RU2824389C2 - Genetically engineered cells sensitive to clostridial neurotoxins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a cell containing an exogenous nucleic acid, which provides expression or overexpression of a clostridial neurotoxin receptor or a variant thereof, or a fragment thereof, which has the ability to bind clostridial neurotoxin, an exogenous nucleic acid which provides expression or overexpression of a ganglioside having a clostridial neurotoxin binding affinity, and an exogenous nucleic acid which provides expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin, as well as a method for production thereof.

EFFECT: invention is effective for identifying the composition of Clostridial neurotoxin for therapeutic and cosmetic use, for characterizing the activity of the composition of Clostridial neurotoxin, as well as for analysis for determining activity of modified or recombinant neurotoxin.

42 cl, 13 dwg, 12 ex

Description

FIELD OF INVENTION

The present invention generally relates to a cell that has been genetically engineered to have increased sensitivity to a clostridial neurotoxin, such as botulinum neurotoxin and tetanus neurotoxin. The invention also relates to a method for producing such a cell and a method for using such a cell in assaying the activity of polypeptides derived from such neurotoxins, such as modified and recombinant versions of such clostridial neurotoxins.

PRIOR ART

The anaerobic Gram-positive bacterium Clostridium botulinum produces various types of neurotoxins, including botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT).

BoNTs are the most potent known toxins, with median lethal dose (LD ₅₀ ) values in mice ranging from 0.5 to 5 ng/kg, depending on the serotype. BoNTs are adsorbed in the gastrointestinal tract and, upon entering the bloodstream, bind to the presynaptic membrane of cholinergic nerve endings and inhibit the release of the neurotransmitter acetylcholine.

BoNT is well known for its ability to induce flaccid muscle paralysis. These muscle relaxant properties have led to the use of BoNT in a number of medical and cosmetic procedures, including the treatment of glabellar frown lines or hyperkinetic facial lines, headache, hemifacial spasm, overactive bladder, hyperhidrosis, nasolabial folds, cervical dystonia, blepharospasm, and spasticity.

There are currently at least eight distinct classes, each with similar structures and modes of action. The different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and this serotype classification correlates with percent sequence identity at the amino acid level. BoNT proteins of a given serotype are classified into different subtypes based on percent amino acid sequence identity.

The different BoNT serotypes differ in the species affected with respect to the severity and duration of paralysis caused. For example, BoNT/A is the most lethal of all known biological agents and, with respect to paralysis, it is 500 times more potent in the rat than BoNT/B. In addition, the duration of paralysis following BoNT/A injection in mice is ten times longer than that following BoNT/E injection.

In nature, clostridial neurotoxins are synthesized as a single-chain polypeptide that is post-translationally modified by proteolytic cleavage to form two polypeptide chains linked by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, located between the cysteine residues that mediate the interchain disulfide bond. It is this double-chain form that is the active form of the toxin. The two chains are referred to as the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain includes a C-terminal targeting component, known as the "targeting group", and an N-terminal translocation component, known as the "translocation domain". The cleavage site is located between the L chain and the translocation component in the open loop region. Following binding of the targeting moiety to its target neuron and internalization of the bound toxin into the cell via an endosome, the translocation domain translocates the L chain across the endosomal membrane into the cytosol.

The L chain includes a protease component known as the "protease domain". It has a non-cytotoxic function and acts by proteolytically cleaving intracellular transport proteins known as SNARE proteins, see Gerard K (2002) "Cell and molecular Biology " ( ^4th edition), John Wiley & Sons, Inc . The acronym SNARE comes from the term Soluble NSF Attachment Receptor , where NSF stands for N -ethylmaleimide Sensitive Factor. The protease domain has zinc - dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins.

Through their respective protease domains, different clostridial neurotoxins cleave different SNARE proteins. BoNT/B, BoNT/D, BoNT/F, BoNT/G, and TeNT cleave synaptobrevin, also known as vesicle-associated membrane protein (VAMP). BoNT/A, BoNT/C, and BoNT/E cleave synaptosome-associated protein of 25 kDa (SNAP-25). BoNT/C cleaves syntaxin.

SNARE proteins are associated with either the secretory vesicle membrane or the cell membrane and facilitate the exocytosis of molecules by mediating the fusion of the secretory vesicle with the cell membrane, thereby allowing the vesicle contents to escape from the cell. Cleavage of such SNARE proteins inhibits such exocytosis and thus suppresses neurotransmitter release from such neurons. As a result, striated muscles are paralyzed and sweat glands cease secreting.

Thus, once delivered to the desired target cell, clostridial neurotoxins are capable of inhibiting cellular secretion from the target cell.

Modification of clostridial neurotoxins to alter their properties is known in the art. Modifications may include amino acid modifications, such as addition, deletion, and/or substitution of amino acid(s), and/or chemical modifications, such as addition of a phosphate or carbohydrate, or formation of disulfide bonds. Modification may also include rearrangement of components of the clostridial neurotoxin, such as flanking a protease component with a translocation component and a targeting component.

It is also known in the art to produce recombinant clostridial neurotoxins that are either genetically identical to a neurotoxin from Clostridia or differ from wild-type clostridial neurotoxins in that they contain fewer amino acids, additional or different amino acids, and/or components arranged in a different order than they are arranged in wild-type clostridial neurotoxins. These recombinant clostridial neurotoxins can also be chemically modified as described above.

However, differences between modified and recombinant clostridial neurotoxins and their wild-type equivalents may affect the desired SNARE protein cleavage property of the neurotoxin. Thus, it may be important to determine whether such differences enhance, reduce, or eliminate such activity.

Various conventional assays are available that allow those skilled in the art to confirm whether these modified or recombinant clostridial neurotoxins possess the desired activity in cleaving the target SNARE protein. These assays involve testing for the presence of products resulting from cleavage of the SNARE protein. For example, after contacting a cell with a modified or recombinant neurotoxin, the cell can be lysed and analyzed by SDS-PAGE to detect the presence of cleavage products. Alternatively, cleavage products can be detected by contacting the cell lysate with antibodies.

Although natural cells can be used in such assays, because such natural cells may have only limited sensitivity to clostridial neurotoxins, the assays typically require the use of high concentrations of such cells. In addition, it is desirable for such assays to use a clonal stable cell line.

Thus, there is a need for a genetically engineered cell that has increased sensitivity to clostridial neurotoxins for use in such assays.

ESSENCE OF THE INVENTION

The present invention relates in part to a cell that has been genetically engineered to express or overexpress a clostridial neurotoxin receptor or a variant or fragment thereof. Such a receptor may be a protein receptor or a ganglioside. Preferably, a cell as described herein (whether with respect to a cell as such as described herein or any method or use that involves a cell as described herein) comprises an exogenous nucleic acid that provides for the expression or overexpression (preferably, overexpression) of the receptor and/or ganglioside, wherein said exogenous nucleic acid is under the control of a constitutive and/or inducible promoter (preferably, a constitutive promoter).

The invention also partially relates to a method for producing such a cell. The method includes introducing into the cell a nucleic acid encoding a clostridial neurotoxin receptor or a variant or fragment thereof that has the ability to bind a clostridial neurotoxin, and/or an enzyme of the ganglioside synthesis pathway, or a variant or fragment thereof that has the catalytic activity of such an enzyme.

The invention also relates in part to an assay for determining the activity of a modified or recombinant neurotoxin. The method comprises contacting the above-mentioned cell with a modified or recombinant neurotoxin under conditions and for a period of time that allow the protease domain of a wild-type clostridial neurotoxin to cleave an indicator protein in the cell, and determining the presence of a product resulting from the cleavage of such an indicator protein.

The invention includes methods for testing/evaluating the activity of a batch of a clostridial neurotoxin for therapeutic/cosmetic use. Such methods are advantageously used in monitoring the activity of the toxin during storage and in tracking the activity over time. Another advantage is the ability to determine optimal storage conditions (e.g., under which the level of activity does not decrease). These methods are particularly useful for characterizing the activity (e.g., cell binding/SNARE cleavage ability) of recombinant clostridial neurotoxins.

One aspect of the invention relates to an in vitro method for characterizing the activity of a clostridial neurotoxin (preferably BoNT) composition or identifying a clostridial neurotoxin (preferably BoNT) composition for therapeutic (and/or cosmetic) use, said method comprising:

a. creating a cell (e.g., a genetically engineered cell) having an exogenous nucleic acid that provides for the expression or overexpression of a receptor and/or ganglioside (preferably a receptor) having a binding affinity for a clostridial neurotoxin, and an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein (preferably a SNARE domain-containing indicator protein) that is cleaved by a clostridial neurotoxin; preferably, wherein the cell does not express the receptor and/or ganglioside (preferably a receptor) in the absence of said exogenous nucleic acid;

b. contact of the said cell with a clostridial neurotoxin composition;

c. comparing the level of cleavage of the indicator protein after exposure (e.g. administration) to the clostridial neurotoxin formulation with the level of cleavage prior to exposure to the clostridial neurotoxin formulation; and

d. identification of (i) a composition of a clostridial neurotoxin as suitable for therapeutic (and/or cosmetic) use where the level of cleavage of an indicator protein is increased after contact, or identification of (ii) the presence of activity where the level of cleavage of an indicator protein is increased after contact; or

e. identification of (i) a clostridial neurotoxin composition as unsuitable for therapeutic (and/or cosmetic) use when the level of indicator protein cleavage does not increase after contact, or identification of (ii) a lack of activity when the level of indicator protein cleavage does not increase after contact.

Another aspect of the invention relates to an in vitro method for characterizing the activity of a clostridial neurotoxin (preferably BoNT) composition or determining a clostridial neurotoxin (preferably BoNT) composition that is suitable for therapeutic (and/or cosmetic) use, said method comprising:

a. operating a cell (e.g., a genetically engineered cell) to insert an exogenous nucleic acid that provides for the expression or overexpression of a receptor and/or ganglioside (preferably a receptor) having binding affinity for a clostridial neurotoxin, and an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein (preferably a SNARE domain-containing indicator protein) cleaved by a clostridial neurotoxin; preferably, wherein the cell does not express the receptor and/or ganglioside (preferably a receptor) in the absence of said exogenous nucleic acid;

b. contact of the said cell with a clostridial neurotoxin composition;

c. comparing the level of cleavage of the indicator protein after exposure (e.g. administration) to the clostridial neurotoxin formulation with the level of cleavage prior to exposure to the clostridial neurotoxin formulation; and

d. identification of (i) a clostridial neurotoxin as suitable for therapeutic (and/or cosmetic) use, where the level of cleavage of an indicator protein is increased after contact, or identification of (ii) the presence of activity, where the level of cleavage of an indicator protein is increased after contact; or

e. identification of (i) a clostridial neurotoxin as unsuitable for therapeutic (and/or cosmetic) use when the level of indicator protein cleavage does not increase after contact, or identification of (ii) a lack of activity when the level of indicator protein cleavage does not increase after contact.

Another aspect of the invention relates to an in vitro method for characterizing the activity of a clostridial neurotoxin (preferably BoNT) composition or determining a clostridial neurotoxin (preferably BoNT) composition that is suitable for therapeutic (and/or cosmetic) use, said method comprising:

a. creating a cell (e.g., a genetically engineered cell) having an exogenous nucleic acid that provides for the expression or overexpression of a receptor and/or ganglioside (preferably a receptor) having a binding affinity for a clostridial neurotoxin, and an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein (preferably a SNARE domain-containing indicator protein) that is cleaved by a clostridial neurotoxin; preferably, wherein the cell does not express the receptor and/or ganglioside (preferably a receptor) in the absence of said exogenous nucleic acid;

b. contact of the said cell with a clostridial neurotoxin composition;

c. comparing the level of cleavage of the indicator protein after exposure (e.g. administration) to the clostridial neurotoxin formulation with the level of cleavage prior to exposure to the clostridial neurotoxin formulation; and

d. identification of (i) a Clostridial neurotoxin formulation as suitable for therapeutic (and/or cosmetic) use where the level of cleavage of the indicator protein is increased or equivalent to the level of cleavage following contact with a control Clostridial neurotoxin formulation, or identification of (ii) the presence of activity where the level of cleavage of the indicator protein following contact is increased or equivalent to the level of cleavage following contact with a control Clostridial neurotoxin formulation; or

e. identification of (i) a clostridial neurotoxin as unsuitable for therapeutic (and/or cosmetic) use when the level of cleavage of the indicator protein after contact is not increased or is not equivalent to the level of cleavage after contact with a control formulation of the clostridial neurotoxin, or identification of (ii) lack of activity when the level of cleavage of the indicator protein after contact is not increased or is not equivalent to the level of cleavage after contact with a control formulation of the clostridial neurotoxin.

The control formulation of the clostridial neurotoxin is a formulation of the clostridial neurotoxin (e.g. a batch of the clostridial neurotoxin) which is known to have cleavage activity and/or is known to be suitable for therapeutic (and/or cosmetic) use (in other words, the control is a positive control). Preferably, the (test) formulation of the clostridial neurotoxin and the control formulation of the neurotoxin are formulations of the clostridial neurotoxin of the same type/serotype, e.g. where the (test) formulation of the clostridial neurotoxin is a BoNT/E formulation, the control clostridial neurotoxin is preferably also a BoNT/E formulation.

Another aspect of the invention relates to a method for constructing a cell suitable for use in an assay for characterizing the activity of a clostridial neurotoxin (preferably BoNT) or determining the composition of a clostridial neurotoxin (preferably BoNT) that is suitable for therapeutic (and/or cosmetic) use, the method comprising:

working with the insertion cell:

(i) an exogenous nucleic acid that provides for the expression or overexpression of a receptor and/or ganglioside having a binding affinity for a clostridial neurotoxin, preferably wherein the cell does not express the receptor and/or ganglioside (preferably the receptor) in the absence of said exogenous nucleic acid; and

(ii) an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein (preferably a SNARE domain-containing indicator protein) that is cleaved by a clostridial neurotoxin.

The following are optional embodiments of any aspect of the invention described herein.

In one embodiment, the cell typically does not express a receptor and/or ganglioside (preferably a receptor, preferably wherein the receptor is SV2A). In other words, in a preferred embodiment, the cell does not express a receptor and/or ganglioside (preferably a receptor, preferably wherein the receptor is SV2A) in the absence of said exogenous nucleic acid.

In one embodiment, the cell is from a cell type that is different from the natural target of the clostridial neurotoxin. In other words, in one embodiment, the cell is not a natural target of the clostridial neurotoxin. For example, the cell may not be a natural target of BoNT/A. Additionally or alternatively, the cell may not be a natural target of BoNT/E.

In one embodiment, the cell is not a nerve cell (e.g., a neuron).

By expressing or overexpressing a receptor and/or a ganglioside having binding affinity for a clostridial neurotoxin, there is an advantage in that the methods and cells of the invention can reduce false negative results (e.g., where low cleavage activity would be incorrectly detected as a result of low clostridial neurotoxin binding affinity and translocation into the cell used in the assay, rather than as a result of low protease domain activity). In addition, the invention relates to a broader range of cell types that can be used in such assays, reducing reliance on, for example, neural cells (e.g., naturally expressing sufficient levels of the receptor/ganglioside), which may be difficult to culture in vitro . These advantages are provided in the context of a cell-based assay (allowing binding, translocation, and protease activity to be characterized) as opposed to a cell-free system (which only characterizes protease activity).

The term "when the level of cleavage of the indicator protein does not increase after contact" means that there is essentially no increase in the level of cleavage. As used herein, the term "essentially" in relation to the term "when the level of cleavage of the indicator protein does not increase after contact" preferably means no more than 0.1%. More preferably, as used herein, the term "when the level of cleavage of the indicator protein does not increase after contact" means that the level of cleavage of the indicator protein does not decrease overall (i.e., the increase in the level of cleavage is 0%).

The level of the receptor and/or ganglioside (preferably the receptor) expressed in the cell described herein is preferably equal to or higher than the level of the receptor and/or ganglioside (preferably the receptor) expressed in the natural target (e.g., nerve cell) of the clostridial neurotoxin. For example, the level of the receptor and/or ganglioside (preferably the receptor) expressed in a cell described herein can be ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or ≥100% relative to the level of the receptor and/or ganglioside (preferably the receptor) expressed in the natural target (e.g., a nerve cell) of the clostridial neurotoxin.

The level of the receptor and/or ganglioside (preferably the receptor) expressed in the cell described herein may preferably be higher than the level of the receptor and/or ganglioside (preferably the receptor) expressed in a cell lacking said exogenous nucleic acid. For example, the level of a receptor and/or ganglioside (preferably a receptor) expressed in a cell described herein may be ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or ≥100% relative to the level of a receptor and/or ganglioside (preferably a receptor) expressed in a cell (e.g., an equivalent cell) lacking said exogenous nucleic acid.

In one embodiment, the term "overexpression" as used in relation to any aspect or embodiment means ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or ≥100% expression relative to the level of the receptor and/or ganglioside (target receptor) that is expressed in the natural target (e.g., a nerve cell) for the clostridial neurotoxin. In one embodiment, the term "overexpression" as used in relation to any aspect or embodiment described herein preferably means ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or ≥100% expression relative to the level of the receptor and/or ganglioside (preferably the receptor) that is expressed in a cell (e.g., any equivalent cell) that lacks said exogenous nucleic acid.

In one embodiment, the clostridial neurotoxin may be BoNT/A (or a BoNT containing a BoNT/AH _CC domain), and preferably the receptor may be SV2A, SV2B and/or SV2C (preferably SV2A).

Additionally or alternatively, the clostridial neurotoxin may be BoNT/B (or BoNT containing the BoNT/BH _CC domain), and preferably the receptor may be Syt-I and/or Syt-II.

Additionally or alternatively, the clostridial neurotoxin may be BoNT/E (or BoNT containing the BoNT/EH _CC domain), and preferably the receptor may be SV2A and/or SV2B (preferably SV2A).

Additionally or alternatively, the clostridial neurotoxin may be BoNT/G (or BoNT containing the BoNT/GH _CC domain), and preferably the receptor may be Syt-I and/or Syt-II.

For further information on suitable receptors/gangliosides, see Binz and Rummel (Journal of Neurochemistry, Volume 109, Issue 6, June 2009, Pages 1584-1595), incorporated herein by reference.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1A shows a Western blot using an anti-SNAP-25 antibody after treatment of N2a cells with BoNT/A at 0.1 nM, 1 nM, or 10 nM for 8 h or at 1 nM, 0.1 nM, or 0.01 nM for 24 h. The presence of the lower band indicates the presence of a cleavage product.

Figure 1B shows a Western blot using an anti-SNAP-25 antibody after treatment of M17 cells with BoNT/A at 0.1 nM, 1 nM, or 10 nM for 8 h or at 1 nM, 0.1 nM, or 0.01 nM for 24 h. The presence of the lower band indicates the presence of a cleavage product.

Figure 1C shows a Western blot using an anti-SNAP-25 antibody after treatment with IMR-32 for 24 h. The presence of the lower band indicates the presence of a cleavage product.

Figure 1D shows a Western blot using an anti-SNAP-25 antibody after 24 h of NG108 treatment. The presence of the lower band indicates the presence of a cleavage product.

Fig. 2A shows a fluorescence micrograph of NG108 cells 24 h after transfection with a plasmid containing the mScarlet-SNAP25-GeNluc construct.

Figure 2B shows a fluorescence micrograph of M17 cells one day after transfection with a plasmid containing the mScarlet-SNAP25-GeNluc construct.

Figure 3 shows a fluorescence micrograph of puromycin-resistant N108 cells stably transfected with a plasmid containing the mScarlet-SNAP25-GeNluc construct.

Fig. 4 is a bar graph showing the average number of cells per HPF for cells that fluoresce green after treatment with 0, 0.1 nM, or 1 nM BoNT/A.

Figure 5A shows a scatterplot of flow cytometry data for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct, showing granularity/complexity on the x-axis and cell size on the y-axis.

Fig. 5B shows a histogram of fluorescence emission intensity measured at 525 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Fig. 5C shows a histogram of fluorescence emission intensity measured at 585 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Fig. 5D shows a histogram of fluorescence emission intensity measured at 617 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Fig. 5E shows a histogram of fluorescence emission intensity measured at 665 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Fig. 5F shows a histogram of fluorescence emission intensity measured at 785 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Fig. 5G shows a scatterplot for flow cytometry data of NG108 cells stably transfected with mScarlet-SNAP-25-GeNluc measured at 665 nm on the x-axis and side scatter (SS) on the y-axis.

Figure 6A shows a scatterplot of flow cytometry data for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct, showing granularity/complexity on the x-axis and cell size on the y-axis.

Figure 6B shows a histogram of fluorescence emission intensity measured at 525 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Fig. 6C shows a histogram of fluorescence emission intensity measured at 585 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Figure 6D shows a histogram of fluorescence emission intensity measured at 617 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Fig. 6E shows a histogram of fluorescence emission intensity measured at 665 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Figure 6F shows a histogram of fluorescence emission intensity measured at 785 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct.

Figure 6G shows a scatterplot of flow cytometry data for M17 cells stably transfected with mScarlet-SNAP-25-GeNluc measured at 665 nm on the x-axis and side scatter (SS) on the y-axis.

Figure 7A shows a histogram of fluorescence emission intensity measured at 525 nm for control NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct.

Fig. 7B shows a histogram of fluorescence emission intensity measured at 525 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 0.1 nM BoNT/A.

Fig. 7C shows a histogram of fluorescence emission intensity measured at 525 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 1.0 nM BoNT/A.

Figure 8A shows a histogram of fluorescence emission intensity measured at 785 nm for control NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct.

Figure 8B shows a histogram of fluorescence emission intensity measured at 785 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 0.1 nM BoNT/A.

Fig. 8C shows a histogram of fluorescence emission intensity measured at 785 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 1.0 nM BoNT/A.

Figure 9 shows a Western blot performed on NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated with no toxin (control), 1 nM or 8 nM BoNT/A, or 0 (control), 1 nM, 10 nM, or 100 nM BoNT/E.

Figure 10A shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and not treated with toxin.

Fig. 10B shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT/A for 72 h.

Fig. 10C shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 1 nM BoNT/A for 72 h.

Fig. 10D shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 0.1 nM BoNT/A for 72 h.

Fig. 10E shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT/E for 72 h.

Fig. 10F shows a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and not treated with toxin.

Fig. 10G shows a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT/A for 72 h.

Fig. 10H shows fluorescence micrographs of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 1 nM BoNT/A for 72 h.

Fig. 10I shows a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 0.1 nM BoNT/A for 72 h.

Fig. 10J shows fluorescence micrographs of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT/E for 72 h.

Figure 11A shows flow cytometry data for wild-type NG108 cells.

Figure 11B shows flow cytometry data for genetically engineered NG108 cells that were selected for high indicator protein expression and sensitivity to BoNT/A at 1000 pM and were not treated with additional BoNT/A.

Fig. 11C shows flow cytometry data for genetically engineered NG108 cells that were selected for high indicator protein expression and sensitivity to BoNT/A at 1000 pM and treated with 100 pM BoNT/A for 48 h.

Fig. 11D shows flow cytometry data for genetically engineered NG108 cells that were selected for high indicator protein expression and sensitivity to BoNT/A at 1000 pM and treated with 100 pM BoNT/A for 96 h.

Figure 11E shows flow cytometry data for genetically engineered NG108 cells that were selected for high indicator protein expression but not for BoNT/A sensitivity and were not further treated with BoNT/A.

Fig. 11F shows flow cytometry data for genetically engineered NG108 cells that were selected for high indicator protein expression but not for BoNT/A sensitivity and treated with 100 pM BoNT/A for 96 h.

Figure 12A shows flow cytometry data for genetically engineered NG108 cells that were selected for high indicator protein expression and sensitivity to BoNT/A at 100 pM and were not treated with additional BoNT/A.

Figure 12B shows flow cytometry data for genetically engineered NG108 cells that were selected for high indicator protein expression and sensitivity to BoNT/A at 100 pM and treated with 100 pM BoNT/A for 96 h.

Fig. 13A is a graph of the percentage of indicator protein cleaved after treatment of NG108 cells genetically engineered to express indicator protein and SV2A or SV2C with varying concentrations of BoNT/A for varying periods of time.

Fig. 13B is a graph of the percentage of indicator protein cleaved after treatment of NG108 cells genetically engineered to express indicator protein and SV2A or SV2C with varying concentrations of BoNT/E for different periods of time.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that the present invention is not limited to the embodiments described herein. In fact, numerous variations, changes and substitutions will be apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be used in practicing the invention.

In the description of the invention, where a range of values is indicated with respect to an embodiment, it should be understood that each intermediate value is encompassed by the embodiment.

As used herein, a "variant" of a protein or polypeptide refers to a protein or polypeptide having an amino acid sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to the amino acid sequence of a reference protein or polypeptide.

"Sequence identity" as used herein refers to the identity between a reference amino acid or nucleotide sequence and a subject amino acid or nucleotide sequence, where the sequence is aligned such that the highest order match is obtained, and which can be calculated using published methods or methods encoded in computer programs, e.g., BLASTP, BLASTN, FASTA (Altschul 1990, J. Mol. Biol. 215:403).

As used herein, a "fragment" of a protein or polypeptide refers to truncated forms of the protein or polypeptide or truncated forms of a variant of the protein or polypeptide.

The present invention relates to a cell that has been genetically engineered to have increased sensitivity to a clostridial neurotoxin.

Clostridial neurotoxins are neurotoxins that are naturally produced by the bacteria Clostridium botulinum .

In certain embodiments of the present invention, the clostridial neurotoxin is a botulinum neurotoxin (BoNT) or a tetanus neurotoxin (TeNT). As used herein, the terms "clostridial neurotoxin,""BoNT'," and "TeNT'," respectively, refer to wild-type clostridial neurotoxins, including those produced by strains other than Clostridium botulinum , as well as modified and recombinant clostridial neurotoxins.

Modified clostridial neurotoxins may contain one or more modifications compared to a wild-type clostridial neurotoxin, including amino acid modifications and/or chemical modifications. Amino acid modifications include deletions, substitutions, or additions of one or more amino acid residues. Chemical modifications include modifications made to one or more amino acids, such as the addition of a phosphate or carbohydrate, or the formation of disulfide bonds.

In certain embodiments, modifications may be made to alter the properties of the clostridial neurotoxin. Modifications to the clostridial neurotoxin may increase or decrease its biological activity.

