIKLENSIRELDMFDMDMAMLVESQGEMIDRIEYNVEHAVDYVER-Ahx-Ahx-CYFQNCPRG-NH ₂
Arginine / vasopressin peptide-45aa syntaxin motif (SEQ ID NO: 59)
CYFQNCPRG-Ahx-Ahx-AEIIKLENSIRELHDMFDMDMAMLVESQGEMIDRIEYNVEHAVDYVE-NH ₂

Combining these peptides with the LcHn part of botulinum toxin A provides an alternative stable combination visualized on the SDS gel of FIG.
Lanes 1 and 2: Syntaxin-AVP was incubated with LcHn-brevin in the presence of SNAP-25 for 60 minutes. The stable complex contains LcHn-SNARE-AVP (AVP is present at the C-terminal end of the SNARE linker).
Lanes 3 and 4: AVP-syntaxin was incubated with LcHn-brevin for 60 minutes in the presence of SNAP-25. The stable complex contains LcHn-AVP-SNARE (AVP is present at the N-terminal end of the SNARE linker).

Example 12
Even peptides shorter than 40 can still form stable complexes (but not SDS resistance). Array used:
Conjugation of biotin -Ahx-EIIRLENSIRELHDMFMDMAMLVESQG-NH ₂ (SEQ ID NO: 60) -27aa Syntaxin 1 peptide Biotin -Ahx-EIIKLENSIRELHDMFMDMAMLVESQGEMID-NH ₂ (SEQ ID NO: 61) -31aa Syntaxin peptide Peptides were immobilized on glutathione beads GST -Observed in pull-down experiments using SNAP25 linker, synaptobrevin protein (triple helix construct). Binding is for 30 minutes at 25 ° C. followed by washing in buffer A (20 mM HEPES, 100 mM NaCl). Proteins and peptides were visualized on an SDS gel (Figure 42). Biotinylated peptide bound to the beads can be seen at the bottom of the SDS gel.

Example 13
A SNARE peptide can chemically crosslink a protein when the protein cannot be expressed by genetic recombination. The peptides retain their ability to form SNARE complexes, and the modified proteins retain their activity. Array used:
45aa syntaxin 1 peptide Ac-AEDAEIIKLENSIRELHDMFDMADMALVESQGEMIDRIEYNVEHAVDYVEC (SEQ ID NO: 62)

The previous peptide was cross-linked to Maleimide-Horse Radish Peroxidase (Sigma-Aldrich Co.).
FIG. 43 is an SDS gel showing maleimide-based crosslinking.
Cross-linked syntaxin-HRP can bind to a GST-triple helix (Snap25-brevin, SB) immobilized on glutathione beads. The results are shown in FIGS. 44 and 45. FIG. 44 shows the absorbance at 650 nm of the TMB substrate. FIG. 45 shows the emission of light visualized by emission on the film. Note, control glutathione beads without GST-triple helix (SB) show only syntaxin-HRP background binding.

Example 14
The 29 amino acid complexin 1 peptide can interact with the SNARE assembly. This peptide can be used for purification after its immobilization or as an additional carrier in SNARE-based assemblies.
Complexin peptide:
Ac-ERKAKYAKMEAEREVMRQGIRDKYGIKKGSGSGGIKVAV-NH ₂ (SEQ ID NO: 63).

FIG. 46 is an SDS gel showing complexin peptide pull-down by following the protein immobilized on Ni2 + beads.
A. Full length HIS-SNAP25 (no SNARE complex)
B. Full length HIS-SNAP25 + full length syntaxin 1 + full length synaptobrevin 2
C. Full length HIS-SNAP25 + full length syntaxin 1 + synaptobrevin 2 45aa peptide Full length HIS-SNAP25 + syntaxin 1 45aa peptide + full length synaptobrevin 2
E. Full length HIS-SNAP25 + syntaxin 1 45aa peptide + synaptobrevin 2 45aa peptide.
In particular, lane E represents the product in which one recombinant protein binds to three synthetic peptides, one of which is a complexin peptide.