The biological activity of clostridial neurotoxin encompasses at least three distinct activities: the first activity is a "proteolytic activity" located in the protease component of the neurotoxin and is responsible for hydrolyzing the peptide bond of one or more SNARE proteins involved in the regulation of cellular membrane fusion. The second activity is a "translocation activity" located in the translocation component of the neurotoxin and is involved in the transport of the neurotoxin across the endosomal membrane and into the cytoplasm. The third activity is a "receptor binding activity" located in the targeting component of the neurotoxin and is involved in the binding of the neurotoxin to the receptor on the target cell.

In certain embodiments, the modification of the neurotoxin may include a truncated component(s) of the clostridial neurotoxin while maintaining the activity of such component(s). For example, the neurotoxin may be modified to include only the portion(s) of the protease component that is necessary for proteolytic activity, only the portion(s) of the translocation component that is necessary for translocation activity, and/or only the portion(s) of the targeting component that is necessary for receptor binding activity.

Clostridial neurotoxin is initially produced as an inactive single-chain polypeptide and is converted to its active double-chain form after cleavage of the neurotoxin at its activation site. This cleavage results in the formation of a double-chain protein with a heavy chain (H chain) containing the translocation and targeting components and a light chain (L chain) containing the protease component.

In certain embodiments, the biological activity of a clostridial neurotoxin is modified by modifying the activation site of the neurotoxin. Thus, the ability of the neurotoxin to be activated can be increased, decreased, or remain unchanged. In certain embodiments, the biological activity of a clostridial neurotoxin is increased or triggered by modifying the activation site so that it is more easily cleaved, thereby activating the neurotoxin. In embodiments where activation is desired only in certain environments or cells, the activation site can be modified so that it is cleaved only by proteases present in such environments or cells. In certain other environments, the biological activity of the neurotoxin is decreased or inactivated by modifying the activation site so that it is less easily cleaved.

In certain embodiments, the biological activity of a clostridial neurotoxin is modified by modifying the protease component of the neurotoxin. The proteolytic activity of the neurotoxin may be increased, decreased, or unchanged. In certain embodiments, the protease component may be replaced with a protease component from another clostridial neurotoxin or a variant or fragment thereof. For example, BoNT/A may be modified by replacing its protease component with the protease component of BoNT/E.

In certain embodiments, the biological activity of a clostridial neurotoxin is modified by modifying a translocation component of the neurotoxin. Thus, the translocation activity of the neurotoxin may be increased, decreased, or unchanged. In certain embodiments, the translocation component may be replaced with a translocation component from another clostridial neurotoxin or a variant thereof, or a fragment thereof. For example, BoNT/A may be modified by replacing its translocation component with a translocation component of BoNT/E.

In certain embodiments, the biological activity of a clostridial neurotoxin is modified by modifying a targeting component of the neurotoxin. Thus, the targeting ability of the neurotoxin may be increased, decreased, or unchanged. In certain embodiments, the targeting component may be replaced with a targeting component from another clostridial neurotoxin or a variant or fragment thereof. For example, BoNT/A may be modified by replacing its targeting component with a targeting component of BoNT/E. In other certain embodiments, the targeting component may be replaced with a non-clostridial polypeptide, such as an antibody.

In addition, the modification may include changing the order of the components of the clostridial neurotoxin, such as flanking the protease component with a translocation component and a targeting component.

Recombinant clostridial neurotoxins are produced genetically. They may be either genetically identical to wild-type clostridial neurotoxins or may differ from wild-type clostridial neurotoxins by having additional amino acids, fewer amino acids, or different amino acids. For example, recombinant clostridial neurotoxins may be produced that replicate any of the modified clostridial neurotoxins noted above. Recombinant clostridial neurotoxins may also have components arranged in an order different from that in wild-type clostridial neurotoxins. Recombinant clostridial neurotoxins may also be chemically modified as described above.

In certain embodiments, the modified or recombinant Clostridial neurotoxin is a polypeptide having an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to a wild-type Clostridial neurotoxin, such as BoNT serotype A, B, C, D, E, F, G, or H, or TeNT.

A number of programs based on algorithms for comparing different sequences are available to those skilled in the art. In this context, the Needleman and Wunsch or Smith and Waterman algorithms provide particularly reliable results. To perform the sequence alignments and calculate the sequence identity values reported in the paper, the commercially available DNASTAR Lasergene MegAlign program, version 7.1.0, based on the Clustal W algorithm, was used over the entire sequence region with the following settings: Pairwise alignment parameters: gap penalty: 10.00, gap length penalty: 0.10, Gonnet amino acid comparison matrix: 250, which, unless otherwise stated, should always be used as the default settings for sequence alignments.

The BoNT/A serotype is divided into at least six subserotypes (also known as subtypes), BoNT/A1 through BoNT/A6, which share between 84% and 98% amino acid sequence identity. BoNT/A proteins within a given subtype have a higher percentage of amino acid sequence identity.

Clostridial neurotoxins target neurons by binding to receptors. Clostridial neurotoxin receptors include protein receptors and plasma membrane gangliosides.

Gangliosides are oligoglycosylceramides derived from lactosylceramide and containing a sialic acid moiety such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), or 3-deoxy-D-glyc-D-galacto-acid (coulosonic acid). Gangliosides are present and concentrated at cell surfaces, with the two hydrocarbon chains of the ceramide group embedded in the plasma membrane and the oligosaccharides located at extracellular surfaces where they provide recognition points for extracellular molecules or the surfaces of adjacent cells. Gangliosides also bind specifically to viruses and bacterial toxins such as clostridial neurotoxins.

Gangliosides are defined by a nomenclature system in which M, D, T, and Q refer to mono-, di-, tri-, and tetrasialogangliosides, respectively, and the numbers 1, 2, 3, etc. refer to the migration order of gangliosides on thin-layer chromatography. For example, the migration order of monosialogangliosides is GM3>GM2>GM1. To indicate variations within the basic structures, additional terms are added, such as GM1a, GD1b, etc. Glycosphingolipids having 0, 1, 2, and 3 sialic acid residues linked to an internal galactose unit are called asialo- (or 0-), a-, b-, and c-series gangliosides, respectively, while gangliosides having sialic acid residues linked to internal N-galactosamine residues are classified as a-series gangliosides. The pathways for the biosynthesis of the 0-, a-, b-, and c-series gangliosides involve the sequential activity of sialyltransferases and glycosyltransferases, as shown, for example, in Ledeen et al., Trends in Biochemical Sciences, 40: 407–418 (2015). Further sialic acidification of each series and at various positions in the carbohydrate chain may occur to form an increasingly complex and heterogeneous series of products, such as the a-series gangliosides with a sialic acid residue(s) linked to an internal N-acetylgalactosamine residue. The gangliosides are transported to the outer leaflet of the plasma membrane by a transport system involving vesicle formation.

To date, approximately 200 gangliosides have been identified in vertebrate tissues. Common gangliosides include: GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, and GQ1.

Clostridial neurotoxins possess two independent binding sites in the HCC domain for gangliosides and neuronal protein receptors. BoNT/A, BoNT/B, BoNT/E, BoNT/F, and BoNT/G have a conserved ganglioside binding site in the HCC domain consisting of an “E(Q)...H(K)...SXWY...G” motif, whereas BoNT/C and BoNT/D exhibit two independent ganglioside binding sites. Lam et al., Progress in Biophysics and Molecular Biology, 117:225–231 (2015).

Most BoNTs bind only to gangliosides that have a 2,3-linked N-acetylneuraminic acid residue (designated Sia5) attached to the Gal4 oligosaccharide core, whereas the corresponding ganglioside binding pocket on TeNT can also bind GM1a, a ganglioside lacking the Sia5 sugar residue. BoNT/D has been found to bind GM1a and GD1a. See Kroken et al., Journal of Biological Chemistry, 286: 26828–26837 (2011). Combining data from ganglioside-deficient mice and biochemical assays, BoNT/A, BoNT/E, BoNT/F and BoNT/G show a preference for the terminal NAcGal-Gal-NAcNeu fragment present in GD1a and GT1b, whereas BoNT/B, BoNT/C, BoNT/D and TeNT require the disialyl motif found in GD1b, GT1b and GQ1b. Abundant complex polysialo-gangliosides such as GD1a, GD1b and GT1b thus appear to be required for the specific accumulation of all BoNT and TeNT serotypes on the neuronal cell surface during the first step of intoxication. See Rummel, Andreas, "Double receptor 10 anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity," Botulinum Neurotoxins, Springer Berlin Heidelberg (2012) 61-90.

In light of the above, in certain embodiments of the present invention, the cell is genetically engineered to express or overexpress a ganglioside. In particular embodiments, the cell is genetically engineered to express or overexpress GM1a, GD1a, GD1b, GT1b, and/or GQ1b. In certain embodiments, the cell is engineered to express or overexpress GD1a, GD1b, and/or GT1b. In certain embodiments, the cell is engineered to express or overexpress GD1b and/or GT1b.

Gangliosides are synthesized starting from ceramide. From ceramide, one pathway involves the addition of a glucose unit by glucosylceramide synthase to form glucosylceramide (GlcCer). β1,4-galactosyltransferase I (GalT-I) then catalyzes the addition of a galactose unit to GlcCer to form lactosylceramide (LacCer). From LacCer, GalNAc transferase (GalNAcT) can add N-acetylgalactosamine to form GA1, or GM3 synthase can add sialic acid to form GM3. From GM3, GD3 can be formed by the addition of an additional sialic acid by GD3 synthase. GT3 can be formed from GD3 by the addition of another sialic acid by GT3 synthase. In a separate pathway, a galactose unit is added to LacCer by galactosylceramide synthase to form galactosylceramide (GalCer). GM4 synthase then adds another carbohydrate group to (ST-V). For example, GD3 forms the "b" series gangliosides GD1b, GT1b, and GQ1b.

The R cells of the present invention can be engineered to express or overexpress a desired ganglioside by being engineered to express or overexpress an enzyme of a biosynthetic pathway that leads to the ganglioside. For example, a cell can be engineered (i.e., by transfection) to contain an exogenous nucleic acid encoding such an enzyme. Thus, in certain embodiments, a cell has been engineered to express or overexpress glucosylceramide synthase, GalT-I, GalNAcT, GM3 synthase, GD3 synthase, GT3 synthase, galactosylceramide synthase, GM4 synthase, GalT-II, ST-IV and/or CT-B.

Those skilled in the art will recognize that variants or fragments of such enzymes that retain the desired catalytic activity may also play a role in synthesizing the gangliosides of interest. Thus, in certain embodiments, a cell has been genetically engineered to express or overexpress a variant or fragment of an enzyme of the ganglioside synthesis pathway that retains the ability of the enzyme. For example, in certain embodiments, the cell has been genetically engineered to express or overexpress a variant or fragment of glucosylceramide synthase that has the ability to add glucose to ceramide, a variant or fragment of GalT-I that has the ability to add a galactose unit to GlcCer, a variant or fragment of GalNAcT that has the ability to add N-acetylgalactosamine to LacCer, a variant or fragment of GM3 synthase that has the ability to add sialic acid to LacCer, a variant or fragment of GD3 synthase that has the ability to add sialic acid to GM3, a variant or fragment of GT3 synthase that has the ability to add sialic acid to GD3, a variant or fragment of galactosylceramide synthase that has the ability to add a galactose unit to LacCer, and/or a variant of GM4 synthase that has the ability to add a carbohydrate group to GalCer.

In certain embodiments, the variant is a protein with an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence of an enzyme of a biosynthetic pathway that results in a ganglioside and that retains the desired catalytic activity of such an enzyme. In other certain embodiments, the variant is a protein with an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to glucosylceramide synthase, GalT-I, LacCer, GalNAcT, GD3 synthase, GT3 synthase, galactosylceramide synthase, GM4 synthase, GalT-II, ST-IV, and/or ST-V and that retains the desired catalytic activity of the enzyme.

A fragment may, for example, contain 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, or 10 amino acids or less.

Assays are known in the art that can be used to determine which variants or fragments have the desired catalytic activity. For example, assays are known to those skilled in the art that can be used to determine whether a variant or fragment of GD3 synthase has the ability to add sialic acid to GM3.

Those skilled in the art will appreciate that the above enzymes may also be encoded by nucleic acids that differ from the above exogenous nucleic acid by conservative substitutions known in the art. Those skilled in the art will also appreciate that variant enzymes may, for example, be encoded by nucleic acids that have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a nucleic acid encoding a wild-type enzyme. Thus, the invention also relates to a cell that has been genetically engineered to contain an exogenous nucleic acid that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with a nucleic acid that encodes one of the above enzymes and/or a nucleic acid that differs from the wild-type nucleic acid and encodes such an enzyme with only conservative substitutions, where the encoded protein is a wild-type enzyme or a variant that retains the catalytic activity of the wild-type enzyme.

In some embodiments, the cell is engineered to express or overexpress an enzyme that serves to catalyze what has been found to be a rate-limiting step in the biosynthesis of a desired ganglioside, or a variant or fragment thereof that has the desired catalytic activity of such an enzyme. For example, GD3 synthase is an enzyme that catalyzes a rate-limiting step in the biosynthesis of "b" series gangliosides, in particular the addition of sialic acid to GM3. Thus, in embodiments where expression or overexpression of GD1b, GT1b, and/or GQ1b is desired, the cell is engineered to express or overexpress GD3 synthase or a variant or fragment thereof that has the ability to add sialic acid to GM3.

For example, a cell can be transfected with a nucleic acid encoding a GD3 synthase, or a nucleic acid as described above, having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to such a nucleic acid, such as one that encodes a variant of a GD3 synthase that retains its catalytic activity, or one that differs from a wild-type GD3 synthase only by conservative substitutions.

Binding of certain clostridial neurotoxins to cells may also depend on binding to protein receptors. BoNT/A, BoNT/D, BoNT/E, BoNT/F, and TeNT bind to synaptic vesicle protein 2 (SV2), with BoNT/A able to bind to all three isoforms (SV2A, SV2B, and SV2C) and BoNT/E able to bind only to SV2A and SV2B. BoNT/B and BoNT/G bind to both isoforms (I and II) of synaptotagmin. Synaptotagmin and SV2 are localized to synaptic vesicles and become available to the extracellular space when the vesicles fuse with the presynaptic membrane. It is during this period that clostridial neurotoxins bind to their protein receptors.

The cells of the present invention can be engineered to express or overexpress a desired protein receptor, such as SV2 (e.g., SV2A, SV2B, and SV2C) or synaptotagmin (e.g., synaptotagmin I and synaptotagmin II). For example, a cell can be engineered (e.g., by transfection) to contain an exogenous nucleic acid encoding such a protein receptor.

The present invention also relates to proteins that are different from such protein receptors, but still retain the ability to bind a clostridial neurotoxin. Such proteins may be a variant or fragment of such a protein receptor that retains the ability of the receptor to bind a clostridial neurotoxin. Thus, in certain embodiments, a cell has been engineered to express or overexpress a variant or fragment of SV2 that binds BoNT/A, BoNT/D, BoNT/E, BoNT/F and/or TeNT. Also, in certain embodiments, a cell has been engineered to express or overexpress a variant or fragment of synaptotagmin that binds BoNT/B and/or BoNT/G.

In certain embodiments, the variant is a protein having an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a sequence of a protein receptor that binds a clostridial neurotoxin. In some embodiments, the variant is a protein having an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SV2 ( e.g. , SV2A (SEQ ID NO: 8), SV2B (SEQ ID NO: 9), and SV2C (SEQ ID NO: 10)) or synaptotagmin ( e.g. , synaptotagmin I (SEQ ID NO: 14) and synaptotagmin II (SEQ ID NO: 15)).

A fragment may, for example, contain 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, or 10 amino acids or less.

In certain embodiments, the variant or fragment comprises domains of wild-type protein receptors that bind a neurotoxin. For example, the variant or fragment can comprise the luminal domain(s) of wild-type SV2 (e.g., SV2A, SV2B, and SV2C) or wild-type synaptotagmin (e.g., synaptotagmin I and synaptotagmin II). In certain other embodiments, the variant or fragment can comprise a fourth luminal domain of wild-type SV2, such as the fourth luminal domain of SV2A (SEQ ID NO: 11), the fourth luminal domain of SV2B (SEQ ID NO: 12), or the fourth luminal domain of SV2C (SEQ ID NO: 13).

Assays are known in the art that can be used to determine which variants or fragments have the desired clostridial neurotoxin binding activity. For example, those skilled in the art know what assay can be used to determine whether a variant or fragment of SV2C has the ability to bind BoNT/A.

Those skilled in the art will appreciate that the above enzymes may also be encoded by nucleic acids that differ from the above exogenous nucleic acid by conservative substitutions known in the art. Those skilled in the art will also appreciate that variants of a protein receptor may, for example, be encoded by nucleic acids that have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a nucleic acid encoding a wild-type protein receptor. Thus, the invention also relates to a cell that has been genetically engineered to contain an exogenous nucleic acid that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with a nucleic acid that encodes one of the above protein receptors and/or a nucleic acid that differs from a wild-type nucleic acid encoding such a protein receptor only by conservative substitutions, where the encoded protein is a wild-type protein receptor or a variant that retains the ability to bind a clostridial neurotoxin.

SV2C is most sensitive to BoNT/A. Thus, in certain embodiments where sensitivity to BoNT/A is desired, the cell is genetically engineered to express or overexpress SV2C or a variant or fragment thereof that is capable of binding BoNT/A. SV2C, however, does not bind BoNT/E, which instead binds SV2A and SV2B. Thus, in certain embodiments where sensitivity to BoNT/E is desired, the cell is genetically engineered to express or overexpress SV2A and/or SV2B, or variants or fragments thereof capable of binding BoNT/E.

The present invention contemplates that a cell may be engineered to express or overexpress two or more protein receptors, two or more enzymes of the ganglioside synthesis pathway, or a protein receptor(s) and an enzyme(s) of the ganglioside synthesis pathway. For example, a cell may be engineered to express or overexpress SV2A and SV2C. Such a cell may, for example, have increased sensitivity to BoNT/A and BoNT/E. Also, a cell may be engineered to express or overexpress GD3 synthase and SV2A and/or SV2C.

In addition, chimeric receptors are known to be capable of binding neurotoxins. For example, chimeric receptors that contain a domain of the above-mentioned protein receptor that binds to a neurotoxin (e.g., the fourth luminal domain of SV2) fused to a transmembrane domain of another receptor, such as the LDL receptor, are known to bind to BoNT and allow its internalization into the cell. The present invention thus also contemplates engineering cells to express such chimeric receptors.

The cell used in the present invention can be any cell, prokaryotic or eukaryotic, capable of expressing a ganglioside and a protein receptor as described above. Examples of such cells include neural cells, neuroendocrine cells (e.g., PC12), embryonic kidney cells (e.g., HEK293 cells), breast cancer cells (e.g., MC7), neuroblastoma cells (e.g., Neuro2a (N2a), M17, IMR-32, N18, and LA-N-2 cells), and neuroblastoma-glioma hybrid cells (e.g., NG108 cells). In certain embodiments, the cell is a neuroblastoma or neuroblastoma-glioma cell. In certain embodiments, the cell is an NG108, M17, or IMR-32 cell. In a specific embodiment, the cell is an NG108 cell.

Cells engineered to express or overexpress clostridial toxin receptors can be further selected for increased sensitivity using directed evolution. In this process, cells are exposed to a clostridial neurotoxin and cells that exhibit sensitivity to a lower concentration of clostridial neurotoxin (as determined, for example, by their cleavage of an indicator protein) are selected for relative to other cells. These cells are expected to have greater sensitivity to the clostridial neurotoxin than cells in general. This process can be repeated with a lower concentration of clostridial neurotoxin, with cells selected for sensitivity to the lower concentration.

The selected cells will have increasing sensitivity to the clostridial neurotoxin with each round.

As discussed previously, a cell of the present invention can be used in an assay for determining the activity of a polypeptide (e.g., a modified or recombinant clostridial neurotoxin). Such an assay involves contacting the cell with the polypeptide and testing for the presence of product(s) resulting from cleavage of the SNARE protein in the cell.

As used herein, the term "contacting" refers to bringing a cell and a clostridial neurotoxin into physical proximity to allow physical and/or chemical interaction. Contacting is performed under conditions and for a time sufficient to allow interaction between the polypeptide and a protein that is susceptible to proteolysis by the wild-type clostridial neurotoxin (e.g., a SNARE protein).

In certain embodiments, such contacting may be accomplished by culturing the cell in a medium containing the polypeptide. Typically, the polypeptide is present in the medium at a concentration of 0.0001 to 10,000 nM, 0.0001 to 1000 nM, 0.0001 to 100 nM, 0.0001 to 10 nM, 0.0001 to 10 nM, 0.0001 to 0.1 nM, 0.0001 to 0.01 nM, or 0.0001 to 0.001 nM. Such culturing may, for example, occur for 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 40 hours or more, or 48 hours or more.

In other specific embodiments, such contacting may be accomplished by transfecting a cell (e.g., transient transfection) with an exogenous nucleic acid encoding the polypeptide.

To enable use in such an assay, the cell contains a protein susceptible to proteolysis by a wild-type clostridial neurotoxin. These proteins will be referred to herein as the "indicator protein(s)". The indicator protein may be endogenous (e.g., an endogenous SNARE protein) or the cell may be genetically engineered to express or overexpress the indicator protein.

As noted above, SNARE proteins such as SNAP-25, synaptobrevin, and syntaxin are known to be susceptible to proteolysis by clostridial neurotoxins. For example, BoNT/A, BoNT/C, and BoNT/E are known to cleave SNAP-25, BoNT/C is also known to cleave syntaxin, and other BoNT serotypes and TeNT are known to cleave synaptobrevin. Accordingly, the invention contemplates that such an indicator protein may comprise the amino acid sequence of such a SNARE protein. The invention also contemplates that the indicator protein may instead comprise the amino acid sequence of a variant or fragment of such a SNARE protein, provided that the variant or fragment is susceptible to proteolysis by a wild-type clostridial neurotoxin. In certain embodiments, the variant may have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a SNARE protein. The portion of the indicator protein having the amino acid sequence of a SNARE protein, or a variant or fragment thereof, will be referred to herein as the "SNARE domain" of the indicator protein.

The term "susceptible to proteolysis" means that the protein is proteolytically cleaved by the protease component of wild-type clostridial neurotoxin. In other words, the protein contains a protease recognition and cleavage site that allows it to be recognized and cleaved by the protease component of wild-type clostridial neurotoxin.

In certain embodiments, the indicator protein is labeled. For example, U.S. Patent No. 8,940,482 to Oyler et al. describes a cell-based assay for assessing Clostridial neurotoxin activity, wherein a cell has been engineered to express a labeled fusion protein comprising a fluorescent protein domain fused to SNAP-25. The fluorescent protein domain is the C-terminal domain of SNAP-25 and becomes part of the C-terminal fragment that results from cleavage of SNAP-25 by the Clostridial neurotoxin. In the assay described by Oyler, the full-length fusion protein is not readily degraded in the cell, but the resulting C-terminal fragment is readily degraded, resulting in degradation of the fluorescent protein. This is due to the presence of residues in SNAP-25 that serve as degrons only when they are exposed by cleavage at the N-terminus of the resulting fragment. Such "N-degrons" are tagged by ubiquitin ligases, and the fragment is thus targeted for degradation in the proteasome.

The present invention thus contemplates embodiments as described by Oyler, wherein the cell is engineered to express a labeled indicator protein that is not readily degraded in its full-length form. In such embodiments, cleavage results in a labeled fragment that is readily degraded in the cell (e.g., due to the presence of an N-degron). The indicator protein has a label on the portion that forms the fragment that is readily degraded, and the label is degraded along with the fragment. In such embodiments, the ability of a polypeptide to cleave a SNARE protein in a cell can be determined by the presence (or absence) of a signal from the label upon contacting the cell with the polypeptide.

In other specific embodiments, the indicator protein also includes a tag on a portion of the indicator protein that, upon cleavage, produces a fragment that is not as easily degraded as another fragment. For example, the indicator protein may be a fusion protein comprising two tags and a SNARE domain with tags flanking the SNARE domain. In embodiments where the full-length indicator protein is not readily cleaved in the cell, but upon cleavage one of the resulting fragments is readily cleaved, cleavage of the SNARE protein may be determined by comparing the signal obtained from the tag on the readily degraded fragment to the signal from the tag on the less readily degraded fragment. For example, in embodiments such as Oyler, where the C-terminal fragment resulting from cleavage is readily cleaved but the N-terminal fragment is not, cleavage can be determined by comparing the signal obtained from a label on the C-terminal fragment with the signal from a label on the N-terminal fragment. In such embodiments, labels that emit fluorescent signals that are more clearly distinguishable from each other (e.g., red and green, or red and blue) can be selected.

As used herein, the term "label" means a detectable marker and includes, for example, a radiolabel, an antibody, and/or a fluorescent label. The amount of the substrate and/or cleavage product of interest can be determined, for example, by autoradiography or spectrometry methods, including methods based on resonance energy transfer between at least two labels, such as FRET analysis (discussed further below). Alternatively, immunological methods such as Western blotting or ELISA can be used for detection.

Examples of labels that can be used in the practice of the present invention include: radioactive isotopes; fluorescent labels; phosphorescent labels; luminescent labels; and compounds capable of binding a labeled binding partner. Examples of fluorescent labels include: yellow fluorescent protein (YFP); blue fluorescent protein (BFP); green fluorescent protein (GFP), such as NeonGreen; red fluorescent protein (RFP), such as mScarlet; cyan fluorescent protein (CFP); and fluorescent mutants thereof. Examples of luminescent labels include: photoproteins; luciferase, such as firefly luciferase, Renilla luciferase, and Gaussia luciferase; chemiluminescent compounds; and electrochemiluminescent (ECL) compounds. In embodiments as noted above, where the N-terminal tag and the C-terminal tag are selected so that the emitted signals are more easily distinguishable from each other, examples of such tag pairs may include RFP and GFP, and RFP and CFP. For example, RFP, such as mScarlet, may serve as an N-terminal tag, and GFP, such as NeonGreen or CFP, may serve as a C-terminal tag.

In some embodiments, the label is a protein label, such as an antibody, a fluorescent protein, a photoprotein, and a luciferase.

As used herein, an "N-terminal tag" refers to a tag, protein or not, located at a region of an indicator protein that is N-terminal to a Clostridial neurotoxin cleavage site, and a "C-terminal tag" refers to a tag, protein or not, located at a region of an indicator protein that is C-terminal to a Clostridial neurotoxin cleavage site. The tag does not have to be at the N-terminus or C-terminus of the indicator protein to be referred to as an N-terminal tag or a C-terminal tag. Rather, these terms refer to the position of the tag relative to the Clostridial neurotoxin cleavage site. In certain embodiments of the present invention, RFP, such as mScarlet, is used as the N-terminal tag, and GFP, such as NeonGreen or CFP, is used as the C-terminal tag.

Another assay is the fluorescence resonance energy transfer (FRET) assay. In such an assay, the indicator protein contains a donor label on one side of the cleavage site and an acceptor label on the other side. The donor label absorbs energy and then transfers it to the acceptor label. The energy transfer results in a decrease in the fluorescence intensity of the donor chromophore and an increase in the emission intensity of the acceptor chromophore. Cleavage of the substrate results in less successful energy transfer. Thus, successful cleavage can be determined based on the limitation of the opportunity for this transfer. In such embodiments, YFP and CFP can be taken as a FRET pair, as can RFP and GFP.

In certain embodiments of the present invention, the indicator protein is a fusion protein that comprises a SNARE domain. The fusion protein may also comprise additional domains, such as a tag domain. The tag domain may have the amino acid sequence of a protein tag. An example of such a fusion protein includes an N-terminal tag domain, such as the amino acid sequence for mScarlet; a SNARE domain, such as the amino acid sequence for SNAP-25; and a C-terminal domain with a tag, such as the amino acid sequence for NeonGreen.

The fusion protein may also include other domains, such as a selectable marker (discussed below). In such embodiments, the selectable marker domain may be separated from the portion of the fusion protein containing the remaining domains (e.g., the SNARE domain and the tag domain(s)) by a linker that can be cleaved to allow separation of the selectable marker and the remainder of the indicator protein after translation. The linker may, for example, be self-cleaving (e.g., self-cleaving peptide 2A).

As discussed previously, the cell can be engineered to express or overexpress the indicator protein. Those skilled in the art will recognize nucleic acids that can be used for such expression and methods for engineering such cells to express such an indicator protein. An example of such a nucleic acid is SEQ ID NO: 1, which expresses a fusion protein having mScarlet as an N-terminal tag, SNAP-25 as a SNARE domain, NeonGreen as a C-terminal tag, luciferase as an additional tag domain, puromycin N-acetyltransferase as a selectable marker, and self-cleaving peptide 2A. Another example of such a nucleic acid is SEQ ID NO: 2, which expresses a fusion protein having mScarlet as an N-terminal tag, SNAP-25 as a SNARE domain, CFP as a C-terminal tag, luciferase as an additional tag domain, puromycin N-acetyltransferase as a selectable marker, and self-cleaving peptide 2A.

The present invention also relates in part to a method for producing the above-mentioned genetically engineered cell. The method comprises introducing into the cell an exogenous nucleic acid encoding a protein of interest. The protein of interest may be, for example, a clostridial neurotoxin receptor or a variant thereof or a fragment thereof having the ability to bind a clostridial neurotoxin; an enzyme of the ganglioside synthesis pathway or a variant thereof or a fragment thereof having the catalytic activity of such an enzyme; and/or an indicator protein.

In certain embodiments, the method comprises transforming a cell with a nucleic acid encoding a protein of interest. Such transformation may be transfection.

In certain embodiments, the nucleic acid encodes a fusion protein comprising two or more domains, wherein each domain has an amino acid sequence for a protein of interest or other components of the fusion protein. For example, the nucleic acid can encode a fusion protein comprising an amino acid sequence for a protein receptor (e.g., SV2A or SV2C) and an amino acid sequence for an enzyme of the ganglioside synthesis pathway (e.g., GD3 synthase). In another example, the nucleic acid can encode a fusion protein comprising an amino acid sequence for a protein receptor, an amino acid sequence for an enzyme of the ganglioside synthesis pathway, and an amino acid sequence for a selectable marker. In yet another example, the nucleic acid can encode a fusion protein comprising an amino acid sequence for a protein receptor, an amino acid sequence for an enzyme of the ganglioside synthesis pathway, an amino acid sequence for an indicator protein, and an amino acid sequence for a selectable marker.

In such embodiments, the domains may be separated from each other by linkers. The linkers may, for example, be cleavable by enzymes in the cell or comprise a self-cleaving peptide (e.g., self-cleaving peptide 2A) that allows the individual domains to form individual proteins in the cell.

The nucleic acid may optionally contain regulatory elements. As used herein, the term "regulatory elements" refers to regulatory elements of gene expression, including transcription and translation, and includes elements such as TATA boxes, promoters, enhancers, ribosome signal binding sites, Shine-Dalgarno sequences, IRES regions, polyadenylation sequences, terminal capping structures, and the like. A regulatory element may contain one or more heterologous regulatory elements or one or more homologous regulatory elements. A "homologous regulatory element" is a regulatory element from a wild-type cell from which a nucleic acid molecule is derived that is involved in regulating the expression of a gene for a nucleic acid molecule or polypeptide in a wild-type cell. A "heterologous regulatory element" is a regulatory element that is not involved in regulating the expression of a gene for a nucleic acid molecule or polypeptide in a wild-type cell. Regulatory elements for inducible expression, such as inducible promoters, can also be used.

The nucleic acid molecule may be, for example, hnRNA, mRNA, RNA, DNA, PNA, LNA, and/or a modified nucleic acid molecule. The nucleic acid molecule may be circular, linear, integrated into a genome, or episomal. The invention also encompasses concatemers encoding fusion proteins containing three, four, five, six, seven, eight, nine, or ten polypeptides. In addition, the nucleic acid molecule may contain sequences encoding signal sequences for intracellular transport, such as signal sequences for transport into an intracellular compartment or for transport through a cell membrane.

Nucleic acid can be engineered to provide high levels of expression in a host cell. Methods for engineering nucleic acid molecules to increase protein expression in host cells are known in the art and include reducing the frequency (number of occurrences) of "slow codons" in the coding sequence of the nucleic acid.

The nucleic acid can be introduced by any method known in the art. For example, it can be included in a vector (e.g., a plasmid) used to introduce the nucleic acid into a cell.

Any vector known in the art that allows expression of a nucleic acid in a cell can be used. The vector can be suitable for expression of the protein of interest in vitro and/or in vivo . The vector can be a vector for transient and/or stable gene expression. The vector can further comprise regulatory elements and/or selectable markers. The vector can be, for example, of artificial or viral origin, of phage origin or of bacterial origin. Examples of vectors for use in the present invention include adenovirus vectors, vaccinia vectors, SV-40 viral vectors, retrovirus vectors, λ-derivatives, and plasmids. Examples of plasmids for use in the present invention include plasmids having the pD2500 or pcDNA3.1 backbone.

Methods for introducing nucleic acid into a cell are known in the art. See Laura Bonetta, "The Inside Scoop-Evaluating Gene Delivery Methods" Nature Methods 2: 875-883 (2005).

The host cell may contain an inducer of expression of the protein of interest. Such an expression inducer may be a nucleic acid molecule or a polypeptide or a chemical compound, including a small chemical compound. The expression inducer may, for example, increase transcription or translation of a nucleic acid molecule encoding the protein of interest. The inducer may, for example, be expressed by recombinant methods known to those skilled in the art. Alternatively, the inducer may be isolated from a cell, for example, a clostridial cell.

In certain embodiments, cells that have been successfully transformed can be identified by the presence of a selectable marker. In such embodiments, the vector containing the exogenous nucleic acid encoding the desired protein can also contain a nucleic acid encoding the selectable marker.

In certain embodiments, the selectable marker is a detectable tag. Examples of such tags include a His tag, a GST tag, a Strep tag, and an SBP tag. The tag can be expressed as part of a fusion protein that also includes the protein of interest. In such embodiments, the tag can be flanked by one or more protease cleavage sites or self-cleaving peptides. This allows the tag to be cleaved from the protein after translation.

In other specific embodiments, the selectable marker confers resistance to an antibiotic. Examples of such selectable markers include puromycin N-acetyltransferase (puromycin resistance), aminoglycoside 3'f3 phosphotransferase (G418 resistance), blasticidin S deaminase (Blasticidin S resistance), and hygromycin B phosphotransferase (hygromycin B resistance). Successful transformation of a cell can thus be determined by exposing the cell to the appropriate antibiotic.

In certain embodiments, successful transformation of cells that are genetically engineered to express or overexpress a ganglioside and/or a clostridial neurotoxin-binding protein receptor can be determined by contacting such cells with a clostridial neurotoxin and determining whether cleavage of the indicator protein occurs therein.

The present invention relates to the use of the above genetically engineered cell in an assay for determining the biological activity of a polypeptide, such as a modified or recombinant clostridial neurotoxin.

The biological activity of such polypeptides can be measured using a variety of tests, all of which are known to those skilled in the art.

As discussed previously, the assay involves contacting a cell with a polypeptide under conditions and for a period of time that would allow the protease domain of a wild-type clostridial neurotoxin to cleave an indicator protein in the cell, and determining the presence of products resulting from cleavage of the indicator protein. The indicator protein may be endogenous to the cell (e.g., an endogenous SNARE protein) or may be an exogenous indicator protein of the type described previously.

Such assays typically also include a step to determine the extent of conversion of the indicator protein to its cleavage product(s). Observation of one or more cleavage products arising upon contact of the polypeptide with the indicator protein, or observation of an increase in the amount of cleavage product(s), indicates proteolytic activity of the polypeptide.

The detection step may include a comparison of the full-length indicator protein and the cleavage product. The comparison may include determining the amount of the full-length indicator protein and/or the amount of one or more cleavage products and may also include calculating the ratio of the full-length indicator protein to the cleavage product. In addition, the assay for determining proteolytic activity may include a step of comparing the cleavage product/products that appear after contact between the test polypeptide and the indicator protein and a control. The control may be, for example, a cleavage product(s) that appears after contact with a clostridial neurotoxin that is known to cleave the same indicator protein.

In certain embodiments, after contacting a cell with a polypeptide, the cell can be lysed and analyzed using gel electrophoresis and Western blotting. For example, a Western blotting can use an anti-SNAP-25 antibody that binds to the N-terminus of SNAP-25 to detect the presence of full-length SNAP-25 and cleaved SNAP-25 (which will migrate in a separate band from full-length SNAP-25).

Methods and approaches used to lyse host cells, such as bacterial cells, are known in the art. Examples include sonication or the use of a French press.

In certain embodiments, the full-length indicator protein is not readily degraded in the cell, but after its cleavage, one of the resulting fragments is readily degraded. This may be, for example, due to the presence of a residue that serves as a degron only when it is exposed by cleavage at the N-terminus of the resulting fragment.

In such embodiments, the indicator protein may be labeled on a portion thereof that is more easily degraded after cleavage. The label should be selected such that when the fragment is degraded, the label is also degraded. In such embodiments, whether cleavage occurs can be determined based on measuring the signal from the label.

The received signal can be compared with the control.

In other specific embodiments, where another fragment formed after cleavage is not as easily degraded, the indicator protein may also include a label on the portion of the indicator protein that forms the fragment after cleavage. In such embodiments, whether cleavage occurs can thus be determined by comparing the signal from the label on the more easily cleaved fragment with the signal from the label on the less easily cleaved fragment, which serves as a control.

The signal(s) from the tag(s) can be analyzed by fluorescence-activated cell sorting (FACS). For example, in an embodiment where the full-length indicator protein and its N-terminal fragment formed after cleavage are not easily degraded inside the cell, but the C-terminal fragment resulting from cleavage is easily degraded, and the N-terminal tag is mScarlet and the C-terminal tag is NeonGreen, FACS analysis of cells after successful cleavage will show lower green emission compared to red emission. In contrast, if cleavage does not occur, red and green fluorescence should be equally dominant.

Alternatively, fluorescence micrographs of the cell may be taken. In the embodiment described above, successful cleavage would result in less green fluorescence being emitted by the cells compared to control cells that were not exposed to the protease. In contrast, red fluorescence should remain the same as in the control.

In addition, in some embodiments, the assay may be a FRET assay. As discussed previously, in such an assay, the indicator protein comprises an N-terminal label and a C-terminal label, wherein one label is a donor label and the other is an acceptor label. Energy transfer between the donor label and the acceptor label results in a decrease in the fluorescence intensity of the donor label and an increase in the emission intensity of the acceptor label. The success of such a transfer depends on the labels remaining in close proximity. Cleavage of the indicator protein tends to make these labels more distant, and thus such a transfer is less successful. Successful cleavage can thus be determined based on a decreased ability to transfer energy.

In addition to the above, any other methods known in the art by which fluorescence from indicator proteins can be analyzed to determine whether cleavage has occurred can be used in the practice of the present invention.

In certain embodiments, a polypeptide is considered proteolytically active if 20% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the indicator protein is converted to cleavage product(s) in less than 1 minute, less than 5 minutes, less than 20 minutes, less than 40 minutes, less than 60 minutes, or less than 120 minutes.

Cleavage can be measured at regular intervals to monitor catalytic activity over time.

All references cited in this specification are herein incorporated by reference with respect to their entire contents and the contents of the invention specifically mentioned in this specification.

EXAMPLES

Example 1 - Selection of the optimal parental cell line for generating an indicator cell line

Neuro2A (N2a; ATCC CCL-131), BE(2)-M17 (M17; ATCC CRL-2267), IMR-32 (ATCC CCL-127), and NG108-15 [108CC15] (ATCC HB-12317) cells were tested to select the optimal parental cell line for the development of a stable transfected cell line.

Following delivery, cells were allowed to recover and grow. Stock cell cultures were then frozen and stored in liquid nitrogen. Once sufficient stock cell culture flasks had been prepared, cells were assayed for their sensitivity to BoNT/A.

The cells were cultured in medium containing BoNT/A (Metabiologics, Inc.) for 8 or 24 h.

Endogenous SNAP-25 cleavage was analyzed by Western blotting using an anti-SNAP-25 antibody (Sigma #S9684) with standard protocols (Fig. 1). NG108 cells showed greater sensitivity to BoNT/A than the other cells, with N2a cells being the least sensitive. Thus, the NG108 cell line was selected as the prime candidate for the development of a stably transfected indicator cell line. The M17 and IMR-32 cell lines showed similar sensitivity to BoNT/A at the concentrations and time points tested. Due to ease of culture and familiarity, the M17 line was chosen as a backup line for NG108.

Example 2 - Transfection of cells with a plasmid containing an indicator construct

The sensitivity of NG108 cells and M17 cells to puromycin (InvivoGen #ANT-PR) and G418 (VWR #97064-358) was determined. Cells were grown to ~50% confluency and then cultured with different concentrations of puromycin and G418. Both cell lines showed similar sensitivity to puromycin and G418.

Plasmids (pD2500; Atum) were constructed to contain a nucleic acid sequence encoding puromycin N-acetyltransferase (PuroR), the chimeric protein, and the self-cleaving peptide 2A. In the expressed product, the self-cleaving peptide 2A was located between PuroR and the chimeric protein. The chimeric protein contained SNAP-25 flanked between the N-terminal and C-terminal fluorescent proteins and luciferase (located at the C-terminus). PuroR confers resistance to puromycin. Luciferase allows luminescent measurements of degradation in addition to fluorescent measurements of degradation by the fluorescent proteins. The N-terminal fluorescent protein was mScarlet, and the C-terminal fluorescent protein was either NeonGreen, green fluorescent protein, or cyan fluorescent protein (CFP). The plasmid insert containing nucleic acids encoding PuroR, self-cleaving peptide 2A, and SNAP-25, CFP, and luciferase (mScarlet-SNAP25-CyanNluc) had the nucleotide sequence of SEQ ID NO: 2.

NeonGreen was chosen for its excitation/emission spectrum and intensity. In the event that NeonGreen is poorly degraded by indicator protein cleavage, CFP was chosen as a backup protein due to previous data indicating that it is rapidly degraded by indicator protein cleavage.

NG108 cells and M17 cells were transfected with plasmids containing the mScarlet-SNAP25-GeNluc construct or the mScarlet-SNAP25-CyanNluc construct. Transfection was performed using Lipofectamine 3000 (ThermoFisher) or polyethyleneimine using standard protocols.

Twenty-four hours after transfection, cells were analyzed by fluorescence microscopy to confirm transfection efficiency and proper expression of the indicator protein (Fig. 2A-B, examples of cells expressing the indicator protein containing NeonGreen are shown). Due to overexpression, most of the indicator protein was cytosolic. Red and green, representing the N- and C-termini of the indicator protein, respectively, were easily detected and indicated that the termini were co-localized. High transient expression was observed in both cell types with transfection efficiency >70%.

After confirming that the cells were efficiently transfected and the indicator proteins were expressed correctly, transfected cells were selected with either 2.5 μg/ml puromycin or shocked with an initial high dose of 20 μg/ml puromycin on day 1 and then cultured in 5-10 μg/ml puromycin. Both treatments yielded a pool of fluorescent, puromycin-resistant cells.

Simultaneously, a series of additional transfections with both indicator constructs and selection for puromycin resistance were performed, generating an additional pool of fluorescent puromycin-resistant cells. This ultimately resulted in approximately six independent pools of fluorescent puromycin-resistant NG108 cells and two independent pools of puromycin-resistant fluorescent M17 cells. Pools were expanded and cultures were frozen and tested for viability upon thawing.

Puromycin-resistant cells were analyzed to confirm stable transfection of the indicator construct (Fig. 3). In NG108 cells stably transfected with the indicator construct containing NeonGreen, both mScarlet (red) and NeonGreen (green) were co-localized, indicating that the full-length intact protein was produced and distributed throughout the cell. Additionally, fluorescence was predominantly observed at the cell membrane, indicating that the protein was correctly localized (due to the presence of SNAP-25).

Example 3 - Confirmation of indicator protein cleavage

Plasmids (pcDNA3,1) were constructed to contain SEQ ID NO: 3, which encodes the BoNT/A light chain, CFP, and an N-terminal SBP tag. Nucleic acid encoding the BoNT/A light chain was synthesized using DNA2.0 (Atum).

Cells from Example 2 stably transfected with indicator constructs (mScarlet-SNAP25-GeNluc or mScarlet-SNAP25-CyanNluc) were transiently transfected with an expression vector containing the CFP-BoNT/A construct. Numerous red, but no green or blue cells, were demonstrated at 24 h and 48 h post-transfection, indicating that the indicator protein was cleaved and the C-terminal fragment was rapidly degraded.

Example 4 - Confirmation of indicator protein cleavage

NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were seeded in 96-well optical plates (ThermoFisher #165305) (20,000-30,000 cells/well) in complete DMEM medium (Corning #50-013-PB) and allowed to attach for 4 h. The medium was then changed to Neurobasal Plus (ThermoFisher #A35829) and the cells were cultured for another 20 h, after which the medium was changed to Neurobasal Plus medium containing 0 (control), 0.1, or 1.0 nM BoNT/A. The cells were cultured for an additional 24 h. Cells were then trypsinized, washed once in medium, and resuspended in DMEM/FBS or DPBS with 10 units of Benzonase/ml at ~2× ¹⁰⁶ cells/ml. Cells were then analyzed on a SY3200 cell sorter (Sony Biotechnologies) using the appropriate lasers/filters for NeonGreen and mScarlet.

Fig. 4 shows the number of green cells per HPF for mScarlet-SNAP25-GeNluc-expressing NG108 cells after treatment with BoNT/A for 24 h. Approximately 25% reduction in green positive cells per HPF was observed for the pool of cells treated with 1 nM BoNT/A.

Example 5 - Confirmation of indicator protein cleavage

Representative NG108 and M17 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were trypsinized, washed once in medium, and resuspended in DMEM/FBS or DPBS with 10 units of Benzonase/ml at ~2× ¹⁰⁶ cells/ml. Cells were then analyzed on a SY3200 cell sorter (Sony Biotechnologies) using the appropriate lasers/filters for NeonGreen and mScarlet.

NG108 cells were excited at 488 nm to directly excite NeonGreen while minimally exciting mScarlet. Fluorescence was measured at different wavelengths. In addition, side and forward scattered light intensity was measured to identify cell subpopulations. Figure 5A is a scatter plot showing side scatter (SS) on the x-axis and forward scatter (FS) on the y-axis: the distribution illustrates the variation of granularity/complexity (SS) and cell size (FS). Figure 5B is a histogram showing the cellular distribution of fluorescence emission intensity measured at 525 nm (FITC filter). Figure 5C is a histogram showing the cellular distribution of fluorescence emission intensity measured at 585 nm (PE filter). Figure 5D is a histogram showing the cellular distribution of fluorescence emission intensity measured at a wavelength of 617 nm (PE-Texas Red filter). Fig. 5E is a histogram showing the cellular distribution of fluorescence emission intensity measured at a wavelength of 665 nm (7AAD filter). Fig. 5F is a histogram showing the cellular distribution of fluorescence emission intensity measured at a wavelength of 785 nm (PE-Cy7 filter). Fig. 5G is a scatter plot showing the fluorescence emission of cells measured at a wavelength of 665 nm (7AAD filter) on the x-axis and the side scatter (SS) on the y-axis. The histograms show two distinct fluorescence peaks, with the smaller fluorescence peak representing non-expressing cells and the larger fluorescence peak representing cells expressing the indicator protein. At all emission measurement wavelengths, fluorescence remains high. This includes the case where emission was measured at 785 nm (Fig. 5F), where most of the emitted light is due to FRET, thus confirming FRET between NeonGreen and mScarlet. The percentage of highly fluorescent cells in the N108 pool ranged from 61% to 93%.

M17 cells were excited at 488 nm to directly excite NeonGreen while minimally exciting mScarlet. Fluorescence was measured at different wavelengths. In addition, side and forward scattered light intensity was measured to identify the cell subpopulation. Figure 6A is a scatter plot showing side scatter (SS) on the x-axis and forward scatter (FS) on the y-axis: the distribution illustrates the variation of granularity/complexity (SS) and cell size (FS). Figure 6B is a histogram showing the cellular distribution of fluorescence emission intensity measured at 525 nm (FITC filter). Figure 6C is a histogram showing the cellular distribution of fluorescence emission intensity measured at 585 nm (PE filter). Figure 6D is a histogram showing the cellular distribution of fluorescence emission intensity measured at a wavelength of 617 nm (PE-Texas Red filter). Fig. 6E is a histogram showing the cellular distribution of fluorescence emission intensity measured at a wavelength of 665 nm (7AAD filter). Fig. 6F is a histogram showing the cellular distribution of fluorescence emission intensity measured at a wavelength of 785 nm (PE-Cy7 filter). Fig. 6G is a histogram showing the cellular distribution of fluorescence emission intensity measured at a wavelength of 665 nm (7AAD filter) on the x-axis and the side scatter (SS) on the y-axis. The histograms show two distinct fluorescence peaks, with the smaller fluorescence peak representing non-expressing cells and the larger fluorescence peak representing cells expressing the indicator protein. At all emission wavelengths measured, fluorescence remains high. This includes the case where emission was measured at 785 nm (Fig. 6F), where most of the emitted light is due to FRET, thus confirming FRET between NeonGreen and mScarlet. The percentage fraction of highly fluorescent cells in the M17 pool ranged from 14% to 24%, i.e., the proportion of cells expressing fluorescent proteins in the M17 cell pool was lower compared to the NG108 cell pool.

NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were treated with BoNT/A at 0 (control), 0.1, or 1.0 nM as described in Example 4.

Fluorescence of a pool of stably transfected NG108 cells was measured using a SY3200 analyzer (Sony Biotechnologies). Cells were excited at 488 nm to directly excite NeonGreen while minimally exciting mScarlet. Fluorescence was measured at 530 nm (FITC filter), which detects NeonGreen fluorescence but not mScarlet fluorescence. Figure 7A is a histogram showing the distribution of fluorescence emission intensity of untreated (control) cells measured at 525 nm. Figure 7B is a histogram showing the distribution of fluorescence emission intensity of cells treated with 0.1 nM BoNT/A measured at 525 nm. Figure 7C is a histogram showing the distribution of fluorescence emission intensity of cells treated with 1 nM BoNT/A measured at 525 nm. NeonGreen fluorescence was reduced by approximately 15% in the cell pool (average fluorescence of cells in the R2 gate) after treatment with 1 nM BoNT/A (Fig. 7C) compared to the untreated control (Fig. 7A), suggesting that the resulting NeonGreen-containing C-terminal fragment is degraded after cleavage.

Flow cytometry was also performed to determine FRET emission. Fluorescence from a stably transfected pool of NG108 cells was measured using a SY3200 analyzer (Sony Biotechnologies). Cells were excited at 488 nm to directly excite NeonGreen while minimally exciting mScarlet. Fluorescence was measured at 785 nm (Cy7 filter), which detects mScarlet fluorescence but not NeonGreen fluorescence. Figure 8A is a histogram showing the distribution of fluorescence emission intensity of untreated (control) cells measured at 785 nm. Figure 8B is a histogram showing the distribution of fluorescence emission intensity of cells treated with 0.1 nM BoNT/A measured at 785 nm. Figure 8C is a histogram showing the distribution of fluorescence emission intensity of cells treated with 1 nM BoNT/A measured at 785 nm. FRET intensity was reduced by 16% (mean fluorescence of cells in gate R6) after treatment with 1 nM BoNT/A (Fig. 8C) compared to the untreated control (Fig. 8A), consistent with the degradation of NeonGreen.

Example 6 - Confirmation of Splitting

Western blotting was performed on NG108 cells from Example 2 stably transfected with the mSclet-SNAP25-GeNluc indicator construct and treated with no toxin (control), 1 nM or 8 nM BoNT/A, or 1 nM, 10 nM, or 100 nM BoNT/E as described in Example 4 (Fig. 9).

Cell lines were tested for cleavage of SNAP-25 (endogenous or exogenous) using standard blotting methods and rabbit anti-SNAP25 primary antibodies.

Cells were lysed using M-PER reagent (ThermoFisher #78501) according to the manufacturer's recommendations. The lysate was cleared at 15 kG for 10 min, and 10 μL of sample was run on a NuPage 12% Bis-Tris gel (ThermoFisher #NP0341BOX) in MOPS buffer (ThermoFisher #NP0001) at 200 V. Proteins were transferred to a PVDF membrane (ThermoFisher #LC2005) using the XCell II blotting system and the Nu-Page transfer protocol. The resulting blot was blocked in 1% BSA/0.05% Tween20/PBS, with 1:3000 anti-SNAP-25 primary antibody, 1:5000 goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (ThermoFisher #31340), and developed in NBT/BCIP substrate (ThermoFisher #34042). The developed blot was scanned, densitometry calculated using ImageJ, and plotted in MS Excel.