Example 15
SNARE assembly can be achieved in the presence of serum. In certain applications, SNARE-based drugs will require new interactions after injection into the blood. GST-triple helix protein (SNAP-25, SB protein conjugated to synaptobrevin) immobilized on glutathione beads with 45 amino acid syntaxin 1 peptide with FITC fluorescent tag in the presence of 100% calf serum Tested for binding. The results are shown in FIG. Note, the presence of the triple helix protein on the bead results in pull-down of the syntaxin fluorescent peptide in the presence of serum. The vertical axis represents relative fluorescence units.
References:

Claims (70)

A complex-forming system for forming a molecular scaffold comprising:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein the four polypeptide helices can form a stable SNARE complex, and wherein the polypeptide helix derived from syntaxin is integrated with the polypeptide helix derived from synaptobrevin or a homologue thereof. The complex formation system. A complex-forming system for forming a molecular scaffold comprising:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein the four polypeptide helices can form a stable SNARE complex, and wherein the polypeptide helices are united to form two helix-containing components. Body forming system.   The system of claim 1, wherein two of the helices are integrated such that both helices can assemble into the same stable SNARE complex.   4. The system of claim 3, wherein the other two helices are integrated so that both helices can assemble into the same stable SNARE complex.   The system of claim 2, wherein three of the helices are integrated such that all three helices can assemble into the same stable SNARE complex.   6. The two polypeptide helices derived from the SNAP protein and one of: a polypeptide helix derived from syntaxin; or a polypeptide helix derived from synaptobrevin or a homologue thereof. system.   The system according to claim 1, wherein one of the helices is immobilized on a substrate.   The system according to any one of claims 1 to 7, wherein the SNAP protein is SNAP-25.   9. The system according to any one of claims 1 to 8, wherein the polypeptide helix is derived from synaptobrevin.   10. The system of any one of claims 1-9, wherein each helix is at least about 40 amino acids in length.   11. A system according to any one of the preceding claims, wherein each sequence of the polypeptide helix has at least about 80% identity with the sequence of the protein or part of the protein from which the polypeptide helix was obtained. .   12. A system according to any one of claims 1 to 11, comprising a cargo moiety comprising a light chain of a botulinum toxin or a functional part thereof and a translocation part of a heavy chain of the botulinum toxin.   13. A system according to any one of claims 1 to 12, comprising a receptor binding part of the heavy chain of botulinum toxin, or a cargo moiety comprising a somatostatin peptide or a functional part thereof.   First and second cargo moieties linked to separate helices or helix-containing components, wherein the first cargo moiety is a translocation of a botulinum toxin light chain or functional portion thereof and a botulinum toxin heavy chain. 14. A system according to any one of the preceding claims comprising a seat and wherein the second cargo moiety comprises a receptor binding part of a heavy chain of botulinum toxin or a somatostatin peptide or a functional part thereof.   15. The system of claim 14, wherein the second cargo moiety comprises a receptor binding portion of a botulinum toxin heavy chain.   16. The system of any one of claims 1-15, further comprising a single polypeptide helix derived from a complexin capable of binding to the SNARE complex.   17. A system according to any one of the preceding claims, further comprising a surfactant.   18. The surfactant is selected from MEGA8, C-HEGA10, C-HEGA11, HEGA9, heptylglucopyranoside, octylglucopyranoside, nonylglucopyranoside, zwittergent 3-08, zwittergent 3-10 and zwittergent 3-12. The system described.   19. A system according to claim 17 or 18, wherein the surfactant is octyl glucopyranoside.   3. A system according to claim 1 or 2, wherein two of the helices are integrated such that both helices cannot assemble into the same NARE complex.   3. A system according to claim 1 or 2, wherein two of the helices are integrated, wherein the system is derived from the same protein as one of the two integrated helices. The system further comprising a single polypeptide helix.   The system of claim 21, wherein the single polypeptide helix is immobilized on a substrate.   23. A multimer produced by the system of any one of claims 20-22.   Use of the complex-forming system according to any one of claims 1 to 22.   Use of the complex-forming system according to any one of claims 1 to 22 in diagnosis. A device having a stable SNARE complex immobilized thereon, wherein the SNARE complex is:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein said apparatus is selected from arrays, assays, microfluidic devices, SPR instruments, QCM instruments, mass spectrometers, electrophoresis instruments, chromatography columns, scanning probe microscopes, and calorimetric instruments. A method of forming a SNARE complex carrying one or more cargo moieties comprising the following steps:
Stable SNARE complex by combining two polypeptide helices derived from SNAP protein, one polypeptide helix derived from syntaxin, and one polypeptide helix derived from synaptobrevin or its homologue. Form the body,
Wherein one or more cargo moieties are attached to the polypeptide helix and wherein:
(I) linking a polypeptide helix derived from syntaxin to a polypeptide helix derived from synaptobrevin or a homolog thereof; or (ii) integrating the polypeptide helices to form two helix-containing components. Said method.   A component for forming a molecular scaffold comprising a polypeptide helix derived from syntaxin linked to a polypeptide helix derived from synaptobrevin or a homologue thereof, wherein the two linked helices are stable Said component capable of forming part of a SNARE complex.   29. The component of claim 28, comprising a sequence selected from SEQ ID NOs: 12, 13, 14, and 73.   A component for forming a molecular scaffold comprising two polypeptide helices derived from a SNAP protein and either a polypeptide helix derived from syntaxin or a polypeptide helix derived from synaptobrevin, wherein Said component in which three helices can be integrated to form a triple helix component and where the three linked helices can form part of a stable SNARE complex.   32. The component of claim 30, comprising the sequence of SEQ ID NO: 15.   32. Use of a component according to any one of claims 28-31.   A kit comprising a component comprising a polypeptide helix derived from syntaxin linked to a polypeptide helix derived from synaptobrevin or a homologue thereof, wherein the two linked helices are stable SNARE complexes Said kit which can form a part of.   34. The kit of claim 33, further comprising two polypeptide helices derived from a SNAP protein capable of forming a stable SNARE complex with a syntaxin / synaptobrevin derived helix.   A kit comprising a component comprising two polypeptide helices derived from a SNAP protein and either a polypeptide helix derived from syntaxin or a polypeptide helix derived from synaptobrevin, wherein the three helices Wherein the three helixes linked together can form part of a stable SNARE complex.   And further comprising a single polypeptide helix derived from a fourth SNARE protein that is either synaptobrevin or a homologue thereof or syntaxin, wherein the single polypeptide helix comprises a stable SNARE complex with a triple helix component. 36. The kit of claim 35, which can be formed. A complex-forming system for forming a molecular scaffold comprising:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein the four polypeptide helices are capable of forming a stable SNARE complex, wherein the SNARE complex is formed in the presence of a surfactant.   38. The system of claim 37, wherein the system does not comprise a Munc18 protein.   Use of detergents to promote assembly of SNARE complexes in the absence of Munc18 protein. A multimer comprising a plurality of stable SNARE complexes linked together, wherein each SNARE complex has the following:
Two polypeptide helices derived from the SNAP protein;
A polypeptide helix derived from syntaxin; and a polypeptide helix derived from synaptobrevin or a homologue thereof,
Where a helix from one SNARE complex is linked to a helix from another SNARE complex to integrate the SNARE complexes, and wherein one or more cargo moieties are Said multimer bound to a polypeptide helix.   41. The multimer of claim 40, further comprising a branch joining three helices from a SNARE complex to helices in three different SNARE complexes. The following steps:
1) providing a first polypeptide helix derived from a first SNARE helix;
2) The second and third polypeptide helices are joined to the first polypeptide helix to form a triple helix complex, wherein the second and third polypeptide helices are second and third Derived from the SNARE helix of
3) A fourth polypeptide helix is attached to the three helix bundle to form a stable SNARE complex, where the fourth polypeptide helix is derived from the fourth SNARE helix, and A fourth polypeptide helix is linked to a fifth polypeptide helix derived from the first SNARE helix; and 4) repeating steps 2) and 3) to form a multimer, wherein 1 Or linking multiple cargo moieties to a polypeptide helix,
A method of producing a multimer comprising:   43. The method of claim 42, wherein the uniqueness of the second, third, fourth and fifth polypeptide helices is maintained in an iterative step.   44. The method of claim 42 or 43, wherein the second and third polypeptide helices are linked together so that they can be assembled together in the same SNARE complex.   45. The method of any one of claims 42 to 44, wherein the first polypeptide helix is immobilized on a substrate.   46. A method according to any one of claims 42 to 45, further comprising a washing step after each binding step to remove all unbound helixes.   Branching is introduced into the multimer by using a sixth polypeptide helix derived from the first SNARE helix, wherein the sixth polypeptide helix is introduced into the second, third, fourth or fifth polyhelix. 47. A method according to any one of claims 42 to 46, wherein the method is coupled to one of the peptide helices.   48. A multimer produced by the method of any one of claims 42 to 47. A complex-forming system for forming a binary compound comprising two cargo moieties, comprising:
Two polypeptide helices derived from the SNAP protein;
A polypeptide helix derived from syntaxin; and a polypeptide helix derived from synaptobrevin or a homologue thereof,
Wherein the four polypeptide helices can form a stable SNARE complex, wherein the first cargo moiety is attached to the first helix and the second cargo moiety is the second Said complex-forming system that binds to two separate helices, where formation of a SNARE complex causes formation of a binary compound.   50. The complex forming system of claim 49, wherein the binary component is a toxin.   51. The complex forming system of claim 50, wherein the toxin is selected from botulinum toxin, diphtheria toxin, tetanus toxin and ricin.   52. The complex forming system of claim 51, wherein the toxin is a botulinum toxin. below:
Two polypeptide helices derived from the SNAP protein;
A polypeptide helix derived from syntaxin; and a polypeptide helix derived from synaptobrevin or a homologue thereof,
Wherein the four polypeptide helices can form a stable SNARE complex, wherein the first cargo moiety is attached to the first helix and the second cargo moiety is the second Bound to two separate helices, wherein the first cargo moiety comprises a botulinum toxin light chain or functional portion thereof and a botulinum toxin heavy chain translocation, and a second cargo moiety 53. The complex forming system of claim 52, wherein said botulinum toxin comprises a heavy chain receptor binding portion of botulinum toxin.   54. Use of the complex-forming system according to any one of claims 49 to 53.   54. Complex formation system according to any one of claims 50 to 53 for use in therapy.   52. The complexing system of claim 51, wherein the toxin is selected from diphtheria toxin and ricin for use in the treatment of neoplastic diseases.   54. A complexing system according to claim 52 or 53 for use in the treatment of a disease or physical condition that is alleviated by inhibition of neural synapses.   Diseases or physical conditions that are alleviated by inhibition of nerve synapses are hyperhidrosis, hypersalivation, dystonia, gastrointestinal disorders, urological disorders, facial spasms, strabismus, cerebral palsy, stuttering, chronic tension headache, hyper lacrimal secretion, multiple 58. The complex forming system of claim 57 selected from the group consisting of sweating, hypopharyngeal constrictor spasm, spastic bladder, pain, migraine, and cosmetic surgery such as wrinkle removal, forehead wrinkle removal, etc. .   54. A method of treating a disease or physical condition that is alleviated by inhibition of neural synapses, comprising the administration of an effective amount of a composition comprising the system of claim 52 or 53. A method of forming a SNARE complex to form a binary compound comprising two cargo moieties, comprising the following steps:
Two polypeptide helices derived from SNAP protein, one polypeptide helix derived from syntaxin, and one polypeptide helix derived from synaptobrevin or a homologue thereof are integrated into a stable SNARE complex. Form,
Wherein the first cargo portion is bound to the first helix and the second cargo portion is bound to the second separate helix, wherein the formation of the SNARE complex is Said method causing the formation of a binary compound.   61. The method of claim 60, wherein the binary component is a toxin.   62. The method of claim 61, wherein the toxin is selected from botulinum toxin, diphtheria toxin, tetanus toxin and ricin.   64. The method of claim 62, wherein the toxin is a botulinum toxin. The following steps:
Two polypeptide helices derived from SNAP protein, one polypeptide helix derived from syntaxin, and one polypeptide helix derived from synaptobrevin or a homologue thereof are integrated into a stable SNARE complex. Form,
Wherein the first cargo portion is coupled to the first helix and the second cargo portion is coupled to the second separate helix, wherein the first cargo portion is botulinum. 64. A toxin light chain or functional portion thereof and a botulinum toxin heavy chain translocation, and wherein the second cargo moiety comprises a botulinum toxin heavy chain receptor binding portion. A method for forming the described botulinum toxin.   A polypeptide helix derived from SNAP protein; syntaxin; or synaptobrevin or a homologue thereof, wherein the polypeptide helix comprises a light chain of a botulinum toxin or a functional part thereof and a translocation of a heavy chain of the botulinum toxin A component for forming a botulinum toxin, which binds a cargo moiety comprising:   A polypeptide helix derived from SNAP protein; syntaxin; or synaptobrevin or a homologue thereof, wherein the polypeptide helix binds a cargo moiety comprising the receptor binding portion of the heavy chain of botulinum toxin Ingredients for forming botulinum toxin.   Use of the component according to claim 65 or 66.   Comprising two polypeptide helices derived from SNAP protein; one polypeptide helix derived from syntaxin; and one polypeptide helix derived from synaptobrevin or a homologue thereof, wherein The peptide helix forms a stable SNARE complex, wherein the first cargo moiety is bound to the first helix and the second cargo moiety is bound to the second separate helix, and Wherein the first cargo portion comprises a botulinum toxin light chain or functional portion thereof and a botulinum toxin heavy chain translocation, and the second cargo portion comprises a botulinum toxin heavy chain receptor binding portion. A kit comprising. A complex-forming system for forming a molecular scaffold comprising:
Two polypeptide helices derived from the SNAP protein;
One polypeptide helix derived from syntaxin;
A polypeptide helix derived from synaptobrevin or a homologue thereof; and one or more cargo moieties attached to the polypeptide helix;
Wherein the four polypeptide helices form a stable SNARE complex, and wherein at least two of the polypeptide helices are less than 50 amino acids in length Formation system.   A method of forming a SNARE complex carrying one or more cargo moieties, comprising the following steps: two polypeptide helices derived from SNAP protein, one polypeptide helix derived from syntaxin, and synaptobrevin Combining a single polypeptide helix derived from the homologue to form a stable SNARE complex, wherein one or more cargo moieties are attached to the polypeptide helix, and wherein The method, wherein at least two of the polypeptide helices are less than 50 amino acids in length.

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