The indicator protein was detected in all lysates. No obvious cleavage products were detected in the control samples. Cells treated with either BoNT/A or BoNT/E produced cleavage products that increased with increasing dosage. Interestingly, cells appeared to be about as sensitive to BoNT/E as to BoNT/A.

Example 7 - Transfection with a receptor construct

Plasmids (pD2500; Atum) were constructed to contain nucleic acid encoding the GD3-SV2C-Syt receptor construct and aminoglycoside 3'-phosphotransferase (Neo) (SEQ ID NO: 4). The nucleic acid expressed a fusion protein containing GD3 synthase, SV2C, syntaxin, and Neo, with each domain separated from each other by self-cleaving 2A peptides. Syntaxin was inserted into the fusion protein for use with other BoNT isoforms. Neo confers resistance to G418.

NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were grown in T75 flasks to ~60% confluency. They were then transfected with the receptor construct containing the plasmid (2 μg/ml) in 5 ml OptiMem/polyethyleneimine overnight according to the standard protocol. In the morning, the cells were washed once with fresh complete DMEM and cultured for an additional 24 to 48 h in complete DMEM. The medium was then replaced with complete DMEM containing 500 μg/ml G418 and the cells were cultured for an additional 1 to 2 weeks with medium/G418 changes as needed. Cells were monitored with onset of cell death 3 days after G418 addition and for ~1 week (~60% cell death). Cells remaining after ~2 weeks were resistant to G418.

Example 8 - Directed evolution of cells

The cells from Example 7 were sorted twice to isolate those cells that had the highest fluorescence and thus the highest expression of the indicator protein.

Example 9 - Directed evolution of cells

Cells selected for high expression of the indicator protein in Example 8 were treated in the same manner as described in Example 4, at 0.1 nM, 1 nM, or 10 nM BoNT/A or 10 nM BoNT/E for 72 hours. Following treatment, cells were washed three times, trypsinized, resuspended in fresh medium, and sorted. Screening of the cells showed a clear dose-dependent response to BoNT/A (Fig. 10A). Fluorescence microscopy showed that the cells also decreased in green fluorescence with treatment dose while maintaining the same level of red fluorescence (Fig. 10B). Although the results clearly showed an increase in sensitivity to BoNT/A, it was noted that a similar increase was not observed for BoNT/E.

Cells sensitive to BoNT/A at 1000 pM (1 nM) were selected.

Example 10 - Directed evolution of cells

Wild-type NG108 cells (i.e., not transfected with reporter or receptor constructs) were sorted (Fig. 11A). These cells show neither green nor red fluorescence.

The cells from Example 9, which were sensitive to BoNT/A at 1000 pM (1 nM), were expanded and treated with 100 pM BoNT/A as described previously or were left untreated (control). After treatment for 48 or 96 hours, the cells were washed three times, trypsinized, resuspended in fresh medium, and sorted. Figs. 11B-D show flow cytometry data for the control, cells treated with 100 pM BoNT/A for 48 hours, and cells treated with 100 pM BoNT/A for 96 hours, respectively.

Cells from Example 8 that were double-sorted for high expression of the indicator construct but not sorted in Example 9 for sensitivity to BoNT/A at 1000 pM were treated with 100 pM BoNT/A in the manner previously described for 96 hours or were left untreated (control). Figs. 11E-F show, respectively, flow cytometry data for the control and cells treated with 100 pM BoNT/A for 96 hours.

In Figures 11A-F, the roughly circular gates select cells that show neither red nor green fluorescence (i.e., cells that do not express the indicator protein), the roughly oval gates select cells that show both red and green fluorescence (i.e., cells that express the indicator protein where the indicator protein has not been cleaved), and the four-sided gates select cells that show red fluorescence but relatively reduced green fluorescence (i.e., cells that express the indicator protein where the indicator protein has been cleaved).

Cells previously selected for sensitivity to 1 nM BoNT/A (Fig. 11D) showed significantly greater sensitivity to BoNT/A at 100 pM (greater cleavage) than cells that were not so selected (Fig. 11F).

Cells that were sensitive to BoNT/A at 100 pM after 96 h of treatment (>2 log more sensitive than wild-type NG108) were selected.

Example 11 - Directed evolution of cells

Cells from Example 10, which were sensitive to BoNT/A at 100 pM for 96 hours of treatment, were expanded and treated with 10 pM BoNT/A for 96 hours as described previously or were left untreated (control). After treatment, cells were washed three times, trypsinized, resuspended in fresh medium, and sorted.

Fig. 12A-B show flow cytometry data for control and 10 pM BoNT/A treated cells, respectively. A noticeable shift in fluorescence of treated cells was observed, although not as significant as at the higher toxin concentration.

Example 12 - Receptor construct for BoNT/E

To confer sensitivity to BoNT/E, NG108 cells from Example 2 that were stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were transfected with a plasmid containing the described GD3-SV2A-Syt receptor construct (SEQ ID NO: 5) using the transfection procedure in Example 2. This plasmid was constructed by modifying a plasmid containing the GD2-SV2C-Syt receptor construct using the HiFi kit (New England Biolabs), oligonucleotides from IDT, and SV2A sequences synthesized by GeneArt.

These cells and the cells from Example 7 expressing the GD3-SV2C-Syt receptor construct were cultured in medium containing either BoNT/A at 0 (control), 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM, or BoNT/E at 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0 nM (control). Cells were treated for 16, 40, 64, or 88 hours. After treatment, cells were lysed and Western blotted using an anti-SNAP-25 antibody. Blot densitometry data were displayed as the percentage of SNAP-25 cleavage (Fig. 13). A marked increase in the sensitivity of SV2A-expressing cells to BoNT/A and BoNT/E was observed compared to SV2C-expressing cells, confirming the requirement of SV2A for conferring sensitivity to BoNT/E.

LIST OF SEQUENCES

DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising an N-terminal mScarlet tag, SNAP-25, a C-terminal NeonGreen tag, and a C-terminal luciferase.

SEQ ID NO: 2 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising an N-terminal mScarlet tag, SNAP-25, a C-terminal CFP tag, and a C-terminal luciferase.

SEQ ID NO: 3 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising an N-terminal CFP and a BoNT/A light chain.

SEQ ID NO: 4 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising domains with amino acid sequences for GD3 synthase, SV2C, syntaxin, and aminoglycoside 3'-phosphotransferase. In the fusion protein, each domain is separated from one another by self-cleaving peptide 2A.

SEQ ID NO: 5 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising domains with amino acid sequences for GD3 synthase, SV2A, syntaxin, and aminoglycoside 3'-phosphotransferase. In the fusion protein, each domain is separated from one another by a self-cleaving peptide 2A.

SEQ ID NO: 6 is the amino acid sequence of GD3 synthase.

SEQ ID NO: 7 is the amino acid sequence for self-cleaving peptide 2A encoded by SEQ ID NOs: 4 and 5.

SEQ ID NO: 8 is the amino acid sequence of SV2A.

SEQ ID NO: 9 is the amino acid sequence of SV2B.

SEQ ID NO: 10 is the amino acid sequence of SV2C.

SEQ ID NO: 11 is the amino acid sequence for the fourth luminal domain of SV2A.

SEQ ID NO: 12 is the amino acid sequence for the fourth luminal domain of SV2B.

SEQ ID NO: 13 is the amino acid sequence for the fourth luminal domain of SV2C.

SEQ ID NO: 14 is the amino acid sequence of synaptotagmin I.

SEQ ID NO: 15 is the amino acid sequence of synaptotagmin II.

SEQ ID NO: 16 is the amino acid sequence of MScarlet.

SEQ ID NO: 17 is the amino acid sequence of NeonGreen.

SEQ ID NO: 18 is the amino acid sequence of CFP.

SEQ ID NO: 19 is the amino acid sequence of SNAP-25.

SEQ ID NO: 20 is the amino acid sequence for aminoglycoside 3'-phosphotransferase (Neo).

SEQ ID NO: 21 is the amino acid sequence for puromycin N-acetyltransferase (PuroR).

SEQ ID NO: 22 is the amino acid sequence of luciferase.

SEQ ID NO:1

ATGGTGTCGAAGGGGGAAGCGGTGATCAAGGAGTTCATGAGGTTTTAAAGTGCATATGGAGGGATCTATGAACGGACACGAGTTTGAGATCGAAGGGGAAGGAGAGGGGCCCATACGAAGGCACCCAGACTGCCAAGCTGAAAGTCACAAAGGGTGGACCCTTGCCCTTCTCGTGGGATATTCTGAGCCCGCAATTCATGTACGGGTCCCGGGCCTTCACCAAGCACCCTGCTGACATTCCG GATTACTATAAGCAGAGCTTCCCGGAAGGCTTCAAATGGGAGCGAGTGATGAACTTCGAGGATGGAGGCGCCGTGACCGTGACTCAGGACACTTCACTGGAAGATGGCACTCTGATCTA CAAGGTCAAGCTGCGGGGCACCAACTTCCCACCGGACGGACCGGTCATGCAGAAAAAGACCATGGGATGGGAGGCCTCCACCGAGCGCCTGTACCCCGAAGATGGAGTCCTCAAGGGGGACATCAAGATGGCCCTGCGGCTCAAGGATGGTGGAAGATACCTGGCTGACTTCAAGACCACGTACAAGGCCAAGAAGCCAGTCCAGATGCCCGGCGCGTACAATGTGGATCGCAAGCTGGACATC ACTTCCCACAACGAGGACTACACCGTGGTGGAGCAGTACGAACGGTCCGAGGGTCGGCACTCCACTGGTGGCATGGACGAGCTGTACAAAATGGCCGAGGATGCAGACATGAGAAACG AACTGGAAGAAATGCAGCGGAGAGCAGACCAGCTCGCGGACGAATCACTGGAATCGACCCGCCGGATGCTTCAACTGGTCGAGGAATCAAAGGACGCGGGTATCCGGACCCTTGTGATGCTGGACGAACAGGGAGAGCAGCTGGAGAGGATCGAAGAGGGAATGGACCAGATTAACAAGGACATGAAGGAAGCGGAAAAGAACCTCACCGACCTTGGAAAGTTCTGCGGGTTGTGCGTGTGTCCG TGCAACAAGCTGAAGTCCTCCGACGCCTACAAGAAGGCCTGGGGAAACAACCAGGACGGTGTCGTGGCTTCCCAACCCGCCACGGGTGGTGGATGAGCGGGAACAGATGGCGATTTCC GGAGGCTTCATTAGACGCGTGACCAACGACGCCCGCGAAAACGAGATGGACGAAAACCTGGAACAAGTGTCGGGAATCATCGGAAACTTGAGACACATGGCCCTCGACATGGGCAACGAAATTGATACACAGAACCGGCAGATTGACCGGATCATGGAAAAGGCAGACTCAAACAAGACTCGGATTGACGAAGCGAACCAGAGGCCACTAAGATGTTGGGTTCCGGGATGGTGTCAAAGGGAGAAGAA GACAACATGGCATCACTGCCCGCCACCCACGAGCTGCACATCTTCGGTTCCATCAACGGGGTCGACTTCGACATGGTCGGCCAGGGAACTGGAAACCCGAATGACGGTTATGA AGAACTGAACCTTAAAATCAACCAAGGGGGACCTTCAGTTCTCGCCCTGGATTTTGGTCCCTCACATTGGATACGGATTCCATCAGTATCTGCCGTACCCCGACGGAATGAGCCCGTTCCAGGCTGCCATGGTGGACGGATCGGGATACCAGGTCCACCGCACCATGCAGTTTGAAGATGGCGCAAGCCTGACCGTGAACTACCGGTATACCTACGAGGGCTCACACATCAAGGGGGAAG CCCAAGTCAAGGGTACCGGCTTCC CGGCCGACGGACCAGTGATGACCAACTCCTTGACCGCCGCCGACTGGTGCCGCAGCAAGAAAACTTACCCCAACGATAAGACAATCATCTCCACTTTCAAGTGGTCCTACACCACGGGCAACGGCAAACGCTACCGAAGCACTGCACGGACCACCTACACTTTCGCGAAGCCTATGGCCGCCAACTACCTGAAGAACCAGCCGATGTACGTGTTCAGAAAGACGGAACTCAAGCACTCCAAAACCGAACTGA ACTTAAGGAGT GGCAGAAGGCTTTCACTGGATTCGAGGACTTTGTCGGCGACTGGCGCCAGACTGCCGGCTACAACCTGGACCAAGTGCTCGAACAGGGGGGTGTCTCCAGCCTCTTCCAAAATCTGGGCGTGTCCGTGACCCCGATCCAGCGGATCGTGCTCAGCGGGGAAAACGGCCTGAAGATCGATATCCACGTCATCATCCCGTACGAGGGACTGAGCGGCGACCAGATGGGTCAGATCGAAAAGATTTTCAAGGTG GTCTATCCCGTG GATGACCACCACTTCAAAGTGATCCTGCATTACGGGACCCTCGTGATCGACGGCGTCACCCCGAACATGATTGATTACTTCGGACGGCCTTATGAAGGGATCGCCGTGTTCGACGGCAAAAAGATCACCGTGACTGGCACCCTGTGGAACGGAAATAAGATCATTGACGAGCGGCTGATCAACCCAGACGGGTCGCTGCTGTTCCGCGTGACCATCAACGGAGTGACCGGCTGGCGGCTGTGCG AGCGCATCCTCGCCTGATAG

SEP ID NO: 2

ATGGTGTCGAAGGGGGAAGCGGTGATCAAGGAGTTCATGAGGTTTTAAAGTGCATATGGAGGGATCTATGAACGGACACGAGTTTGAGATCGAAGGGGAAGGAGAGGGGCCCATACGAAGGCACCCAGACTGCCAAGCTGAAAGTCACAAAGGGTGGACCCTTGCCCTTCTCGTGGGATATTCTGAGCCCGCAATTCATGTACGGGTCCCGGGCCTTCACCAAGCACCCTGCTGACATTCCGGA TTACTATAAGCAGAGCTTCCCGGAAGGCTTCAAATGGGAGCGAGTGATGAACTTCGAGGATGGAGGCGC CGTGACCGTGACTCAGGACACTTCACTGGAAGATGGCACTCTGATCTACAAGGTCAAGCTGCGGGGCACCAACTTCCCACCGGACGGACCGGTCATGCAGAAAAAGACCATGGGATGGGAGGCCTCCACCGAGCGCCTGTACCCCGAAGATGGAGTCCTCAAGGGGGACATCAAGATGGCCCTGCGGCTCAAGGATGGTGGAAGATACCTGGCTGACTTCAAGACCACGTACAAGGCCAAGAGCCAG TCCAGATGCCCGGCGCGTACAATGTGGATCGCAAGCTGGACATCACTTCCCACAACGAGGACTACAC CGTGGTGGAGCAGTACGAACGGTCCGAGGGTCGGCACTCCACTGGTGGCATGGACGAGCTGTACAAAATGGCCGAGGATGCAGACATGAGAAACGAACTGGAAGAAATGCAGCGGAGAGCAGACCAGCTCGCGGACGAATCACTGGAATCGACCCGCCGGATGCTTCAACTGGTCGAGGAATCAAAGGACCGCGGGTATCCGGACCCTTGTGATGCTGGACGAACAGGGAGAGCAGCTGGAGAGGA TCGAAGAGGGAATGGACCAGATTAACAAGGACATGAAGGAAGCGGAAAAGAACCTCACCGACCTTGGAA AGTTCTGCGGGTTGTGCGTGTGTCCGTGCAACAAGCTGAAGTCCTCCGACGCCTACAAGAAGGCCTGGGGAAACAACCAGGACGGTGTCGTGGCTTCCCAACCCGCACGGGTGGTGGATGAGCGGGAACAGATGGCGATTTCCGGAGGCTTCATTAGACGCGTGACCAACGACGCCCGCGAAAACGAGATGGACGAAAACCTGGAACAAGTGTCGGGAATCATCGGAAACTTGAGACA CATGGGCCCTCGACATGGGCAACGAAATTGATACACAGAACCGGCAGATTGACCGGATCATGGAAAAGGCAGACTCAA ACAAGACTCGGATTGACGAAGCGAACCAGAGGGCCACTAAGATGTTGGGTTCCGGGATGGTGTCAAAGGGAGAAGAACTGTTCACTGGAGTGGTGCCCATCCTGGTGGAGCTGGATGGCGATGTGAACGGCCATAAATTCTCAGTCAGCGGAGAGGGAGAGGGCGATGCGACTTACGGAAAGCTGACTTTGAAGTTTATCTGCACTACCGGAAAGCTGCCTGTGCCATGGCCTACCCTCGTGAC CACCCTGTCCTGGGGCGTCCAATGTTTCGCACGCTACCCTGACCATATGAAGCAGCACGACTTCTTCAAG TCCGCCATGCCCGAGGGCTACGTGCAGGAACGCACCATCTTCTTCAAGGACGACGGGAACTACAAAACCAGGGCTGAAGTGAAGTTCGAGGGAGACACCCTGGTCAATCGGATTGAATTGAAGGGAATCGATTTCAAGGAAGATGGAAACATCCTGGGACATAAGCTTGAGTACAACTACTTCTCCGACAACGTGTACATCACGGCCGATAAGCAGAAGAACGGAATCAAAGCTAACTTCAAGATTCGGC ACAACATTGAGGACGGCGGCGTCCAGCTGGCGGACCATTATCAGCAGAATACCCCTATTGGGGAT GGACCGGTGCTGCTCCCGGACAACCATTACCTGTCCACCCAATCTAAGCTGAGCAAGGACCCAAACGAGAAGCGCGATCACATGGTGCTGCTCGAGTTCGTGACTGCCGCCGGGCTTCACACACTTGAGGACTTTGTCGGCGACTGGCGCCAGACTGCCGGCTACAACCTGGACCAAGTGCTCGAACAGGGGGGTGTCTCCAGCCTCTTCCAAAATCTGGGCGTGTCCGTGACCCCGATCCAGC GGATCGTGCTCAGCGGGAAAACGGCCTGAAGATCGATATCCACGTCATCATCCCGTACGAGGGACTGAGC GGCGACCAGATGGGTCAGATCGAAAAGATTTTCAAGGTGGTCTATCCCGTGGATGACCACCACTTCAAAGTGATCCTGCATTACGGGACCCTCGTGATCGACGGCGTCACCCCGAACATGATTGATTACTTCGGACGGCCTTATGAAGGGATCGCCGTGTTCGACGGCAAAAAGATCACCGTGACTGGCACCCTGTGGAACGGAAATAAGATCATTGACGAGCGGCTGATCAACCCAGACGGGTC GCTGCTGTTCCGCGTGACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATCCTCGCCTGATAG

SEP ID NO: 3

ATGACCATGGATGAGCAGCAATCGCAGGCTGTAGCCCCGGTATATGTCGGTGGTATGGATGAGAAAACGACTGGGTGGCGGGGTGGACACGTCGTCGAGGGCCTGGCAGGCGAACTTGAACAACTGCGGGCTCGCTTGGAGCACCACCCGCAAGGACAGCGCGAGCCGTCCATGGTGTCAAAGGGGGAGGAACTGTTT ACTGGGGTCGTCCCTATCTTGGTGGAACTCGACGGGGATGTGAACGGACACAAGTTTTCGGTATCCGGGGAAGGCGAGGGGGATGCCACgTATGGAAAGCTCACACTTAAGTTCATCTGCACGACAGGGAAGCTCCCAGTGCCTTGGCCCACGTTGGTGACTACGCTCACATGGGGTGTCCAGTGCTTCGCACGGTATCCCGACCAcATGAAGCAGCATGATTTCTTTAAGTCAGCCATGCC GGAGGGATATGTAC AAGAAAGGACCATCTTCTTCAAAGATGACGGTAACTACAAGACCAGAGCCGAGGTAAAGTTTGAAGGCGACACTCTCGTGAACAGGATTGAGCTGAAGGGAATTGATTTCAAAGAGGATGGGAACATCCTTGGTCACAAATTGGAGTACAATGCCATTTCGGATAACGTGTACATTACAGCGGATAAGCAGAAGAATGGGATCAAAGCGAATTTCAAAATCAGGCATAACATCGAGGACGGGTCGGTGCAGC TCGCC GACCATTACCAGCAGAATACGCCCATCGGAGATGGACCCGTACTTCTGCCCGACAATCATTATCTGTCAACGCAATCAGCGCTTAGCAAAGATCCCAATGAGAAAAGGGACCACATGGTGCTCCTCGAATTtGTGACGGCAGCGGGAATTACCCTCGGGATGGACGAACTGTACAAAAGCGGGTTGAGACTCGAGCGCTGAACTCGAGATGCCTTTTGTCAACAAGCAGTTTAACTATAAGGATCCCG TGAATGGT GTGGACATTGCCTACATCAAGATTCCAAACGCTGGACAAATGCAGCCCGTCAAGGCTTTCAAAATTCACAACAAGATCTGGGTGATCCCGGAGAGGGACACCTTTACCAATCCAGAAGAGGGCGACCTTAACCCTCCGCCAGAGGCCAAACAGGTGCCCGTGAGCTATTACGACTCAACTTATCTCTCCACCGACAACGAAAAGGACAATTACCTCAAGGGAGTCACCAAGCTGTTCGAACGGATCTACT CTACCGA TCTCGGCAGGATGCTCCTGACTTCTATCGTGCGGGGCATCCCCTTCTGGGGTGGGAGCACCATTGACACCGAACTGAAGGTGATTGATACCAATTGCATCAACGTCATCCAGCCAGACGGTTCCTACCGGTCTGAGGAGCTCAATCTTGTGATTATTGGCCCGTCAGCTGATATCATCCAGTTCGAATGCAAGTCTTTCGGACACGACGTGCTTAATCTCACCCGCAATGGTTACGGAAGC ACCCAGTACATCAGA TTCTCTCCGGACTTTACTTTCGGATTTGAAGAGTCACTGGAAGTCGACACCAATCCTGCTCGGAGCCGGAAAGTTCGCCACCGACCCTGCAGTGACCCTTGCTCACGAGCTGATTCATGCAGAGCATCGCCTGTACGGGATCGCCATCAATCCTAACCGCGTGTTTAAGGTCAATACCAACGCTTACTATGAAATGAGCGGACTGGAGGTGTCCTTCGAGGAACTGCGCACCTTCGGA GGTCATGACGTAAGTT CATCGACTCACTGCAAGAGAATGAGTTCCGGCTGTACTATTACAACAAGTTTAAGGATGTCGCCTCAACTCTGAACAAGGCCAAAAGCATCATCGGCACCACCGCCAGCCTGCAATACATGAAAAACGTGTTCAAGGAAAAGTACCTTCTTAGCGAAGATACTTCCGGGAAGTTTTCAGTCGACAAACTGAAGTTCGACAAGCTGTACAAGATGCTCACCGAAATCTACACCGAGGACAATTTTG TGAACTTCTTC AAAGTGATTAACAGAAAGACCTACTGAACTTCGACAAAGCCGTGTTCCGGATTAACATTGTGCCCGATGAGAACTACACTATCAAGGACGGGTTCAACCTTAAGGGTGCAAATCTTTCAACTAATTTCAACGGACAGAATACTGAGATCAATTCAAGGAACTTCACTAGACTCAAGAATTTCACTGGGCTTTTCGAGTTCTATAAGCTGCTGTGTGTCCGCGGAATTATCCCCTTCAAGTGAAGCTTCG TCAATGA

SEP ID NO: 4

ATGTCCCCATGTGGACGAGCGCGCAGACAGACCTCAAGAGGAGCGATGGCCGTGCTGGCCTGGAAGTTCCCGAGGACTCGCCTGCCCATGGGAGCCTCTGCTCTGTGTGTGGTCGTGCTGTGTTGGCTGTACATCTTCCCGGTGTACCGGCTGCCTAACGAAAAGGAAATTGTGCAGGGCGTGCTCCAGCAGGGGACCGCTTGGCGGCGCAACCAGACCGCTGCGAGGGCTTTTCGGAAG CAGATGGAAGATTGTTGCGACCCCGCCCATCTTTTCGCGATGACCAAGATGAACAGCCCGATGGGAAAGTCCATGTGGTACGACGGAGAGTTCCTGTATTCCTTCACCATTGACAACAGCACTTACTCACTGTTCCCGC AAGCCACCCCCTTCCAACTGCCGCTTAAGAAGTGCGCCGTCGTGGGGAACGGCGGCATCCTCAAGAAGTCCGGATGCGGGCGCCAGATTGATGAAGCCAACTTCGTGATGCGGTGCAATCTCCCGCCACTCTCGTCCGAGTACACCAAGGACGTGGGGTCAAAGTCGCAGCTCGTCACCGCCAACCCTTCGATCATCAGACAACGGTTCCAGAACCTTCTGTGGAGCCGGAAAACATTTG TGGATAACATGAAGATCTACAACCATTCCTACATCTATATGCCTGCCTTCTCCATGAAAACTGGAACCGAACCCTCCCTGAGAGTGTACTACACCCTGTCCGACGTGGGCGCAAACCAGACCGTCCTTTTCGCCAACCCC AACTTCCTGCGCTCCATCGGAAAGTTCTGGAAGTCCAGAGGCATTCACGCGAAACGCTTGTCCACTGGATTGTTCTTGGTGTCCGCCGCTCTGGGCCTGTGCGAGGAAGTGGCCATATACGGATTCTGGCCTTTCTCCGTCAACATGCACGAGCAGCCCATCTCCCACCATTATTACGACAATGTCCTGCCTTTCTCGGGATTTCACGCGATGCCCGAGGAGTTCTTGCAACTGTGGTACCTTCA CAAGATCGGTGCCCTGCGGATGCAGCTGGACCCTTGCGAGGACACCTCGCTGCAACCCACCTCGGAGCAGAAACTCATTTCCGAAGAGGATCTGAACGGGGAGCAGAAGCTCATCTCCGAGGAGGACCTGAACG GAGAACAGAAGCTGATTAGCGAAGAGGACCTGGGCAGCGGTGCCACCAATTTTTCTCTGCTCAAGCAGGCCGGAGATGTGGAAGAGAACCCGGGTCCCATGGAGGACTCCTACAAAGATCGGACTTCTCTGATGAAGGGAGCCAAGGACATCGCCAGGGAAGTGAAGAAGCAAACCGTCAAGAAGGTCAACCAGGCCGTGGACAGAGCCCAGGACGAGTACACCCAGCGGTCGTACTCGCG GTTCCAGGATGAAGAGGATGACGACGACTACTACCCTGCCGGCGAAACCTATAATGGGGAAGCCAACGATGACGAAGGCTCCAGCGAAGCCACTGAAGGACACGACGAGGACGACGAAATCTACGAAGGAGAATACCAG GGCATCCCTTCGATGAATCAGGCCAAAGATTCAATTGTGTCAGTGGGACAGCCTAAGGGCGACGAGTACAAGGACCGGAGAGAGCTCGAAAGCGAGCGGAGGGCCGACGAAGAGGAACTGGCACAACAGTACGAGCTGATCATCCAGGAGTGTGGGCACGGCCGGTTCCAGTGGGCGCTGTTCTTCGTGCTCGGAATGGCACTGATGGCCGACGGCGTGGAAGTGTTCGTGGTCGGATTC GTGCTGCCCTCGGCCGAAACCGACCTCTGCATTCCCAACTCCGGCTCGGGATGGCTGGGGTCCATCGTGTACCTGGGAATGATGGTCGGCGCCTTCTTCTGGGGTGGCCTGGCAGACAAGGTCGGCCGGAAGCAGTCCC TCTTGATCTGCATGAGCGTCAACGGATTTTTCGCCTTCCTGTCATCATTCGTGCAAGGTTACGGGTTCTTCCTTTTCTGCCGCCTGCTGTCCGGCTTTGGGATCGGCGGGGCTATTCCGACTGTGTTCTCCTACTTTGCCGAAGTGCTGGCTCGCGAAAAACGGGGCGAACACCTTTCCTGGCTGTGTATGTTCTGGATGATCGGCGGCATCTACGCCTCGGCCATGGCCTGGGCTATTATC CCGCATTATGGGTGGTCCTTCTCAATGGGAAGCGCATACCAGTTCCATTCGTGGCGGGTGTTCGTGATCGTGTGCGCCCTCCCGTGTGTGTCCTCCGTGGTGGCTCTGACATTCATGCCGGAGTCACCTCGGTTCTTG TTGGAAGTCGGGAAGCACGACGAAGCCTGGAATGATTCTGAAGCTGATCCACGACACTAATATGCGGGCCCGGGGACAGCCTGAGAAAGTGTTCACCGTCAACAAGATTAAGACCCCGAAGCAAATCGATGAACTGATTGAAATTGAGTCCGACACGGAACTTGGTACCGCCGGTGCTTCGTGCGGATTCGCACCGAGCTGTACGGAATCTGGCTCACCTTCATGCGCTGCTTCAACTAC CCCGTGCGCGACAACACCATCAAGCTGACCATCGTGTGGTTCACTCTGTCTTTCGGCTACTATGGGCTGTCAGTGTGGTTCCCGGATGTCATCAAGCCGCTCCAATCCGATGAATACGCCCTGCTGACCCGCAATGTGG AGAGAGACAAATACGCCAACTTCACCATCAATTTCACCATGGAAAACCAGATTCACACCGGAATGGAGTACGACAATGGACGATTCATCGGAGTGAAGTTCAAGAGCGTGACCTTCAAGGACTCGGTGTTCAAGTCCTGTACCTTCGAGGACGTGACCAGCGTGAACACTTATTTTAAATTGCACCTTCATCGATACTGTGTTCGATAACACCGACTTCGAGCCCTATAAGTTCATTGACTCG GAGTTCAAGAACTGTTCATTCTTCCACAACAAGACTGGTTGCCAGATCACCTTCGATGACGACTACAGCGCCTACTGGATCTACTTTGTGAACTTTTTGGGAACTCTCGCAGTGCTTCCTGGCAACATTGTGTCC GCACTCCTGATGGATCGGATTGGCAGGCTCACGATGCTTGGGGGGTCCATGGTCCTCTCCGGGATCTCGTGCTTCTTCCTGTGGTTCGGCACCTCGGAGTCCATGATGATCGGAATGTTGTGCCTGTACAACGGTCTGACCATCAGCGCCTGGAACAGCCTCGACGTGGTCACCGTCGAGCTGTATCCTACCGACCGGCGCGCGCGACAGGCTTCGGATTCCTGAACGCACTGTGCAAGGC AGCCGCGGTCCTGGGAAATCTGATCTTTGGTTCGCTGGTGTCCATCACTAAGAGCATCCCTATTCTGCTCGCCTCCACGGTGCTCGTGTGTGGTGGCCTG GTCGGGCTGTGCCTGCCCGACACTCGCACCCAAGTGCTCATGGACTACAAGGATGACGATGATAAGGGAGACTACAAGGACGATGACGACAAGGGGGATTACAAGGACGACGATGACAAAGGAAGCGGCGCCACTAACTTTTCCCTGCTGAAGCAGGCCGGGGACGTCGAAGAAAACCCCGGGCCAATGCGCAACATTTTCAAGCGGAATCAGGAGCCTATCGTGGCCCCGGCCACCACTACCGCCACTATGC CTATTGGACCTGTCGACAACTCCACGGAATCAGGCGGCGCCGGCGAATCCCAAGAGGACATGTTCGCCAAGCTGAAGGAGAAACTGT TCAACGAAATCAACAAGATTCCCCTCCCGCCGTGGGCCCTGATCGCTATCGCTGTCGTCGCCGGACTGCTGCTGCTTACTTGCTGCTTCTGCATTTGCAAGAAGTGTTGTTGCAAGAAAAAGAAAAACAAGAAGGAAAAGGGGAAGGGAATGAAGAACGCCATGAATATGAAGGACATGAAGGGCGGACAGGATGATGATGATGCTGAAACTGGGCTGACTGAGGGCGAAGGAGAGGGCGAAGAGGAGA AGGAACCTGAGAACCTGGGAAAGCTCCAATTCTCCCTGGATTACGACTTCCAAGCCAACCAGCTGACTGTGGGAGTGTTGCAAGCCGCCGA GCTGCCAGCCCTGGACATGGGCGGCACCTCCGACCCCTATGTGAAGGTGTTCTTGCTGCCTGACAAGAAGAAGAAGTACGAAACCAAGGTGCACCGCAAGACCCTGAACCCCGCTTTCAACGAACCCTTCACTTTCAAAGTGCCCTACCAAGAGCTCGGGGGAAAGACTCTCGTGATGGCGATCTACGACTTCGACCGGTTCAGCAAGCACGATATCATCGGGGAGGTCAAGGTCCCGATGA ACACCGTGGACCTTGGCCAACCGATTGAAGAATGGCGCGATCTCCAGGGTGGCGAAAAGGAGGAGCCCGAGAAACTGGGTGACATCTGTACATCACTG CGCTACGTGCCGACCGCCGGGAAGCTCACTGTCTGCATCCTGGAGGCCAAGAACCTGAAGAAAATGGACGTGGGCGGGCTCTCCGACCCTTACGTGAAGATCCACCTGATGCAGAACGGAAAGCGGCTGAAGAAGAAGAAAACCACTGTGAAGAAAAAGACTCTGAACCCCTACTTCAACGAGTCGTTCTCCTTCGAAATCCCGTTTGAGCAAATCCAGAAGGTCCAAGTGGTCGTGACTGTG CTTGACTACGACAAGCTCGAAAGAACGAGGCCATTGGCAAAATCTTCGTGGGATCGAACGCAACTGGCACCGAGCTGAGACACTGGTCTGACATG CTCGCCAACCCAAGGCGGCCGATTGCTCAGTGGCACTCCTTGAAACCTGAGGAAGAAGTGGATGCCCTTCTTGGAAAGAACAAGATGTACCCCTACGACGTCCCTGATTACGCGGGATACCCGTACGATGTGCCTGACTATGCCGGCTACCCGTACGATGTGCCAGACTACGCTGGCTCCGGAGCCACGAACTTTTCGCTGCTGAAACAGGCCGGCGACGTGGAAGAAAATCCCGGTCCAAT GATTGAACAAGATGGATTGCACGCTGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATG CCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAG TATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACATGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCG GATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAGGAACATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATT GCTGAGGAACTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCTTCTATCGCCTTCTT GACGAGTTCTTCTGATAG

SEP ID NO: 5

ATGTCCCCATGTGGACGAGCGCGCAGACAGACCTCAAGAGGAGCGATGGCCGTGCTGGCCTGGAAGTTCCCGAGGACTCGCCTGCCCATGGGAGCCTCTGCTCTGTGTGTGGTCGTGCTGTGTTGGCTGTACATCTTCCCGGTGTACCGGCTGCCTAACGAAAAGGAAATTGTGCAGGGCGTGCTCCAGCAGGGGACCGCTTGGCGGCGCAACCAGACCGCTGCGAGGGCTTTTCGGA AGCAGATGGAAGATTGTTGCGACCCCGCCCATCTTTTCGCGATGACCAAGATGAACAGCCCGATGGGAAAGTCCATGTGGTACGACGGAGAGTTCCTGTATTCCTTCACCATTGACAACAG CACTTACTCACTGTTCCCGCAAGCCACCCCCTTCCAACTGCCGCTTAAGAAGTGCGCCGTCGTGGGGAACGGCGGCATCCTCAAGAAGTCCGGATGCGGGCGCCAGATTGATGAAGCCAACTTCGTGATGCGGTGCAATCTCCCGCCACTCTCGTCCGAGTACACCAAGGACGTGGGGTCAAAGTCGCAGCTCGTCACCGCCAACCCTTCGATCATCAGACAACGGTTCCAGAACCT TCTGTGGAGCCGGAAAACATTTGTGGATAACATGAAGATCTACAACCATTCCTACATCTATATGCCTGCCTTCTCCATGAAAACTGGAACCGAACCCTCCCTGAGAGTGTACTACACCCTGTC CGACGTGGGCGCAAACCAGACCGTCCTTTTCGCCAACCCCAACTTCCTGCGCTCCATCGGAAAGTTCTGGAAGTCCAGAGGCATTCACGCGAAACGCTTGTCCACTGGATTGTTCTTGGTGTCCGCCGCTCTGGGCCTGTGCGAGGAAGTGGCCATATACGGATTCTGGCCTTTCTCCGTCAACATGCACGAGCAGCCCATCTCCCACCATTATTACGACAATGTCCTGCCTTTCTCGGGATTTCA CGCGATGCCCGAGGAGTTCTTGCAACTGTGGTACCTTCACAAAGATCGGTGCCCTGCGGATGCAGCTGGACCCTTGCGAGGACACCTCGCTGCAACCCACCTCGGAGCAGAAAC TCATTTCCGAAGAGGATCTGAACGGGGAGCAGAAGCTCATCTCCGAGGAGGACCTGAACGGAGAACAGAAGCTGATTAGCGAAGAGGACCTGGGCAGCGGTGCCACCAATTTTTCTCTGCTCAAGCAGGCCGGAGATGTGGAAGAGAACCCGGGTCCCATGGAGGACTCCTACAAAGATCGGACTTCTCTGATGAAGGGAGCCAAGGACATCGCCAGGGAAGTGAAGAAGCAAACCGTCAAGA AGGTCAACCAGGCCGTGGACAGAGCCCAGGACGAGTACACCCAGCGGTCGTACTCCGGGTTCCAGGATGAAGAGGATGACGACGACTACTACCCTGCCGGCGAAACCTAATGGGG AAGCCAACGATGACGAAGGCTCCAGCGAAGCCACTGAAGGACACGACGAGGACGACGAAATCTACGAAGGAATACCAGGGCATCCCTTCGATGAATCAGGCCAAAGATTCAATTGTGTCAGTGGGACAGCCTAAGGGCGACGAGTACAAGGACCGGAGAGAGCTCGAAAGCGAGCGGAGGGCCGACGAAGAGGAACTGGCACAACAGTACGAGCTGATCATCCAGGAGTGTGGGCACGGCCGG TTCCAGTGGGCGCTGTTCTTCGTGCTCGGAATGGCACTGATGGCCGACGGCGTGGAAGTGTTCGTGGTCGGATTCGTGCTGCCCTCGGCCGAAACCGACCTCTGCATTCCCAAC TCCGGCTCGGGATGGCTGGGGTCCATCGTGTACCTGGGAATGATGGTCGGCGCCTTCTTCTGGGGTGGCCTGGCAGACAAGGTCGGCCGGAAGCAGTCCCTCTTGATCTGCATGAGCGTCAACGGATTTTTCGCCTTCCTGTCATCATTCGTGCAAGGTTACGGGTTCTTCCTTTTCTGCCGCCTGCTGTCCGGCTTTGGGATCGGCGGGGCTATTCCGACTGTGTTCTCCTACTTTG CCGAAGTGCTGGCTCGCGAAAAACGGGGCGAACACCTTTCCTGGCTGTGTATGTTCTGGATGATCGGCGGCATCTACGCCTCGGCCATGGCCTGGGCTATTATCCCGCATTATGGGTGGTCC TTCTCAATGGGAAGCGCATACCAGTTCCATTCGTGGCGGGTGTTCGTGATCGTGTGCGCCCTCCCGTGTGTGTCCTCCGTGGTGGCTCTGACATTCATGCCGGAGTCACCTCGGTTCTTGTTGGAAGTCGGGAAGCACGACGAAGCCTGGATGATTCTGAAGCTGATCCACGACACTAATATGCGGGCCCGGGGACAGCCTGAGAAAGTGTTCACCGTCAACAAGATTAAGACCCCG AAGCAAATCGATGAACTGATTGAAATTGAGTCCGACACCGGAACTTGGTACCGCCGGTGCTTCGTGCGGATTCGCACCGAGCTGTACGGAATCTGGCTCACCTTCATGCGCTGCTTCAACTAC CCCGTGCGCGACAACACCATCAAGCTGACCATCGTGTGGTTCACTCTGTCTTTCGGCTACTATGGGCTGTCAGTGTGGTTCCCGGATGTCATCAAGCCGCTCCAATCCGATGAATACGCCCTGCTGACCCGCAATGTGGAGAGACAAATACGCCAACTTCACCATCAATTTTCACCATGGAAAACCAGATTCACACCGGAATGGAGTACGACAATGGACGATTCATCGGAGTGAAGTTCA AGAGCGTGACCTTCAAGGACTCGGTGTTCAAGTCCTGTACCTTCGAGGACGTGACCAGCGTGAACACTTATTTTAAGAATTGCACCTTCATCGATACTGTGTTCGATAACACCGACTTC GAGCCCTATAAGTTCATTGACTCGGAGTTCAAGAACTGTTCATTCTTCCACAACAAGACTGGTTGCCAGATCACCTTCGATGACGACTACAGCGCCTACTGGATCTACTTTGTGAACTTTTTGGGAACTCTCGCAGTGCTTCCTGGCAACATTGTGTCCGCACTCCTGATGGATCGGATTGGCAGGCTCACGATGCTTGGGGGGTCCATGGTCCTCTCCGGGATCTCGTGCTTCTTCCTGTGGT TCGGCACCTCGGAGTCCATGATGATCGGAATGTTGTGCCTGTACAACGGTCTGACCATCAGCGCCTGGAACAGCCTCGACGTGGTCACCGTCGAGCTGTATCCTACCGACCGGCG CGCGACAGGCTTCGGATTCCTGAACGCACTGTGCAAGGCAGCCGCGGTCCTGGGAAATCTGATCTTTGGTTCGCTGGTGTCCATCACTAAGAGCATCCCTATTCTGCTCGCCTCCACGGTGCTCGTGTGTGGTGGCCTGGTCGGGCTGTGCCTGCCCGACACTCGCACCCAAGTGCTCATGGACTACAAGGATGACGATGATAAGGGAGACTACAAGGACGATGACGACAAGGGGGATTACAAGGA CGACGATGACAAAGGAAGCGGCGCCACTAACTTTTCCCTGCTGAAGCAGGCCGGGGACGTCGAAGAAAACCCCGGGCCAATGCGCAACATTTTCAAGCGGAATCAGGAGCCTAT CGTGGCCCCGGCCACCACTACCGCCACTATGCCTATTGGACCTGTCGACAACTCCACGGAATCAGGCGGCGCCGGCGAATCCCAAGAGGACATGTTCGCCAAGCTGAAGGAGAAACTGTTCAACGAAATCAACAAGATTCCCCTCCCGCCGTGGGCCCTGATCGCTATCGCTGTCGTCGCCGGACTGCTGCTGCTTACTTGCTGCTTCTGCATTTGCAAGAAGTGTTGTTGCAAGAAAAAGAAAAAA CAAGAAGGAAAAGGGGAAGGGAATGAAGAACGCCATGAATATGAAGGACATGAAGGGCGGACAGGATGATGATGATGCTGAAACTGGGCTGACTGAGGGCGAAGGAGAGGGCG AAGAGGAGAAGGAACCTGAGAACCTGGGAAAGCTCCAATTCTCCCTGGATTACGACTTCCAAGCCAACCAGCTGACTGTGGGAGTGTTGCAAGCCGCCGAGCTGCCAGCCCTGGACATGGGCGGCACCTCCGACCCCTATGTGAAGGTGTTCTTGCTGCCTGACAAGAAGAAGAAGTACGAAACCAAGGTGCACCGCAAGACCCTGAACCCCGCTTTCAACGAAACCTTCACTTTCAAAG TGCCCTACCAAGAGCTCGGGGGAAAGACTCTCGTGATGGCGATCTACGACTTCGACCGGTTCAGCAAGCACGATTCATCGGGGAGGTCAAGGTCCCGATGAACACCGTGGACCTTGGCC AACCGATTGAAGAATGGCGCGATCTCCAGGGTGGCGAAAAGGAGGAGCCCGAGAAACTGGGTGACATCTGTACATCACTGCGCTACGTGCCGACCGCCGGGAAGCTCACTGTCTGCATCCTGGAGGCCAAGAACCTGAAGAAAATGGACGTGGGCGGGCTCTCCGACCCTTACGTGAAGATCCACCTGATGCAGAACGGAAAGCGGCTGAAGAAGAAGAAAACCACTGTGAAGAAAAAGACTCTGAAC CCCTACTTCAACGAGTCGTTCTCCTTCGAAATCCCGTTTGAGCAAATCCAGAAGGTCCAAGTGGTCGTGACTGTGCTTGACTACGACAAGCTCGAAAGAACGAGGCCATT GGCAAAATCTTCGTGGGATCGAACGCAACTGGCACCGAGCTGAGACACTGGTCTGACATGCTCGCCAACCAAGGCGGCCGATTGCTCAGTGGCACTCCTTGAAACCTGAGGAAGAAGTGGATGCCCTTCTTGGAAAGAACAAGATGTACCCCTACGACGTCCCTGATTACGCGGGATACCCGTACGATGTGCCTGACTATGCCGGCTACCCGTACGATGTGCCAGACTACGCTGGCTCCGGAG CCACGAACTTTTCGCTGCTGAAACAGGCCGGCGACGTGGAAGAAAATCCCGGTCCAATGATTGAACAAGATGGATTGCACGCTGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTC GGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCA GGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACATGCCCATTCGACCACCAAGCGAAACATCGC ATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAGGAACATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCG TTGGCTACCCGTGATATTGCTGAGGAACTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCTTCTATCGCCTTCTT GACGAGTTCTTCTGATAG

SEQ ID NO:6

MSPCGRARRQTSRGAMAVLAWKFPRTRLPMGASALCVWLCWLYIFPVYRLPNEKEIVQGVLQQGTAWRRNQTAARAFRKQMEDCCDPAHLFAMTKMNSPMGKSMWYDGEFLYSFTIDNSTYSLFPQATPFQLPLKKCAWGNGGILKKSGCGRQIDEANFVMRCNLPPLSSEYTKDVGSKSQLVTANPSIIRQRFQNLL WSRKTFVDNMKIYNHSYIYMPAFSMKTGTEPSLRVYYTLSDVGANQTVLFANPNFLRSIGKFWKSRGIHAKRLSTGLFLVSAALGLCEEVAIYGFWPFSVNMHEQPISHHYYDNVLPFSGFHAMPEEFLQLWYLHKIGALRMQLDPCEDTSLQPTS

SEP ID NO: 7

GSGATNFSLLKQAGDVEENPGP

SEP ID NP: 8

MEEGFRDRAAFIRGAKDIAKEVKKHAAKKWKGLDRVQDEYSRRSYSRFEEEDDDDDFPAPSDGYYRGEGTQDEEEGGASSDATEGHDEDDEIYEGEYQGIPRAESGGKGERMADGAPLAGVRGGLSDGEGPPGGRGEAQRRKEREELAQQYEAILRECGHGRFQWTLYFVLGLALMADGVEVFVVGFVLPSAEKDMC LSDSNKGMLGLIVYLGMMVGAFLWGGLADRLGRRQCLLISLSVNSVFAFFSSFVQGYGTFLFCRLLSGVGIGGSIPIVFSYFSEFLAQEKRGEHLSWLCMFWMIGGVYAAAMAWAIIPHYGWSFQMGSAYQFHSWRVFVLVCAFPSVFAIGALTTQPESPRFFLENGKHDEAW MVLKQVHDTNMRAKGHPERVFSVTHIKTIHQEDELIEIQSDTGTWYQRWGVRALSLGGQVWGNFLSCFGPEYRRITLMMMGVWFTMSFSYYGLTVWFPDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCTFINTVFYNTDLFEYKFV NSRLINSTFLHNKEGCPLDVTGTGEGAYMVYFVSFLGTLAVLPGNIVSALLMDKIGRLRMLAGSSVMSCVSCFFLSFGNSESAMIALLCLFGGVSIASWNALDVLTVELYPSDKRTTAFGFLNALCKLAAVLGISIFTSFVGITKAAPILFASAALALGSSLALKLPETRGQVLQ

SEP ID NP: 9

MEDSYKDRTSLMKGAKDIAREVKKQTVKKVNQAVDRAQDEYTQRSYSRFQDEEDDDDYYPAGETYNGEANDDEGSSEATEGHDEDDEIYEGEYQGIPSMNQAKDSIVSVGQPKGDEYKDRRELESERRADEEELAQQYELIIQECGHGRFQWALFFVLGMALMADGVEVFWGFVLPSAETDLCIPNSGSGWL GSIVYLGMMVGAFFWGGLADKVGRKQSLLICMSVNGFFAFLSSFVQGYGFFLFCRLLSGFGIGGAIPTVFSYFAEVLAREKRGEHLSWLCMFWMIGGIYASAMAWIIPHYGWSFSMGSAYQFHSWRVFVIVCALPCVSSWALTFMPESPRFLLEVGKHDEAWMILKLIH DTNMRARGQPEKVFTVNKIKTPKQIDELIEIESDTGTWYRRCFVRIRTELYGIWLTFMRCFNYPVRDNTIKLTIVWFTLSFGYYGLSVWFPDVIKPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFF HNKTGCQITFDDDYSAYWIYFVNFLGTLAVLPGNIVSALLMDRIGRLTMLGGSMVLSGISCFFLWFGTSESMMIGMLCLYNGLTISAWNSLDWTVELYPTDRRATGFGFLNALCKAAAVLGNLIFGSLVSITKSIPILLASTVLVCGGLVGLCLPDTRTQVLM

SEP ID NO: 10

MDDYKYQDNYGGYAPSDGYYRGNESNPEEDAQSDVTEGHDEEDEIYEGEYQGIPHPDDVKAKQAKMAPSRMDSLRGQTDLMAERLEDEEQLAHQYETIMDECGHGRFQWILFFVLGLALMADGVEVFWSFALPSAEKDMCLSSSKKGMLGMIVYLGMMAGAFILGGLADKLGRKRVLSMSLAVNASFASLSSFV QGYGAFLFCRLIS GIGIGGALPIVFAYFSEFLSREKRGEHLSWLGIFWMTGGLYASAMAWSIIPHYGWGFSMGTNYHFHSWRVFVIVCALPCTVSMVALKFMPESPRFLLEMGKHDEAWMILKQVHDTNMRAKGTPEKVFTVSNIKTPKQMDEFIEIQSSTGTWYQRWLVRFKTIFKQVWDNALYCVMGPYRMNTLILAWWFAMAFSY YGLTVWFPDMIRYFQDEEYKSKMKVFFGEHVYGATINFTME NQIHQHGKLVNDKFTRMYFKHVLFEDTFFDECYFEDVTSTDTYFKNCTIESTIFYNTDLYEHKFINCRFINSTFLEQKEGCHMDLEQDNDFLIYLVSFLGSLSVLPGNIISALLMDRIGRLKMIGGSMLISAVCCFFLFFGNSESAMIGWQCLFCGTSIAAWNALDVITVELYPTNQRATAFGILNGLCKFGAILGNTIFASF VGITKWPILLAAASLVGGGLIALRLPETREQVLM

SEP ID NO: 11

FPDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCTFINTVFYNTDLFEYKFVNSRLINSTFLHNKEGCPLDVTGTGEGAY

SEP ID NP: 12

WFPDMIRYFQDEEYKSKMKVFFGEHVYGATINFTMENQIHQHGKLVNDKFTRMYFKHVLFEDTFFDECYFEDVTSTDTYFKNCTIESTIFYNTDLYEHKFINCRFINSTFLEQKEGCHMDLEQDNDFLIY

SEP ID NP: 13

WFPDVIKPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYSAY

SEP ID NP: 14

MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPWALIAIAIVAVLLVLTCCFCICKKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKV FLLPDKKKKFETKVHRKTLNPV FNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDIIGEFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTWILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQWVTVLDYDKIGKNDAIGKVFVGYNSTGAEL RHWSDMLANPRRPIAQWHTLQVEEEVD AMLAVKK

SEP ID NP: 15

MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLPPWALIAIAWAGLLLLTCCFCICKKCCCKKKKNKKEKGKGMKNAMNMKDMKGGQDDDDAETGLTEGEGEGEEEKEPENLGKLQFSLDYDFQANQLTVGVLQAAELPALDMGGTSDPYVKVFLLPDKKKKYETK VHRKTLNPAFNETFT FKVPYQELGGKTLVMAIYDFDRFSKHDIIGEVKVPMNTVDLGQPIEEWRDLQGGEKEEPEKLGDICTSLRYVPTAGKLTVCILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTVKKKTLNPYFNESFSFEIPFEQIQKVQWVTVLDYDKLGKNEAIGKIFVGSNATGTELRHWSDMLANPR RPIAQWHSLKPEEEVDALLGKNK

SEP ID NO: 16

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMP GAYNVDRKLDITSHNEDYTWEQYERSEGRHSTGGMDELYK

SEP ID NO: 17

MASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFR KTELKHSKTELNFKEWQKAFTGFEDFVGDWRQTAGYNLDQVLEQGGVSSLFQ

SEP ID NP: 18

LFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLST QSKLSKDPNEK RDHMVLLEFVTAAGL

SEP ID NP: 19

MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGWASQPARWDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG

SEP ID NP: 20

MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDWTEAGRDWLLLGEVPGQDLLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQGLAPAELFARLKARMPDGEDLWTHGDACLPNIMVENGRFSGFIDCG RLGVADRYQDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF

SEP ID NP: 21

MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAWLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA

SEP ID NO: 22

HTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILA

--->

LIST OF SEQUENCES

<110> SYNAPTIC RESEARCH, LLC

<120> GENETICALLY ENGINEERED CELLS SENSITIVE TO

CLOSTRIDIAL NEUROTOXINS

<130> P67202WO

<150> US 62/858,384

<151> 2019-06-07

<160> 22

<170> PatentIn version 3.5

<210> 1

<211> 2502

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 1

atggtgtcga agggggaagc ggtgatcaag gagttcatga ggtttaaagt gcatatggag 60

ggatctatga acggacacga gtttgagatc gaaggggaag gagaggggcg cccatacgaa 120

ggcacccaga ctgccaagct gaaagtcaca aagggtggac ccttgccctt ctcgtgggat 180

attctgagcc cgcaattcat gtacgggtcc cggggccttca ccaagcaccc tgctgacatt 240

ccggattact ataagcagag cttcccggaa ggcttcaaat gggagcgagt gatgaacttc 300

gaggatggag gcgccgtgac cgtgactcag gacacttcac tggaagatgg cactctgatc 360

tacaaggtca agctgcgggg caccaacttc ccaccggacg gaccggtcat gcagaaaaag 420

accatgggat gggaggcctc caccgagcgc ctgtaccccg aagatggagt cctcaagggg 480

gacatcaaga tggccctgcg gctcaaggat ggtggaagat acctggctga cttcaagacc 540

acgtacaagg ccaagaagcc agtccagatg cccggcgcgt acaatgtgga tcgcaagctg 600

gacatcactt cccacaacga ggactacacc gtggtggagc agtacgaacg gtccgaggt 660

cggcactcca ctggtggcat ggacgagctg tacaaaatgg ccgaggatgc agacatgaga 720

aacgaactgg aagaaatgca gcggagagca gaccagctcg cggacgaatc actggaatcg 780

acccgccgga tgcttcaact ggtcgaggaa tcaaaggacg cgggtatccg gacccttgtg 840

atgctggacg aacagggaga gcagctggag aggatcgaag agggaatgga cgattaac 900

aaggacatga aggaagcgga aaagaacctc accgaccttg gaaagttctg cgggttgtgc 960

gtgtgtccgt gcaacaagct gaagtcctcc gacgcctaca agaaggcctg gggaaacaac 1020

caggacggtg tcgtggcttc ccaacccgca cgggtggtgg atgagcggga acagatggcg 1080

atttccggag gcttcattag acgcgtgacc aacgacgccc gcgaaaacga gatggacgaa 1140

aacctggaac aagtgtcggg aatcatcgga aacttgagac acatggccct cgacatgggc 1200

aacgaaattg atacacagaa ccggcagatt gaccggatca tggaaaaggc agactcaaac 1260

aagactcgga ttgacgaagc gaaccagagg gccactaaga tgttgggttc cgggatggtg 1320

tcaaagggag aagaagacaa catggcatca ctgcccgcca cccacgagct gcacatcttc 1380

ggttccatca acggggtcga cttcgacatg gtcggccagg gaactggaaa cccgaatgac 1440

ggttatgaag aactgaacct taaatcaacc aagggggacc ttcagttctc gccctggatt 1500

ttggtccctc acattggata cggattccat cagtatctgc cgtaccccga cggaatgagc 1560

ccgttccagg ctgccatggt ggacggatcg ggataccagg tccaccgcac catgcagttt 1620

gaagatggcg caagcctgac cgtgaactac cggtatacct acgagggctc acacatcaag 1680

ggggaagccc aagtcaaggg taccggcttc ccggccgacg gaccagtgat gaccaactcc 1740

ttgaccgccg ccgactggtg ccgcagcaag aaaacttacc ccaacgataa gacaatcatc 1800

tccactttca agtggtccta caccacgggc aacggcaaac gctaccgaag cactgcacgg 1860

accacctaca ctttcgcgaa gcctatggcc gccaactacc tgaagaacca gccgatgtac 1920

gtgttcagaa agacggaact caagcactcc aaaaccgaac tgaactttaa ggagtggcag 1980

aaggctttca ctggattcga ggactttgtc ggcgactggc gccagactgc cggctacaac 2040

ctggaccaag tgctcgaaca ggggggtgtc tccagcctct tccaaaatct gggcgtgtcc 2100

gtgaccccga tccagcggat cgtgctcagc ggggaaaacg gcctgaagat cgatatccac 2160

gtcatcatcc cgtacgaggg actgagcggc gaccagatgg gtcagatcga aaagattttc 2220

aaggtggtct atcccgtgga tgaccaccac ttcaaagtga tcctgcatta cgggaccctc 2280

gtgatcgacg gcgtcacccc gaacatgatt gattacttcg gacggcctta tgaagggatc 2340

gccgtgttcg acggcaaaaa gatcaccgtg actggcaccc tgtggaacgg aaataagatc 2400

attgacgagc ggctgatcaa cccagacggg tcgctgctgt tccgcgtgac catcaacgga 2460

gtgaccggct ggcggctgtg cgagcgcatc ctcgcctgat ag 2502

<210> 2

<211> 2517

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 2

atggtgtcga agggggaagc ggtgatcaag gagttcatga ggtttaaagt gcatatggag 60

ggatctatga acggacacga gtttgagatc gaaggggaag gagaggggcg cccatacgaa 120

ggcacccaga ctgccaagct gaaagtcaca aagggtggac ccttgccctt ctcgtgggat 180

attctgagcc cgcaattcat gtacgggtcc cggggccttca ccaagcaccc tgctgacatt 240

ccggattact ataagcagag cttcccggaa ggcttcaaat gggagcgagt gatgaacttc 300

gaggatggag gcgccgtgac cgtgactcag gacacttcac tggaagatgg cactctgatc 360

tacaaggtca agctgcgggg caccaacttc ccaccggacg gaccggtcat gcagaaaaag 420

accatgggat gggaggcctc caccgagcgc ctgtaccccg aagatggagt cctcaagggg 480

gacatcaaga tggccctgcg gctcaaggat ggtggaagat acctggctga cttcaagacc 540

acgtacaagg ccaagaagcc agtccagatg cccggcgcgt acaatgtgga tcgcaagctg 600

gacatcactt cccacaacga ggactacacc gtggtggagc agtacgaacg gtccgaggt 660

cggcactcca ctggtggcat ggacgagctg tacaaaatgg ccgaggatgc agacatgaga 720

aacgaactgg aagaaatgca gcggagagca gaccagctcg cggacgaatc actggaatcg 780

acccgccgga tgcttcaact ggtcgaggaa tcaaaggacg cgggtatccg gacccttgtg 840

atgctggacg aacagggaga gcagctggag aggatcgaag agggaatgga cgattaac 900

aaggacatga aggaagcgga aaagaacctc accgaccttg gaaagttctg cgggttgtgc 960

gtgtgtccgt gcaacaagct gaagtcctcc gacgcctaca agaaggcctg gggaaacaac 1020

caggacggtg tcgtggcttc ccaacccgca cgggtggtgg atgagcggga acagatggcg 1080

atttccggag gcttcattag acgcgtgacc aacgacgccc gcgaaaacga gatggacgaa 1140

aacctggaac aagtgtcggg aatcatcgga aacttgagac acatggccct cgacatgggc 1200

aacgaaattg atacacagaa ccggcagatt gaccggatca tggaaaaggc agactcaaac 1260

aagactcgga ttgacgaagc gaaccagagg gccactaaga tgttgggttc cgggatggtg 1320

tcaaagggag aagaactgtt cactggagtg gtgcccatcc tggtggagct ggatggcgat 1380

gtgaacggcc ataaattctc agtcagcgga gagggagagg gcgatgcgac ttacggaaag 1440

ctgactttga agtttatctg cactaccgga aagctgcctg tgccatggcc taccctcgtg 1500

accaccctgt cctggggcgt ccaatgtttc gcacgctacc ctgaccatat gaagcagcac 1560

gacttcttca agtccgccat gcccgagggc tacgtgcagg aacgcaccat cttcttcaag 1620

gacgacggga actacaaaac cagggctgaa gtgaagttcg agggagacac cctggtcaat 1680

cggattgaat tgaagggaat cgatttcaag gaagatggaa acatcctggg acataagctt 1740

gagtacaact acttctccga caacgtgtac atcacggccg ataagcagaa gaacggaatc 1800

aaagctaact tcaagattcg gcacaacatt gaggacggcg gcgtccagct ggcggaccat 1860

tatcagcaga atacccctat tggggatgga ccggtgctgc tcccggacaa ccattacctg 1920

tccacccaat ctaagctgag caaggaccca aacgagaagc gcgatcacat ggtgctgctc 1980

gagttcgtga ctgccgccgg gcttcacaca cttgaggact ttgtcggcga ctggcgccag 2040

actgccggct acaacctgga ccaagtgctc gaacaggggg gtgtctccag cctcttccaa 2100

aatctgggcg tgtccgtgac cccgatccag cggatcgtgc tcagcgggga aaacggcctg 2160

aagatcgata tccacgtcat catcccgtac gagggactga gcggcgacca gatgggtcag 2220

atcgaaaaga ttttcaaggt ggtctatccc gtggatgacc accacttcaa agtgatcctg 2280

cattacggga ccctcgtgat cgacggcgtc accccgaaca tgattgatta cttcggacgg 2340

ccttatgaag ggatcgccgt gttcgacggc aaaaagatca ccgtgactgg caccctgtgg 2400

aacggaaata agatcattga cgagcggctg atcaacccag acgggtcgct gctgttccgc 2460

gtgaccatca acggagtgac cggctggcgg ctgtgcgagc gcatcctcgc ctgatag 2517

<210> 3

<211> 2250

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 3

atgaccatgg atgagcagca atcgcaggct gtagccccgg tatatgtcgg tggtatggat 60

gagaaaacga ctgggtggcg gggtggacac gtcgtcgagg gcctggcagg cgaacttgaa 120

caactgcggg ctcgcttgga gcaccacccg caaggacagc gcgagccgtc catggtgtca 180

aagggggagg aactgtttac tggggtcgtc cctatcttgg tggaactcga cggggatgtg 240

aacggacaca agttttcggt atccggggaa ggcgaggggg atgccacgta tggaaagctc 300

acacttaagt tcatctgcac gacagggaag ctcccagtgc cttggcccac gttggtgact 360

acgctcacat ggggtgtcca gtgcttcgca cggtatcccg accacatgaa gcagcatgat 420

ttctttaagt cagccatgcc ggagggatat gtacaagaaa ggaccatctt cttcaaagat 480

gacggtaact acaagaccag agccgaggta aagtttgaag gcgacactct cgtgaacagg 540

attgagctga agggaattga tttcaaagag gatgggaaca tccttggtca caaattggag 600

tacaatgcca tttcggataa cgtgtacatt acagcggata agcagaagaa tgggatcaaa 660

gcgaatttca aaatcaggca taacatcgag gacgggtcgg tgcagctcgc cgaccattac 720

cagcagaata cgcccatcgg agatggaccc gtacttctgc ccgacaatca ttatctgtca 780

acgcaatcag cgcttagcaa agatcccaat gagaaaaggg accacatggt gctcctcgaa 840

tttgtgacgg cagcgggaat taccctcggg atggacgaac tgtacaaaag cgggttgaga 900

ctcgagcgct gaactcgaga tgccttttgt caacaagcag tttaactata aggatcccgt 960

gaatggtgtg gacattgcct acatcaagat tccaaacgct ggacaaatgc agcccgtcaa 1020

ggctttcaaa attcacaaca agatctgggt gatcccggag agggacacct ttaccaatcc 1080

agaagaggc gaccttaacc ctccgccaga ggccaaacag gtgcccgtga gctattacga 1140

ctcaacttat ctctccaccg acaacgaaaa ggacaattac ctcaagggag tcaccaagct 1200

gttcgaacgg atctactcta ccgatctcgg caggatgctc ctgacttcta tcgtgcgggg 1260

catccccttc tggggtggga gcaccattga caccgaactg aaggtgattg ataccaattg 1320

catcaacgtc atccagccag acggttccta ccggtctgag gagctcaatc ttgtgattat 1380

tggcccgtca gctgatatca tccagttcga atgcaagtct ttcggacacg acgtgcttaa 1440

tctcacccgc aatggttacg gaagcaccca gtacatcaga ttctctccgg actttacttt 1500

cggatttgaa gagtcactgg aagtcgacac caatcctctg ctcggagccg gaaagttcgc 1560

caccgaccct gcagtgaccc ttgctcacga gctgattcat gcagagcatc gcctgtacgg 1620

gatcgccatc aatcctaacc gcgtgtttaa ggtcaatacc aacgcttact atgaaatgag 1680

cggactggag gtgtccttcg aggaactgcg caccttcgga ggtcatgacg ctaagttcat 1740

cgactcactg caagagaatg agttccggct gtactattac aacaagttta aggatgtcgc 1800

ctcaactctg aacaaggcca aaagcatcat cggcaccacc gccagcctgc aatacatgaa 1860

aaacgtgttc aaggaaaagt accttcttag cgaagatact tccgggaagt tttcagtcga 1920

caaactgaag ttcgacaagc tgtacaagat gctcaccgaa atctacaccg aggacaattt 1980

tgtgaacttc ttcaaagtga ttaacagaaa gacctatctg aacttcgaca aagccgtgtt 2040

ccggattaac attgtgcccg atgagaacta cactatcaag gacgggttca accttaaggg 2100

tgcaaatctt tcaactaatt tcaacggaca gaatactgag atcaattcaa ggaacttcac 2160

tagactcaag aatttcactg ggcttttcga gttctataag ctgctgtgtg tccgcggaat 2220

tatccccttc aagtgaagct tcgtcaatga 2250

<210> 4

<211> 5772

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 4

atgtccccat gtggacgagc gcgcagacag acctcaagag gagcgatggc cgtgctggcc 60

tggaagttcc cgaggactcg cctgcccatg ggagcctctg ctctgtgtgt ggtcgtgctg 120

tgttggctgt acatcttccc ggtgtaccgg ctgcctaacg aaaaggaaat tgtgcagggc 180

gtgctccagc aggggaccgc ttggcggcgc aaccagaccg ctgcgaggc ttttcggaag 240

cagatggaag attgttgcga ccccgcccat cttttcgcga tgaccaagat gaacagcccg 300

atgggaaagt ccatgtggta cgacggagag ttcctgtatt ccttcaccat tgacaacagc 360

acttactcac tgttcccgca agccaccccc ttccaactgc cgcttaagaa gtgcgccgtc 420

gtggggaacg gcggcatcct caagaagtcc ggatgcgggc gccagattga tgaagccaac 480

ttcgtgatgc ggtgcaatct cccgccactc tcgtccgagt acaccaagga cgtggggtca 540

aagtcgcagc tcgtcaccgc caacccttcg atcatcagac aacggttcca gaaccttctg 600

tggagccgga aaacatttgt ggataacatg aagatctaca accattccta catctatatg 660

cctgccttct ccatgaaaac tggaaccgaa ccctccctga gagtgtacta caccctgtcc 720

gacgtgggcg caaaccagac cgtccttttc gccaacccca acttcctgcg ctccatcgga 780

aagttctgga agtccagagg cattcacgcg aaacgcttgt ccactggatt gttcttggtg 840

tccgccgctc tgggcctgtg cgaggaagtg gccatatacg gattctggcc tttctccgtc 900

aacatgcacg agcagcccat ctcccaccat tattacgaca atgtcctgcc tttctcggga 960

tttcacgcga tgcccgagga gttcttgcaa ctgtggtacc ttcacaagat cggtgccctg 1020

cggatgcagc tggacccttg cgaggacacc tcgctgcaac ccacctcgga gcagaaactc 1080

atttccgaag aggatctgaa cggggagcag aagctcatct ccgaggagga cctgaacgga 1140

gaacagaagc tgattagcga agaggacctg ggcagcggtg ccaccaattt ttctctgctc 1200

aagcaggccg gagatgtgga agagaacccg ggtcccatgg aggactccta caaagatcgg 1260

acttctctga tgaagggagc caaggacatc gccagggaag tgaagaagca aaccgtcaag 1320

aaggtcaacc aggccgtgga cagagcccag gacgagtaca cccagcggtc gtactcgcgg 1380

ttccaggatg aagaggatga cgacgactac taccctgccg gcgaaaccta taatggggaa 1440

gccaacgatg acgaaggctc cagcgaagcc actgaaggac acgacgagga cgacgaaatc 1500

tacgaaggag aataccaggg catcccttcg atgaatcagg ccaaagattc aattgtgtca 1560

gtgggacagc ctaagggcga cgagtacaag gaccggagag agctcgaaag cgagcggagg 1620

gccgacgaag aggaactggc acaacagtac gagctgatca tccaggagtg tgggcacggc 1680

cggttccagt gggcgctgtt cttcgtgctc ggaatggcac tgatggccga cggcgtggaa 1740

gtgttcgtgg tcggattcgt gctgccctcg gccgaaaccg acctctgcat tcccaactcc 1800

ggctcgggat ggctggggtc catcgtgtac ctgggaatga tggtcggcgc cttcttctgg 1860

ggtggcctgg cagacaaggt cggccggaag cagtccctct tgatctgcat gagcgtcaac 1920

ggatttttcg ccttcctgtc atcattcgtg caaggttacg ggttcttcct tttctgccgc 1980

ctgctgtccg gctttgggat cggcggggct attccgactg tgttctccta ctttgccgaa 2040

gtgctggctc gcgaaaaacg gggcgaacac ctttcctggc tgtgtatgtt ctggatgatc 2100

ggcggcatct acgcctcggc catggcctgg gctattatcc cgcattatgg gtggtccttc 2160

tcaatgggaa gcgcatacca gttccattcg tggcgggtgt tcgtgatcgt gtgcgccctc 2220

ccgtgtgtgt cctccgtggt ggctctgaca ttcatgccgg agtcacctcg gttcttgttg 2280

gaagtcggga agcacgacga agcctggatg attctgaagc tgatccacga cactaatatg 2340

cgggcccggg gacagcctga gaaagtgttc accgtcaaca agattaagac cccgaagcaa 2400

atcgatgaac tgattgaaat tgagtccgac accggaactt ggtaccgccg gtgcttcgtg 2460

cggattcgca ccgagctgta cggaatctgg ctcaccttca tgcgctgctt caactacccc 2520

gtgcgcgaca acaccatcaa gctgaccatc gtgtggttca ctctgtcttt cggctactat 2580

gggctgtcag tgtggttccc ggatgtcatc aagccgctcc aatccgatga atacgccctg 2640

ctgacccgca atgtggagag agacaaatac gccaacttca ccatcaattt caccatggaa 2700

aaccagattc acaccggaat ggagtacgac aatggacgat tcatcggagt gaagttcaag 2760

agcgtgacct tcaaggactc ggtgttcaag tcctgtacct tcgaggacgt gaccagcgtg 2820

aacacttatt ttaagaattg caccttcatc gatactgtgt tcgataacac cgacttcgag 2880

ccctataagt tcattgactc ggagttcaag aactgttcat tcttccacaa caagactggt 2940

tgccagatca ccttcgatga cgactacagc gcctactgga tctactttgt gaactttttg 3000

ggaactctcg cagtgcttcc tggcaacatt gtgtccgcac tcctgatgga tcggattggc 3060

aggctcacga tgcttggggg gtccatggtc ctctccggga tctcgtgctt cttcctgtgg 3120

ttcggcacct cggagtccat gatgatcgga atgttgtgcc tgtacaacgg tctgaccatc 3180

agcgcctgga acagcctcga cgtggtcacc gtcgagctgt atcctaccga ccggcgcgcg 3240

acaggcttcg gattcctgaa cgcactgtgc aaggcagccg cggtcctggg aaatctgatc 3300

tttggttcgc tggtgtccat cactaagagc atccctattc tgctcgcctc cacggtgctc 3360

gtgtgtggtg gcctggtcgg gctgtgcctg cccgacactc gcacccaagt gctcatggac 3420

tacaaggatg acgatgataa gggagactac aaggacgatg acgacaaggg ggattacaag 3480

gacgacgatg acaaaggaag cggcgccact aacttttccc tgctgaagca ggccggggac 3540

gtcgaagaaa accccggggcc aatgcgcaac attttcaagc ggaatcagga gcctatcgtg 3600

gccccggcca ccactaccgc cactatgcct attggacctg tcgacaactc cacggaatca 3660

ggcggcgccg gcgaatccca agaggacatg ttcgccaagc tgaaggagaa actgttcaac 3720

gaaatcaaca agattcccct cccgccgtgg gccctgatcg ctatcgctgt cgtcgccgga 3780

ctgctgctgc ttacttgctg cttctgcatt tgcaagaagt gttgttgcaa gaaaaagaaa 3840

aacaagaagg aaaaggggaa gggaatgaag aacgccatga atatgaagga catgaagggc 3900

ggacaggatg atgatgatgc tgaaactggg ctgactgagg gcgaaggaga gggcgaagag 3960

gagaaggaac ctgagaacct gggaaagctc caattctccc tggattacga cttccaagcc 4020

aaccagctga ctgtgggagt gttgcaagcc gccgagctgc cagccctgga catgggcggc 4080

acctccgacc cctatgtgaa ggtgttcttg ctgcctgaca agaagaagaa gtacgaaacc 4140

aaggtgcacc gcaagaccct gaaccccgct ttcaacgaaa ccttcacttt caaagtgccc 4200

taccaagagc tcgggggaaa gactctcgtg atggcgatct acgacttcga ccggttcagc 4260

aagcacgata tcatcgggga ggtcaaggtc ccgatgaaca ccgtggacct tggccaaccg 4320

attgaagaat ggcgcgatct ccagggtggc gaaaaggagg agcccgagaa actgggtgac 4380

atctgtacat cactgcgcta cgtgccgacc gccgggaagc tcactgtctg catcctggag 4440

gccaagaacc tgaagaaaat ggacgtgggc gggctctccg acccttacgt gaagatccac 4500

ctgatgcaga acggaaagcg gctgaagaag aagaaaacca ctgtgaagaa aaagactctg 4560

aacccctact tcaacgagtc gttctccttc gaaatcccgt ttgagcaaat ccagaaggtc 4620

caagtggtcg tgactgtgct tgactacgac aagctcggaa agaacgaggc cattggcaaa 4680

atcttcgtgg gatcgaacgc aactggcacc gagctgagac actggtctga catgctcgcc 4740

aacccaaggc ggccgattgc tcagtggcac tccttgaaac ctgaggaaga agtggatgcc 4800

cttcttggaa agaacaagat gtacccctac gacgtccctg attacgcggg atacccgtac 4860

gatgtgcctg actatgccgg ctacccgtac gatgtgccag actacgctgg ctccggagcc 4920

acgaactttt cgctgctgaa acaggccggc gacgtggaag aaaatcccgg tccaatgatt 4980

gaacaagatg gattgcacgc tggttctccg gccgcttggg tggagaggct attcggctat 5040

gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag 5100

gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac 5160

gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 5220

gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 5280

ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 5340

ctgcatacgc ttgatccggc tacatgccca ttcgaccacc aagcgaaaca tcgcatcgag 5400

cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaggaacat 5460

caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag 5520

gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 5580

ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 5640

ttggctaccc gtgatattgc tgaggaactt ggcggcgaat gggctgaccg cttcctcgtg 5700

ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 5760

ttcttctgat ag 5772

<210> 5

<211> 5772

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 5

atgtccccat gtggacgagc gcgcagacag acctcaagag gagcgatggc cgtgctggcc 60

tggaagttcc cgaggactcg cctgcccatg ggagcctctg ctctgtgtgt ggtcgtgctg 120

tgttggctgt acatcttccc ggtgtaccgg ctgcctaacg aaaaggaaat tgtgcagggc 180

gtgctccagc aggggaccgc ttggcggcgc aaccagaccg ctgcgaggc ttttcggaag 240

cagatggaag attgttgcga ccccgcccat cttttcgcga tgaccaagat gaacagcccg 300

atgggaaagt ccatgtggta cgacggagag ttcctgtatt ccttcaccat tgacaacagc 360

acttactcac tgttcccgca agccaccccc ttccaactgc cgcttaagaa gtgcgccgtc 420

gtggggaacg gcggcatcct caagaagtcc ggatgcgggc gccagattga tgaagccaac 480

ttcgtgatgc ggtgcaatct cccgccactc tcgtccgagt acaccaagga cgtggggtca 540

aagtcgcagc tcgtcaccgc caacccttcg atcatcagac aacggttcca gaaccttctg 600

tggagccgga aaacatttgt ggataacatg aagatctaca accattccta catctatatg 660

cctgccttct ccatgaaaac tggaaccgaa ccctccctga gagtgtacta caccctgtcc 720

gacgtgggcg caaaccagac cgtccttttc gccaacccca acttcctgcg ctccatcgga 780

aagttctgga agtccagagg cattcacgcg aaacgcttgt ccactggatt gttcttggtg 840

tccgccgctc tgggcctgtg cgaggaagtg gccatatacg gattctggcc tttctccgtc 900

aacatgcacg agcagcccat ctcccaccat tattacgaca atgtcctgcc tttctcggga 960

tttcacgcga tgcccgagga gttcttgcaa ctgtggtacc ttcacaagat cggtgccctg 1020

cggatgcagc tggacccttg cgaggacacc tcgctgcaac ccacctcgga gcagaaactc 1080

atttccgaag aggatctgaa cggggagcag aagctcatct ccgaggagga cctgaacgga 1140

gaacagaagc tgattagcga agaggacctg ggcagcggtg ccaccaattt ttctctgctc 1200

aagcaggccg gagatgtgga agagaacccg ggtcccatgg aggactccta caaagatcgg 1260

acttctctga tgaagggagc caaggacatc gccagggaag tgaagaagca aaccgtcaag 1320

aaggtcaacc aggccgtgga cagagcccag gacgagtaca cccagcggtc gtactcgcgg 1380

ttccaggatg aagaggatga cgacgactac taccctgccg gcgaaaccta taatggggaa 1440

gccaacgatg acgaaggctc cagcgaagcc actgaaggac acgacgagga cgacgaaatc 1500

tacgaaggag aataccaggg catcccttcg atgaatcagg ccaaagattc aattgtgtca 1560

gtgggacagc ctaagggcga cgagtacaag gaccggagag agctcgaaag cgagcggagg 1620

gccgacgaag aggaactggc acaacagtac gagctgatca tccaggagtg tgggcacggc 1680

cggttccagt gggcgctgtt cttcgtgctc ggaatggcac tgatggccga cggcgtggaa 1740

gtgttcgtgg tcggattcgt gctgccctcg gccgaaaccg acctctgcat tcccaactcc 1800

ggctcgggat ggctggggtc catcgtgtac ctgggaatga tggtcggcgc cttcttctgg 1860

ggtggcctgg cagacaaggt cggccggaag cagtccctct tgatctgcat gagcgtcaac 1920

ggatttttcg ccttcctgtc atcattcgtg caaggttacg ggttcttcct tttctgccgc 1980

ctgctgtccg gctttgggat cggcggggct attccgactg tgttctccta ctttgccgaa 2040

gtgctggctc gcgaaaaacg gggcgaacac ctttcctggc tgtgtatgtt ctggatgatc 2100

ggcggcatct acgcctcggc catggcctgg gctattatcc cgcattatgg gtggtccttc 2160

tcaatgggaa gcgcatacca gttccattcg tggcgggtgt tcgtgatcgt gtgcgccctc 2220

ccgtgtgtgt cctccgtggt ggctctgaca ttcatgccgg agtcacctcg gttcttgttg 2280

gaagtcggga agcacgacga agcctggatg attctgaagc tgatccacga cactaatatg 2340

cgggcccggg gacagcctga gaaagtgttc accgtcaaca agattaagac cccgaagcaa 2400

atcgatgaac tgattgaaat tgagtccgac accggaactt ggtaccgccg gtgcttcgtg 2460

cggattcgca ccgagctgta cggaatctgg ctcaccttca tgcgctgctt caactacccc 2520

gtgcgcgaca acaccatcaa gctgaccatc gtgtggttca ctctgtcttt cggctactat 2580

gggctgtcag tgtggttccc ggatgtcatc aagccgctcc aatccgatga atacgccctg 2640

ctgacccgca atgtggagag agacaaatac gccaacttca ccatcaattt caccatggaa 2700

aaccagattc acaccggaat ggagtacgac aatggacgat tcatcggagt gaagttcaag 2760

agcgtgacct tcaaggactc ggtgttcaag tcctgtacct tcgaggacgt gaccagcgtg 2820

aacacttatt ttaagaattg caccttcatc gatactgtgt tcgataacac cgacttcgag 2880

ccctataagt tcattgactc ggagttcaag aactgttcat tcttccacaa caagactggt 2940

tgccagatca ccttcgatga cgactacagc gcctactgga tctactttgt gaactttttg 3000

ggaactctcg cagtgcttcc tggcaacatt gtgtccgcac tcctgatgga tcggattggc 3060

aggctcacga tgcttggggg gtccatggtc ctctccggga tctcgtgctt cttcctgtgg 3120

ttcggcacct cggagtccat gatgatcgga atgttgtgcc tgtacaacgg tctgaccatc 3180

agcgcctgga acagcctcga cgtggtcacc gtcgagctgt atcctaccga ccggcgcgcg 3240

acaggcttcg gattcctgaa cgcactgtgc aaggcagccg cggtcctggg aaatctgatc 3300

tttggttcgc tggtgtccat cactaagagc atccctattc tgctcgcctc cacggtgctc 3360

gtgtgtggtg gcctggtcgg gctgtgcctg cccgacactc gcacccaagt gctcatggac 3420

tacaaggatg acgatgataa gggagactac aaggacgatg acgacaaggg ggattacaag 3480

gacgacgatg acaaaggaag cggcgccact aacttttccc tgctgaagca ggccggggac 3540

gtcgaagaaa accccggggcc aatgcgcaac attttcaagc ggaatcagga gcctatcgtg 3600

gccccggcca ccactaccgc cactatgcct attggacctg tcgacaactc cacggaatca 3660

ggcggcgccg gcgaatccca agaggacatg ttcgccaagc tgaaggagaa actgttcaac 3720

gaaatcaaca agattcccct cccgccgtgg gccctgatcg ctatcgctgt cgtcgccgga 3780

ctgctgctgc ttacttgctg cttctgcatt tgcaagaagt gttgttgcaa gaaaaagaaa 3840

aacaagaagg aaaaggggaa gggaatgaag aacgccatga atatgaagga catgaagggc 3900

ggacaggatg atgatgatgc tgaaactggg ctgactgagg gcgaaggaga gggcgaagag 3960

gagaaggaac ctgagaacct gggaaagctc caattctccc tggattacga cttccaagcc 4020

aaccagctga ctgtgggagt gttgcaagcc gccgagctgc cagccctgga catgggcggc 4080

acctccgacc cctatgtgaa ggtgttcttg ctgcctgaca agaagaagaa gtacgaaacc 4140

aaggtgcacc gcaagaccct gaaccccgct ttcaacgaaa ccttcacttt caaagtgccc 4200

taccaagagc tcgggggaaa gactctcgtg atggcgatct acgacttcga ccggttcagc 4260

aagcacgata tcatcgggga ggtcaaggtc ccgatgaaca ccgtggacct tggccaaccg 4320

attgaagaat ggcgcgatct ccagggtggc gaaaaggagg agcccgagaa actgggtgac 4380

atctgtacat cactgcgcta cgtgccgacc gccgggaagc tcactgtctg catcctggag 4440

gccaagaacc tgaagaaaat ggacgtgggc gggctctccg acccttacgt gaagatccac 4500

ctgatgcaga acggaaagcg gctgaagaag aagaaaacca ctgtgaagaa aaagactctg 4560

aacccctact tcaacgagtc gttctccttc gaaatcccgt ttgagcaaat ccagaaggtc 4620

caagtggtcg tgactgtgct tgactacgac aagctcggaa agaacgaggc cattggcaaa 4680

atcttcgtgg gatcgaacgc aactggcacc gagctgagac actggtctga catgctcgcc 4740

aacccaaggc ggccgattgc tcagtggcac tccttgaaac ctgaggaaga agtggatgcc 4800

cttcttggaa agaacaagat gtacccctac gacgtccctg attacgcggg atacccgtac 4860

gatgtgcctg actatgccgg ctacccgtac gatgtgccag actacgctgg ctccggagcc 4920

acgaactttt cgctgctgaa acaggccggc gacgtggaag aaaatcccgg tccaatgatt 4980

gaacaagatg gattgcacgc tggttctccg gccgcttggg tggagaggct attcggctat 5040

gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag 5100

gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac 5160

gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 5220

gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 5280

ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 5340

ctgcatacgc ttgatccggc tacatgccca ttcgaccacc aagcgaaaca tcgcatcgag 5400

cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaggaacat 5460

caggggctcg cgccagccga actngttcgcc aggctcaagg cgcgcatgcc cgacggcgag 5520

gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 5580

ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 5640

ttggctaccc gtgatattgc tgaggaactt ggcggcgaat gggctgaccg cttcctcgtg 5700

ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 5760

ttcttctgat ag 5772

<210> 6

<211> 356

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 6

Met Ser Pro Cys Gly Arg Ala Arg Arg Gln Thr Ser Arg Gly Ala Met

1 5 10 15

Ala Val Leu Ala Trp Lys Phe Pro Arg Thr Arg Leu Pro Met Gly Ala

20 25 30

Ser Ala Leu Cys Val Val Val Leu Cys Trp Leu Tyr Ile Phe Pro Val

35 40 45

Tyr Arg Leu Pro Asn Glu Lys Glu Ile Val Gln Gly Val Leu Gln Gln

50 55 60

Gly Thr Ala Trp Arg Arg Asn Gln Thr Ala Ala Arg Ala Phe Arg Lys

65 70 75 80

Gln Met Glu Asp Cys Cys Asp Pro Ala His Leu Phe Ala Met Thr Lys

85 90 95

Met Asn Ser Pro Met Gly Lys Ser Met Trp Tyr Asp Gly Glu Phe Leu

100 105 110

Tyr Ser Phe Thr Ile Asp Asn Ser Thr Tyr Ser Leu Phe Pro Gln Ala

115 120 125

Thr Pro Phe Gln Leu Pro Leu Lys Lys Cys Ala Val Val Gly Asn Gly

130 135 140

Gly Ile Leu Lys Lys Ser Gly Cys Gly Arg Gln Ile Asp Glu Ala Asn

145 150 155 160

Phe Val Met Arg Cys Asn Leu Pro Pro Leu Ser Ser Glu Tyr Thr Lys

165 170 175

Asp Val Gly Ser Lys Ser Gln Leu Val Thr Ala Asn Pro Ser Ile Ile

180 185 190

Arg Gln Arg Phe Gln Asn Leu Leu Trp Ser Arg Lys Thr Phe Val Asp

195 200 205

Asn Met Lys Ile Tyr Asn His Ser Tyr Ile Tyr Met Pro Ala Phe Ser

210 215 220

Met Lys Thr Gly Thr Glu Pro Ser Leu Arg Val Tyr Tyr Thr Leu Ser

225 230 235 240

Asp Val Gly Ala Asn Gln Thr Val Leu Phe Ala Asn Pro Asn Phe Leu

245 250 255

Arg Ser Ile Gly Lys Phe Trp Lys Ser Arg Gly Ile His Ala Lys Arg

260 265 270

Leu Ser Thr Gly Leu Phe Leu Val Ser Ala Ala Leu Gly Leu Cys Glu

275 280 285

Glu Val Ala Ile Tyr Gly Phe Trp Pro Phe Ser Val Asn Met His Glu

290 295 300

Gln Pro Ile Ser His His Tyr Tyr Asp Asn Val Leu Pro Phe Ser Gly

305 310 315 320

Phe His Ala Met Pro Glu Glu Phe Leu Gln Leu Trp Tyr Leu His Lys

325 330 335

Ile Gly Ala Leu Arg Met Gln Leu Asp Pro Cys Glu Asp Thr Ser Leu

340 345 350

Gln Pro Thr Ser

355

<210> 7

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 7

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 8

<211> 742

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 8

Met Glu Glu Gly Phe Arg Asp Arg Ala Ala Phe Ile Arg Gly Ala Lys

1 5 10 15

Asp Ile Ala Lys Glu Val Lys Lys His Ala Ala Lys Lys Val Val Lys

20 25 30

Gly Leu Asp Arg Val Gln Asp Glu Tyr Ser Arg Arg Ser Tyr Ser Arg

35 40 45

Phe Glu Glu Glu Asp Asp Asp Asp Asp Phe Pro Ala Pro Ser Asp Gly

50 55 60

Tyr Tyr Arg Gly Glu Gly Thr Gln Asp Glu Glu Glu Gly Gly Ala Ser

65 70 75 80

Ser Asp Ala Thr Glu Gly His Asp Glu Asp Asp Glu Ile Tyr Glu Gly

85 90 95

Glu Tyr Gln Gly Ile Pro Arg Ala Glu Ser Gly Gly Lys Gly Glu Arg

100 105 110

Met Ala Asp Gly Ala Pro Leu Ala Gly Val Arg Gly Gly Leu Ser Asp

115 120 125

Gly Glu Gly Pro Pro Gly Gly Arg Gly Glu Ala Gln Arg Arg Lys Glu

130 135 140

Arg Glu Glu Leu Ala Gln Gln Tyr Glu Ala Ile Leu Arg Glu Cys Gly

145 150 155 160

His Gly Arg Phe Gln Trp Thr Leu Tyr Phe Val Leu Gly Leu Ala Leu

165 170 175

Met Ala Asp Gly Val Glu Val Phe Val Val Gly Phe Val Leu Pro Ser

180 185 190

Ala Glu Lys Asp Met Cys Leu Ser Asp Ser Asn Lys Gly Met Leu Gly

195 200 205

Leu Ile Val Tyr Leu Gly Met Met Val Gly Ala Phe Leu Trp Gly Gly

210 215 220

Leu Ala Asp Arg Leu Gly Arg Arg Gln Cys Leu Leu Ile Ser Leu Ser

225 230 235 240

Val Asn Ser Val Phe Ala Phe Phe Ser Ser Phe Val Gln Gly Tyr Gly

245 250 255

Thr Phe Leu Phe Cys Arg Leu Leu Ser Gly Val Gly Ile Gly Gly Ser

260 265 270

Ile Pro Ile Val Phe Ser Tyr Phe Ser Glu Phe Leu Ala Gln Glu Lys

275 280 285

Arg Gly Glu His Leu Ser Trp Leu Cys Met Phe Trp Met Ile Gly Gly

290 295 300

Val Tyr Ala Ala Ala Met Ala Trp Ala Ile Ile Pro His Tyr Gly Trp

305 310 315 320

Ser Phe Gln Met Gly Ser Ala Tyr Gln Phe His Ser Trp Arg Val Phe

325 330 335

Val Leu Val Cys Ala Phe Pro Ser Val Phe Ala Ile Gly Ala Leu Thr

340 345 350

Thr Gln Pro Glu Ser Pro Arg Phe Phe Leu Glu Asn Gly Lys His Asp

355 360 365

Glu Ala Trp Met Val Leu Lys Gln Val His Asp Thr Asn Met Arg Ala

370 375 380

Lys Gly His Pro Glu Arg Val Phe Ser Val Thr His Ile Lys Thr Ile

385 390 395 400

His Gln Glu Asp Glu Leu Ile Glu Ile Gln Ser Asp Thr Gly Thr Trp

405 410 415

Tyr Gln Arg Trp Gly Val Arg Ala Leu Ser Leu Gly Gly Gln Val Trp

420 425 430

Gly Asn Phe Leu Ser Cys Phe Gly Pro Glu Tyr Arg Arg Ile Thr Leu

435 440 445

Met Met Met Gly Val Trp Phe Thr Met Ser Phe Ser Tyr Tyr Gly Leu

450 455 460

Thr Val Trp Phe Pro Asp Met Ile Arg His Leu Gln Ala Val Asp Tyr

465 470 475 480

Ala Ser Arg Thr Lys Val Phe Pro Gly Glu Arg Val Glu His Val Thr

485 490 495

Phe Asn Phe Thr Leu Glu Asn Gln Ile His Arg Gly Gly Gln Tyr Phe

500 505 510

Asn Asp Lys Phe Ile Gly Leu Arg Leu Lys Ser Val Ser Phe Glu Asp

515 520 525

Ser Leu Phe Glu Glu Cys Tyr Phe Glu Asp Val Thr Ser Ser Asn Thr

530 535 540

Phe Phe Arg Asn Cys Thr Phe Ile Asn Thr Val Phe Tyr Asn Thr Asp

545 550 555 560

Leu Phe Glu Tyr Lys Phe Val Asn Ser Arg Leu Ile Asn Ser Thr Phe

565 570 575

Leu His Asn Lys Glu Gly Cys Pro Leu Asp Val Thr Gly Thr Gly Glu

580 585 590

Gly Ala Tyr Met Val Tyr Phe Val Ser Phe Leu Gly Thr Leu Ala Val

595 600 605

Leu Pro Gly Asn Ile Val Ser Ala Leu Leu Met Asp Lys Ile Gly Arg

610 615 620

Leu Arg Met Leu Ala Gly Ser Ser Val Met Ser Cys Val Ser Cys Phe

625 630 635 640

Phe Leu Ser Phe Gly Asn Ser Glu Ser Ala Met Ile Ala Leu Leu Cys

645 650 655

Leu Phe Gly Gly Val Ser Ile Ala Ser Trp Asn Ala Leu Asp Val Leu

660 665 670

Thr Val Glu Leu Tyr Pro Ser Asp Lys Arg Thr Thr Ala Phe Gly Phe

675 680 685

Leu Asn Ala Leu Cys Lys Leu Ala Ala Val Leu Gly Ile Ser Ile Phe

690 695 700

Thr Ser Phe Val Gly Ile Thr Lys Ala Ala Pro Ile Leu Phe Ala Ser

705 710 715 720

Ala Ala Leu Ala Leu Gly Ser Ser Leu Ala Leu Lys Leu Pro Glu Thr

725 730 735

Arg Gly Gln Val Leu Gln

740

<210> 9

<211> 727

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 9

Met Glu Asp Ser Tyr Lys Asp Arg Thr Ser Leu Met Lys Gly Ala Lys

1 5 10 15

Asp Ile Ala Arg Glu Val Lys Lys Gln Thr Val Lys Lys Val Asn Gln

20 25 30

Ala Val Asp Arg Ala Gln Asp Glu Tyr Thr Gln Arg Ser Tyr Ser Arg

35 40 45

Phe Gln Asp Glu Glu Asp Asp Asp Tyr Tyr Pro Ala Gly Glu Thr

50 55 60

Tyr Asn Gly Glu Ala Asn Asp Asp Glu Gly Ser Ser Glu Ala Thr Glu

65 70 75 80

Gly His Asp Glu Asp Asp Glu Ile Tyr Glu Gly Glu Tyr Gln Gly Ile

85 90 95

Pro Ser Met Asn Gln Ala Lys Asp Ser Ile Val Ser Val Gly Gln Pro

100 105 110

Lys Gly Asp Glu Tyr Lys Asp Arg Arg Glu Leu Glu Ser Glu Arg Arg

115 120 125

Ala Asp Glu Glu Glu Leu Ala Gln Gln Tyr Glu Leu Ile Ile Gln Glu

130 135 140

Cys Gly His Gly Arg Phe Gln Trp Ala Leu Phe Phe Val Leu Gly Met

145 150 155 160

Ala Leu Met Ala Asp Gly Val Glu Val Phe Val Val Gly Phe Val Leu

165 170 175

Pro Ser Ala Glu Thr Asp Leu Cys Ile Pro Asn Ser Gly Ser Gly Trp

180 185 190

Leu Gly Ser Ile Val Tyr Leu Gly Met Met Val Gly Ala Phe Phe Trp

195 200 205

Gly Gly Leu Ala Asp Lys Val Gly Arg Lys Gln Ser Leu Leu Ile Cys

210 215 220

Met Ser Val Asn Gly Phe Phe Ala Phe Leu Ser Ser Phe Val Gln Gly

225 230 235 240

Tyr Gly Phe Phe Leu Phe Cys Arg Leu Leu Ser Gly Phe Gly Ile Gly

245 250 255

Gly Ala Ile Pro Thr Val Phe Ser Tyr Phe Ala Glu Val Leu Ala Arg

260 265 270

Glu Lys Arg Gly Glu His Leu Ser Trp Leu Cys Met Phe Trp Met Ile

275 280 285

Gly Gly Ile Tyr Ala Ser Ala Met Ala Trp Ala Ile Ile Pro His Tyr

290 295 300

Gly Trp Ser Phe Ser Met Gly Ser Ala Tyr Gln Phe His Ser Trp Arg

305 310 315 320

Val Phe Val Ile Val Cys Ala Leu Pro Cys Val Ser Ser Val Val Ala

325 330 335

Leu Thr Phe Met Pro Glu Ser Pro Arg Phe Leu Leu Glu Val Gly Lys

340 345 350

His Asp Glu Ala Trp Met Ile Leu Lys Leu Ile His Asp Thr Asn Met

355 360 365

Arg Ala Arg Gly Gln Pro Glu Lys Val Phe Thr Val Asn Lys Ile Lys

370 375 380

Thr Pro Lys Gln Ile Asp Glu Leu Ile Glu Ile Glu Ser Asp Thr Gly

385 390 395 400

Thr Trp Tyr Arg Arg Cys Phe Val Arg Ile Arg Thr Glu Leu Tyr Gly

405 410 415

Ile Trp Leu Thr Phe Met Arg Cys Phe Asn Tyr Pro Val Arg Asp Asn

420 425 430

Thr Ile Lys Leu Thr Ile Val Trp Phe Thr Leu Ser Phe Gly Tyr Tyr

435 440 445

Gly Leu Ser Val Trp Phe Pro Asp Val Ile Lys Pro Leu Gln Ser Asp

450 455 460

Glu Tyr Ala Leu Leu Thr Arg Asn Val Glu Arg Asp Lys Tyr Ala Asn

465 470 475 480

Phe Thr Ile Asn Phe Thr Met Glu Asn Gln Ile His Thr Gly Met Glu

485 490 495

Tyr Asp Asn Gly Arg Phe Ile Gly Val Lys Phe Lys Ser Val Thr Phe

500 505 510

Lys Asp Ser Val Phe Lys Ser Cys Thr Phe Glu Asp Val Thr Ser Val

515 520 525

Asn Thr Tyr Phe Lys Asn Cys Thr Phe Ile Asp Thr Val Phe Asp Asn

530 535 540

Thr Asp Phe Glu Pro Tyr Lys Phe Ile Asp Ser Glu Phe Lys Asn Cys

545 550 555 560

Ser Phe Phe His Asn Lys Thr Gly Cys Gln Ile Thr Phe Asp Asp Asp

565 570 575

Tyr Ser Ala Tyr Trp Ile Tyr Phe Val Asn Phe Leu Gly Thr Leu Ala

580 585 590

Val Leu Pro Gly Asn Ile Val Ser Ala Leu Leu Met Asp Arg Ile Gly

595 600 605

Arg Leu Thr Met Leu Gly Gly Ser Met Val Leu Ser Gly Ile Ser Cys

610 615 620

Phe Phe Leu Trp Phe Gly Thr Ser Glu Ser Met Met Ile Gly Met Leu

625 630 635 640

Cys Leu Tyr Asn Gly Leu Thr Ile Ser Ala Trp Asn Ser Leu Asp Val

645 650 655

Val Thr Val Glu Leu Tyr Pro Thr Asp Arg Arg Ala Thr Gly Phe Gly

660 665 670

Phe Leu Asn Ala Leu Cys Lys Ala Ala Ala Val Leu Gly Asn Leu Ile

675 680 685

Phe Gly Ser Leu Val Ser Ile Thr Lys Ser Ile Pro Ile Leu Leu Ala

690 695 700

Ser Thr Val Leu Val Cys Gly Gly Leu Val Gly Leu Cys Leu Pro Asp

705 710 715 720

Thr Arg Thr Gln Val Leu Met

725

<210> 10

<211> 683

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 10

Met Asp Asp Tyr Lys Tyr Gln Asp Asn Tyr Gly Gly Tyr Ala Pro Ser

1 5 10 15

Asp Gly Tyr Tyr Arg Gly Asn Glu Ser Asn Pro Glu Glu Asp Ala Gln

20 25 30

Ser Asp Val Thr Glu Gly His Asp Glu Glu Asp Glu Ile Tyr Glu Gly

35 40 45

Glu Tyr Gln Gly Ile Pro His Pro Asp Asp Val Lys Ala Lys Gln Ala

50 55 60

Lys Met Ala Pro Ser Arg Met Asp Ser Leu Arg Gly Gln Thr Asp Leu

65 70 75 80

Met Ala Glu Arg Leu Glu Asp Glu Glu Gln Leu Ala His Gln Tyr Glu

85 90 95

Thr Ile Met Asp Glu Cys Gly His Gly Arg Phe Gln Trp Ile Leu Phe

100 105 110

Phe Val Leu Gly Leu Ala Leu Met Ala Asp Gly Val Glu Val Phe Val

115 120 125

Val Ser Phe Ala Leu Pro Ser Ala Glu Lys Asp Met Cys Leu Ser Ser

130 135 140

Ser Lys Lys Gly Met Leu Gly Met Ile Val Tyr Leu Gly Met Met Ala

145 150 155 160

Gly Ala Phe Ile Leu Gly Gly Leu Ala Asp Lys Leu Gly Arg Lys Arg

165 170 175

Val Leu Ser Met Ser Leu Ala Val Asn Ala Ser Phe Ala Ser Leu Ser

180 185 190

Ser Phe Val Gln Gly Tyr Gly Ala Phe Leu Phe Cys Arg Leu Ile Ser

195 200 205

Gly Ile Gly Ile Gly Gly Ala Leu Pro Ile Val Phe Ala Tyr Phe Ser

210 215 220

Glu Phe Leu Ser Arg Glu Lys Arg Gly Glu His Leu Ser Trp Leu Gly

225 230 235 240

Ile Phe Trp Met Thr Gly Gly Leu Tyr Ala Ser Ala Met Ala Trp Ser

245 250 255

Ile Ile Pro His Tyr Gly Trp Gly Phe Ser Met Gly Thr Asn Tyr His

260 265 270

Phe His Ser Trp Arg Val Phe Val Ile Val Cys Ala Leu Pro Cys Thr

275 280 285

Val Ser Met Val Ala Leu Lys Phe Met Pro Glu Ser Pro Arg Phe Leu

290 295 300

Leu Glu Met Gly Lys His Asp Glu Ala Trp Met Ile Leu Lys Gln Val

305 310 315 320

His Asp Thr Asn Met Arg Ala Lys Gly Thr Pro Glu Lys Val Phe Thr

325 330 335

Val Ser Asn Ile Lys Thr Pro Lys Gln Met Asp Glu Phe Ile Glu Ile

340 345 350

Gln Ser Ser Thr Gly Thr Trp Tyr Gln Arg Trp Leu Val Arg Phe Lys

355 360 365

Thr Ile Phe Lys Gln Val Trp Asp Asn Ala Leu Tyr Cys Val Met Gly

370 375 380

Pro Tyr Arg Met Asn Thr Leu Ile Leu Ala Val Val Trp Phe Ala Met

385 390 395 400

Ala Phe Ser Tyr Tyr Gly Leu Thr Val Trp Phe Pro Asp Met Ile Arg

405 410 415

Tyr Phe Gln Asp Glu Glu Tyr Lys Ser Lys Met Lys Val Phe Phe Gly

420 425 430

Glu His Val Tyr Gly Ala Thr Ile Asn Phe Thr Met Glu Asn Gln Ile

435 440 445

His Gln His Gly Lys Leu Val Asn Asp Lys Phe Thr Arg Met Tyr Phe

450 455 460

Lys His Val Leu Phe Glu Asp Thr Phe Phe Asp Glu Cys Tyr Phe Glu

465 470 475 480

Asp Val Thr Ser Thr Asp Thr Tyr Phe Lys Asn Cys Thr Ile Glu Ser

485 490 495

Thr Ile Phe Tyr Asn Thr Asp Leu Tyr Glu His Lys Phe Ile Asn Cys

500 505 510

Arg Phe Ile Asn Ser Thr Phe Leu Glu Gln Lys Glu Gly Cys His Met

515 520 525

Asp Leu Glu Gln Asp Asn Asp Phe Leu Ile Tyr Leu Val Ser Phe Leu

530 535 540

Gly Ser Leu Ser Val Leu Pro Gly Asn Ile Ile Ser Ala Leu Leu Met

545 550 555 560

Asp Arg Ile Gly Arg Leu Lys Met Ile Gly Gly Ser Met Leu Ile Ser

565 570 575

Ala Val Cys Cys Phe Phe Leu Phe Phe Gly Asn Ser Glu Ser Ala Met

580 585 590

Ile Gly Trp Gln Cys Leu Phe Cys Gly Thr Ser Ile Ala Ala Trp Asn

595 600 605

Ala Leu Asp Val Ile Thr Val Glu Leu Tyr Pro Thr Asn Gln Arg Ala

610 615 620

Thr Ala Phe Gly Ile Leu Asn Gly Leu Cys Lys Phe Gly Ala Ile Leu

625 630 635 640

Gly Asn Thr Ile Phe Ala Ser Phe Val Gly Ile Thr Lys Val Val Pro

645 650 655

Ile Leu Leu Ala Ala Ala Ser Leu Val Gly Gly Gly Leu Ile Ala Leu

660 665 670

Arg Leu Pro Glu Thr Arg Glu Gln Val Leu Met

675 680

<210> 11

<211> 128

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 11

Phe Pro Asp Met Ile Arg His Leu Gln Ala Val Asp Tyr Ala Ser Arg

1 5 10 15

Thr Lys Val Phe Pro Gly Glu Arg Val Glu His Val Thr Phe Asn Phe

20 25 30

Thr Leu Glu Asn Gln Ile His Arg Gly Gly Gln Tyr Phe Asn Asp Lys

35 40 45

Phe Ile Gly Leu Arg Leu Lys Ser Val Ser Phe Glu Asp Ser Leu Phe

50 55 60

Glu Glu Cys Tyr Phe Glu Asp Val Thr Ser Ser Asn Thr Phe Phe Arg

65 70 75 80

Asn Cys Thr Phe Ile Asn Thr Val Phe Tyr Asn Thr Asp Leu Phe Glu

85 90 95

Tyr Lys Phe Val Asn Ser Arg Leu Ile Asn Ser Thr Phe Leu His Asn

100 105 110

Lys Glu Gly Cys Pro Leu Asp Val Thr Gly Thr Gly Glu Gly Ala Tyr

115 120 125

<210> 12

<211> 130

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 12

Trp Phe Pro Asp Met Ile Arg Tyr Phe Gln Asp Glu Glu Tyr Lys Ser

1 5 10 15

Lys Met Lys Val Phe Phe Gly Glu His Val Tyr Gly Ala Thr Ile Asn

20 25 30

Phe Thr Met Glu Asn Gln Ile His Gln His Gly Lys Leu Val Asn Asp

35 40 45

Lys Phe Thr Arg Met Tyr Phe Lys His Val Leu Phe Glu Asp Thr Phe

50 55 60

Phe Asp Glu Cys Tyr Phe Glu Asp Val Thr Ser Thr Asp Thr Tyr Phe

65 70 75 80

Lys Asn Cys Thr Ile Glu Ser Thr Ile Phe Tyr Asn Thr Asp Leu Tyr

85 90 95

Glu His Lys Phe Ile Asn Cys Arg Phe Ile Asn Ser Thr Phe Leu Glu

100 105 110

Gln Lys Glu Gly Cys His Met Asp Leu Glu Gln Asp Asn Asp Phe Leu

115 120 125

Ile Tyr

130

<210> 13

<211> 128

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 13

Trp Phe Pro Asp Val Ile Lys Pro Leu Gln Ser Asp Glu Tyr Ala Leu

1 5 10 15

Leu Thr Arg Asn Val Glu Arg Asp Lys Tyr Ala Asn Phe Thr Ile Asn

20 25 30

Phe Thr Met Glu Asn Gln Ile His Thr Gly Met Glu Tyr Asp Asn Gly

35 40 45

Arg Phe Ile Gly Val Lys Phe Lys Ser Val Thr Phe Lys Asp Ser Val

50 55 60

Phe Lys Ser Cys Thr Phe Glu Asp Val Thr Ser Val Asn Thr Tyr Phe

65 70 75 80

Lys Asn Cys Thr Phe Ile Asp Thr Val Phe Asp Asn Thr Asp Phe Glu

85 90 95

Pro Tyr Lys Phe Ile Asp Ser Glu Phe Lys Asn Cys Ser Phe Phe His

100 105 110

Asn Lys Thr Gly Cys Gln Ile Thr Phe Asp Asp Asp Tyr Ser Ala Tyr

115 120 125

<210> 14

<211> 422

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 14

Met Val Ser Glu Ser His His Glu Ala Leu Ala Ala Pro Pro Val Thr

1 5 10 15

Thr Val Ala Thr Val Leu Pro Ser Asn Ala Thr Glu Pro Ala Ser Pro

20 25 30

Gly Glu Gly Lys Glu Asp Ala Phe Ser Lys Leu Lys Glu Lys Phe Met

35 40 45

Asn Glu Leu His Lys Ile Pro Leu Pro Pro Trp Ala Leu Ile Ala Ile

50 55 60

Ala Ile Val Ala Val Leu Leu Val Leu Thr Cys Cys Phe Cys Ile Cys

65 70 75 80

Lys Lys Cys Leu Phe Lys Lys Lys Asn Lys Lys Lys Gly Lys Glu Lys

85 90 95

Gly Gly Lys Asn Ala Ile Asn Met Lys Asp Val Lys Asp Leu Gly Lys

100 105 110

Thr Met Lys Asp Gln Ala Leu Lys Asp Asp Asp Ala Glu Thr Gly Leu

115 120 125

Thr Asp Gly Glu Glu Lys Glu Glu Pro Lys Glu Glu Glu Lys Leu Gly

130 135 140

Lys Leu Gln Tyr Ser Leu Asp Tyr Asp Phe Gln Asn Asn Gln Leu Leu

145 150 155 160

Val Gly Ile Ile Gln Ala Ala Glu Leu Pro Ala Leu Asp Met Gly Gly

165 170 175

Thr Ser Asp Pro Tyr Val Lys Val Phe Leu Leu Pro Asp Lys Lys Lys

180 185 190

Lys Phe Glu Thr Lys Val His Arg Lys Thr Leu Asn Pro Val Phe Asn

195 200 205

Glu Gln Phe Thr Phe Lys Val Pro Tyr Ser Glu Leu Gly Gly Lys Thr

210 215 220

Leu Val Met Ala Val Tyr Asp Phe Asp Arg Phe Ser Lys His Asp Ile

225 230 235 240

Ile Gly Glu Phe Lys Val Pro Met Asn Thr Val Asp Phe Gly His Val

245 250 255

Thr Glu Glu Trp Arg Asp Leu Gln Ser Ala Glu Lys Glu Glu Gln Glu

260 265 270

Lys Leu Gly Asp Ile Cys Phe Ser Leu Arg Tyr Val Pro Thr Ala Gly

275 280 285

Lys Leu Thr Val Val Ile Leu Glu Ala Lys Asn Leu Lys Lys Met Asp

290 295 300

Val Gly Gly Leu Ser Asp Pro Tyr Val Lys Ile His Leu Met Gln Asn

305 310 315 320

Gly Lys Arg Leu Lys Lys Lys Lys Thr Thr Ile Lys Lys Asn Thr Leu

325 330 335

Asn Pro Tyr Tyr Asn Glu Ser Phe Ser Phe Glu Val Pro Phe Glu Gln

340 345 350

Ile Gln Lys Val Gln Val Val Val Thr Val Leu Asp Tyr Asp Lys Ile

355 360 365

Gly Lys Asn Asp Ala Ile Gly Lys Val Phe Val Gly Tyr Asn Ser Thr

370 375 380

Gly Ala Glu Leu Arg His Trp Ser Asp Met Leu Ala Asn Pro Arg Arg

385 390 395 400

Pro Ile Ala Gln Trp His Thr Leu Gln Val Glu Glu Glu Val Asp Ala

405 410 415

Met Leu Ala Val Lys Lys

420

<210> 15

<211> 419

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 15

Met Arg Asn Ile Phe Lys Arg Asn Gln Glu Pro Ile Val Ala Pro Ala

1 5 10 15

Thr Thr Thr Ala Thr Met Pro Ile Gly Pro Val Asp Asn Ser Thr Glu

20 25 30

Ser Gly Gly Ala Gly Glu Ser Gln Glu Asp Met Phe Ala Lys Leu Lys

35 40 45

Glu Lys Leu Phe Asn Glu Ile Asn Lys Ile Pro Leu Pro Pro Trp Ala

50 55 60

Leu Ile Ala Ile Ala Val Val Ala Gly Leu Leu Leu Leu Thr Cys Cys

65 70 75 80

Phe Cys Ile Cys Lys Lys Cys Cys Cys Lys Lys Lys Lys Asn Lys Lys

85 90 95

Glu Lys Gly Lys Gly Met Lys Asn Ala Met Asn Met Lys Asp Met Lys

100 105 110

Gly Gly Gln Asp Asp Asp Asp Ala Glu Thr Gly Leu Thr Glu Gly Glu

115 120 125

Gly Glu Gly Glu Glu Glu Lys Glu Pro Glu Asn Leu Gly Lys Leu Gln

130 135 140

Phe Ser Leu Asp Tyr Asp Phe Gln Ala Asn Gln Leu Thr Val Gly Val

145 150 155 160

Leu Gln Ala Ala Glu Leu Pro Ala Leu Asp Met Gly Gly Thr Ser Asp

165 170 175

Pro Tyr Val Lys Val Phe Leu Leu Pro Asp Lys Lys Lys Lys Tyr Glu

180 185 190

Thr Lys Val His Arg Lys Thr Leu Asn Pro Ala Phe Asn Glu Thr Phe

195 200 205

Thr Phe Lys Val Pro Tyr Gln Glu Leu Gly Gly Lys Thr Leu Val Met

210 215 220

Ala Ile Tyr Asp Phe Asp Arg Phe Ser Lys His Asp Ile Ile Gly Glu

225 230 235 240

Val Lys Val Pro Met Asn Thr Val Asp Leu Gly Gln Pro Ile Glu Glu

245 250 255

Trp Arg Asp Leu Gln Gly Gly Glu Lys Glu Glu Pro Glu Lys Leu Gly

260 265 270

Asp Ile Cys Thr Ser Leu Arg Tyr Val Pro Thr Ala Gly Lys Leu Thr

275 280 285

Val Cys Ile Leu Glu Ala Lys Asn Leu Lys Lys Met Asp Val Gly Gly

290 295 300

Leu Ser Asp Pro Tyr Val Lys Ile His Leu Met Gln Asn Gly Lys Arg

305 310 315 320

Leu Lys Lys Lys Lys Thr Thr Val Lys Lys Lys Thr Leu Asn Pro Tyr

325 330 335

Phe Asn Glu Ser Phe Ser Phe Glu Ile Pro Phe Glu Gln Ile Gln Lys

340 345 350

Val Gln Val Val Val Thr Val Leu Asp Tyr Asp Lys Leu Gly Lys Asn

355 360 365

Glu Ala Ile Gly Lys Ile Phe Val Gly Ser Asn Ala Thr Gly Thr Glu

370 375 380

Leu Arg His Trp Ser Asp Met Leu Ala Asn Pro Arg Arg Pro Ile Ala

385 390 395 400

Gln Trp His Ser Leu Lys Pro Glu Glu Glu Val Asp Ala Leu Leu Gly

405 410 415

Lys As Lys

<210> 16

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 16

Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys

1 5 10 15

Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly

20 25 30

Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys

35 40 45

Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro

50 55 60

Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile

65 70 75 80

Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg

85 90 95

Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr

100 105 110

Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr

115 120 125

Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

130 135 140

Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly

145 150 155 160

Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala

165 170 175

Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly

180 185 190

Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp

195 200 205

Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr

210 215 220

Gly Gly Met Asp Glu Leu Tyr Lys

225 230

<210> 17

<211> 248

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 17

Met Ala Ser Leu Pro Ala Thr His Glu Leu His Ile Phe Gly Ser Ile

1 5 10 15

Asn Gly Val Asp Phe Asp Met Val Gly Gln Gly Thr Gly Asn Pro Asn

20 25 30

Asp Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr Lys Gly Asp Leu Gln

35 40 45

Phe Ser Pro Trp Ile Leu Val Pro His Ile Gly Tyr Gly Phe His Gln

50 55 60

Tyr Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe Gln Ala Ala Met Val

65 70 75 80

Asp Gly Ser Gly Tyr Gln Val His Arg Thr Met Gln Phe Glu Asp Gly

85 90 95

Ala Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His Ile

100 105 110

Lys Gly Glu Ala Gln Val Lys Gly Thr Gly Phe Pro Ala Asp Gly Pro

115 120 125

Val Met Thr Asn Ser Leu Thr Ala Ala Asp Trp Cys Arg Ser Lys Lys

130 135 140

Thr Tyr Pro Asn Asp Lys Thr Ile Ile Ser Thr Phe Lys Trp Ser Tyr

145 150 155 160

Thr Thr Gly Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr Thr Tyr

165 170 175

Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln Pro Met

180 185 190

Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn

195 200 205

Phe Lys Glu Trp Gln Lys Ala Phe Thr Gly Phe Glu Asp Phe Val Gly

210 215 220

Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln

225 230 235 240

Gly Gly Val Ser Ser Leu Phe Gln

245

<210> 18

<211> 223

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 18

Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val

1 5 10 15

Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr

20 25 30

Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro

35 40 45

Val Pro Trp Pro Thr Leu Val Thr Thr Leu Ser Trp Gly Val Gln Cys

50 55 60

Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser

65 70 75 80

Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp

85 90 95

Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr

100 105 110

Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly

115 120 125

Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Phe Ser Asp Asn Val

130 135 140

Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys

145 150 155 160

Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr

165 170 175

Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn

180 185 190

His Tyr Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys

195 200 205

Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Leu

210 215 220

<210> 19

<211> 206

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 19

Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg

1 5 10 15

Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met

20 25 30

Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val

35 40 45

Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met

50 55 60

Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp

65 70 75 80

Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys

85 90 95

Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val

100 105 110

Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala

115 120 125

Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn

130 135 140

Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu

145 150 155 160

Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg

165 170 175

Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile

180 185 190

Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly

195 200 205

<210> 20

<211> 264

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 20

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210> 21

<211> 199

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 21

Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val

1 5 10 15

Pro Arg Ala Val Arg Thr Leu Ala Ala Ala Phe Ala Asp Tyr Pro Ala

20 25 30

Thr Arg His Thr Val Asp Pro Asp Arg His Ile Glu Arg Val Thr Glu

35 40 45

Leu Gln Glu Leu Phe Leu Thr Arg Val Gly Leu Asp Ile Gly Lys Val

50 55 60

Trp Val Ala Asp Asp Gly Ala Ala Val Ala Val Trp Thr Thr Pro Glu

65 70 75 80

Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala

85 90 95

Glu Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu

100 105 110

Leu Ala Pro His Arg Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val

115 120 125

Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val

130 135 140

Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu

145 150 155 160

Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe

165 170 175

Thr Val Thr Ala Asp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys

180 185 190

Met Thr Arg Lys Pro Gly Ala

195

<210> 22

<211> 169

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 22

His Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr

1 5 10 15

Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln

20 25 30

Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly

35 40 45

Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly

50 55 60

Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val

65 70 75 80

Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr

85 90 95

Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg

100 105 110

Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr

115 120 125

Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn

130 135 140

Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly

145 150 155 160

Trp Arg Leu Cys Glu Arg Ile Leu Ala

165

<---

Claims (80)

1. A cell for the identification of a clostridial neurotoxin composition for therapeutic and cosmetic use, wherein said cell has been genetically engineered to contain: a. an exogenous nucleic acid that provides for the expression or overexpression of a clostridial neurotoxin receptor or a variant thereof, or a fragment thereof, that has the ability to bind a clostridial neurotoxin; b. an exogenous nucleic acid that provides for the expression or overexpression of a ganglioside having binding affinity for a clostridial neurotoxin, and c. an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin. 2. An in vitro method for identifying the composition of a clostridial neurotoxin for therapeutic and cosmetic use, wherein said method comprises: a. creation of a cell that has i. an exogenous nucleic acid that provides for the expression or overexpression of a clostridial neurotoxin receptor or a variant thereof, or a fragment thereof, that has the ability to bind a clostridial neurotoxin; ii. an exogenous nucleic acid that provides for the expression or overexpression of a ganglioside having binding affinity for a clostridial neurotoxin, and iii. an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin; b. contacting the said cell with a clostridial neurotoxin composition; c. comparing the level of cleavage of the indicator protein after exposure to the clostridial neurotoxin formulation with the level of cleavage before exposure to the clostridial neurotoxin formulation; and d. identification of a clostridial neurotoxin formulation as suitable for therapeutic and/or cosmetic use where the level of degradation of the indicator protein is increased following exposure; or e. identification of a clostridial neurotoxin composition as unsuitable for therapeutic and/or cosmetic use when the level of indicator protein breakdown does not increase following exposure. 3. An in vitro method for identifying a composition of a clostridial neurotoxin that is suitable for therapeutic and cosmetic use, said method comprising: a. creation of a cell that has i. an exogenous nucleic acid that provides for the expression or overexpression of a Clostridial neurotoxin receptor or a variant thereof, or a fragment thereof, that has the ability to bind a Clostridial neurotoxin; ii. an exogenous nucleic acid that provides for the expression or overexpression of a ganglioside having binding affinity for a Clostridial neurotoxin, and iii. an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin; b. contacting the said cell with a clostridial neurotoxin composition; c. comparing the level of cleavage of the indicator protein following exposure to the clostridial neurotoxin formulation with the level of cleavage following exposure to a control clostridial neurotoxin formulation, wherein the control clostridial neurotoxin formulation is a clostridial neurotoxin formulation known to have cleavage activity and/or known to be suitable for therapeutic and/or cosmetic use; and d. identification of a clostridial neurotoxin formulation as suitable for therapeutic and/or cosmetic use when the level of cleavage of the indicator protein following exposure is increased or equivalent to the level of cleavage following exposure to a control clostridial neurotoxin formulation; or e. identifying a clostridial neurotoxin as unsuitable for therapeutic and/or cosmetic use when the level of degradation of the indicator protein after exposure is not increased or is not equivalent to the level of degradation after exposure to a control clostridial neurotoxin formulation. 4. An in vitro method for characterizing the activity of a clostridial neurotoxin composition, said method comprising: a. creation of a cell that has i. an exogenous nucleic acid that provides for the expression or overexpression of a clostridial neurotoxin receptor or a variant thereof, or a fragment thereof, that has the ability to bind a clostridial neurotoxin; ii. an exogenous nucleic acid that provides for the expression or overexpression of a ganglioside having binding affinity for a clostridial neurotoxin, and iii. an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin; b. contacting the said cell with a clostridial neurotoxin composition; c. comparing the level of cleavage of the indicator protein following exposure to the clostridial neurotoxin formulation with the level of cleavage following exposure to the clostridial neurotoxin formulation; and d. a characteristic of the presence of activity in the composition with an increase in the level of cleavage of the indicator protein after contact; or e. the characteristic of the absence of activity in the composition in the absence of an increase in the level of cleavage of the indicator protein after contact. 5. An in vitro method for characterizing the activity of a clostridial neurotoxin composition, said method comprising: a. creation of a cell that has i. an exogenous nucleic acid that provides for the expression or overexpression of a clostridial neurotoxin receptor or a variant thereof, or a fragment thereof, that has the ability to bind a clostridial neurotoxin; ii. an exogenous nucleic acid that provides for the expression or overexpression of a ganglioside having binding affinity for a clostridial neurotoxin, and iii. an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin; b. contacting the said cell with a clostridial neurotoxin composition; c. comparing the level of cleavage of the indicator protein after exposure to the clostridial neurotoxin formulation with the level of cleavage prior to exposure to a control clostridial neurotoxin formulation, where the control clostridial neurotoxin formulation is a clostridial neurotoxin formulation known to have cleavage activity; and d. a characteristic of the presence of activity in the formulation where the level of cleavage of the indicator protein after contact is increased or equivalent to the level of cleavage after contact with a control formulation of the clostridial neurotoxin, or e. a characteristic of inactivity where the level of cleavage of the indicator protein after exposure is not increased or is not equivalent to the level of cleavage after exposure to a control clostridial neurotoxin composition. 6. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell that overexpresses the receptor and ganglioside expresses more of the receptor and ganglioside compared to the natural target of the clostridial neurotoxin. 7. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell that overexpresses the receptor and the ganglioside expresses more of the receptor and ganglioside compared to the cell without the exogenous nucleic acid. 8. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell is a neural cell, a non-neuronal cell, a neuroendocrine cell, an embryonic kidney cell, a breast cancer cell, a neuroblastoma cell, or a neuroblastoma-glioma hybrid cell; preferably, a non-neuronal cell. 9. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell is a neuroblastoma cell or a neuroblastoma-glioma cell. 10. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell is a neuroblastoma-glioma cell. 11. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell is an NG108 cell. 12. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to overexpress the ganglioside. 13. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress GM1a, GD1a, GD1b, GT1b and/or GQ1b. 14. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress GD1a, GD1b and/or GT1b. 15. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress GD1b and/or GT1b. 16. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress a ganglioside synthesis pathway enzyme or a variant thereof, or a fragment thereof, having the catalytic activity of such an enzyme. 17. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress glucosylceramide synthase, GalT-I, GalNAcT, GM3 synthase, GD3 synthase, GT3 synthase, galactosylceramide synthase, GM4 synthase, GalT-II, ST-IV, or ST-V, or a variant or fragment thereof having the catalytic activity of such an enzyme. 18. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress GD3 synthase, or a variant or fragment thereof having the catalytic activity of GD3 synthase. 19. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress GD3 synthase. 20. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to overexpress a protein receptor or variant thereof, or fragment thereof, that has the ability to bind a clostridial neurotoxin. 21. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress SV2 or synaptotagmin, or a variant or fragment thereof, that has the ability to bind a clostridial neurotoxin. 22. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress SV2 or a variant or fragment thereof that has the ability to bind a clostridial neurotoxin. 23. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress SV2A or SV2C (preferably SV2A), or a variant or fragment thereof, that has the ability to bind a clostridial neurotoxin. 24. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress the fourth luminal domain of SV2A or SV2C. 25. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress the indicator protein. 26. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress a SNARE domain-containing indicator protein. 27. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress an indicator protein, wherein the indicator protein comprises an amino acid sequence of syntaxin, synaptobrevin, or SNAP-25, or a variant or fragment thereof, that is susceptible to proteolysis by the protease component of a wild-type clostridial neurotoxin. 28. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress a labeled indicator protein. 29. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress an indicator protein comprising an N-terminal tag and a C-terminal tag. 30. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress an indicator protein comprising the amino acid sequence of a fluorescent protein label. 31. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress an indicator protein comprising the amino acid sequence of mScarlet and the amino acid sequence of NeonGreen. 32. The cell of claim 1 or the method of any one of claims 2-5, wherein the cell has been genetically engineered to express or overexpress an indicator protein comprising mScarlet as an N-terminal tag and NeonGreen as a C-terminal tag. 33. A method for producing a cell according to any one of claims 1 or 6-32, wherein the method comprises introducing into the cell a nucleic acid encoding a receptor for a clostridial neurotoxin or a variant thereof, or a fragment thereof that has the ability to bind a clostridial neurotoxin, a nucleic acid encoding an enzyme of the ganglioside synthesis pathway, or a variant thereof, or a fragment thereof that has the catalytic activity of such an enzyme, and a nucleic acid that provides expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin. 34. The method according to claim 33, wherein the method further comprises introducing into the cell a nucleic acid encoding an indicator protein. 35. The method according to claims 33, 34, wherein the nucleic acid is introduced by transfection. 36. A method of assaying for determining the activity of a modified or recombinant neurotoxin, comprising contacting a cell according to any one of claims 1 or 6-32 with a modified or recombinant neurotoxin under conditions and for a period of time that allows the protease domain of a wild-type clostridial neurotoxin to cleave an indicator protein in the cell, and determining the activity of the modified or recombinant neurotoxin upon detection of a product resulting from the cleavage of such indicator protein. 37. The method according to claim 36, wherein the full-length indicator protein is difficult to destroy in the cell, but after its cleavage, one of the resulting fragments is easily destroyed. 38. The method according to any one of claims 36-37, wherein the indicator protein is labeled. 39. The method according to any one of claims 36-38, wherein the indicator protein comprises a C-terminal tag, and the full-length indicator protein is difficult to degrade in the cell, but after its cleavage the resulting C-terminal fragment is easily degraded, and the destruction of the C-terminal fragment leads to the destruction of the C-terminal tag. 40. The method according to any one of claims 36-39, wherein the indicator protein comprises a C-terminal tag, and the full-length indicator protein is difficult to degrade in the cell, but after its cleavage the resulting C-terminal fragment is easily degraded, and the destruction of the C-terminal fragment leads to the destruction of the C-terminal tag, and the cleavage of the indicator protein is determined by measuring the signal from the C-terminal tag after contacting the cell with a modified or recombinant neurotoxin. 41. The method according to claim 36, wherein after such contact the cell is lysed and the resulting cell lysate is contacted with antibodies during Western blotting. 42. A method for constructing a cell suitable for determining the composition of a clostridial neurotoxin that is suitable for therapeutic and/or cosmetic use, wherein the method comprises: a. work with the insertion cell: i. an exogenous nucleic acid that provides for the expression or overexpression of a clostridial neurotoxin receptor or a variant thereof, or a fragment thereof, that has the ability to bind a clostridial neurotoxin; ii. an exogenous nucleic acid that provides for the expression or overexpression of a ganglioside having binding affinity for a clostridial neurotoxin, and iii. an exogenous nucleic acid that provides expression or overexpression of an indicator protein cleaved by a clostridial neurotoxin.

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